[
  {
    "key": "2Q7HFERL",
    "title": "Convalescent Plasma Therapy for COVID-19: State of the Art",
    "abstract": "SUMMARY Convalescent plasma (CP) therapy has been used since the early 1900s to treat emerging infectious diseases; its ef\ufb01cacy was later associated with the evidence that polyclonal neutralizing antibodies can reduce the duration of viremia. Recent large outbreaks of viral diseases for which effective antivirals or vaccines are still lacking has renewed the interest in CP as a life-saving treatment. The ongoing COVID-19 pandemic has led to the scaling up of CP therapy to unprecedented levels. Compared with historical usage, pathogen reduction technologies have now added an extra layer of safety to the use of CP, and new manufacturing approaches are being explored. This review summarizes historical settings of application, with a focus on betacoronaviruses, and surveys current approaches for donor selection and CP collection, pooling technologies, pathogen inactivation systems, and banking of CP. We additionally list the ongoing registered clinical trials for CP throughout the world and discuss the trial results published thus far.",
    "full_text": "REVIEW\ncrossm\n\nConvalescent Plasma Therapy for COVID-19: State of the Art\nDaniele Focosi,a Arthur O. Anderson,b Julian W. Tang,c Marco Tuccorid,e\naNorth-Western Tuscany Blood Bank, Pisa University Hospital, Pisa, Italy bDepartment of Respiratory Mucosal Immunity, US Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USA cRespiratory Sciences, University of Leicester, Leicester, United Kingdom dDivision of Pharmacology and Pharmacovigilance, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy eUnit of Adverse Drug Reaction Monitoring, Pisa University Hospital, Pisa, Italy\n\nSUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 CP DONOR RECRUITMENT STRATEGIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 CONVALESCENT PLASMA AND PATHOGEN INACTIVATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3\nTechnologies To Virally Reduce Plasma (Pathogen Inactivation) . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5\nLarge-pool products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 MPFS into immunoglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 CP BANKING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 LESSONS FROM SARS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 LESSONS FROM MERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 CONVALESCENT PLASMA FOR COVID-19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 MONITORING RESPONSE TO CP TREATMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 CONCERNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 SIDE BENEFITS FROM CP IN COVID-19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 AUTHOR BIOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17\n\nSUMMARY Convalescent plasma (CP) therapy has been used since the early 1900s to treat emerging infectious diseases; its ef\ufb01cacy was later associated with the evidence that polyclonal neutralizing antibodies can reduce the duration of viremia. Recent large outbreaks of viral diseases for which effective antivirals or vaccines are still lacking has renewed the interest in CP as a life-saving treatment. The ongoing COVID-19 pandemic has led to the scaling up of CP therapy to unprecedented levels. Compared with historical usage, pathogen reduction technologies have now added an extra layer of safety to the use of CP, and new manufacturing approaches are being explored. This review summarizes historical settings of application, with a focus on betacoronaviruses, and surveys current approaches for donor selection and CP collection, pooling technologies, pathogen inactivation systems, and banking of CP. We additionally list the ongoing registered clinical trials for CP throughout the world and discuss the trial results published thus far.\nKEYWORDS Ebola virus disease, Middle East respiratory syndrome, antibodydependent enhancement, convalescent blood product, convalescent plasma, convalescent whole blood, coronavirus disease 2019, enzyme-linked immunosorbent assay, intravenous immunoglobulins, plaque reduction neutralization test, SARS\n\nINTRODUCTION\nThe recent COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1) has demonstrated the fragility of our health systems in tackling emergency situations related to the spread of new infectious agents that require the rapid development of effective care strategies. Unfortunately, there are several potentially pandemic viruses, such as \ufb02aviviruses (e.g., West Nile virus [WNV],\n\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nClinical Microbiology Reviews\n\nCitation Focosi D, Anderson AO, Tang JW, Tuccori M. 2020. Convalescent plasma therapy for COVID-19: state of the art. Clin Microbiol Rev 33:e00072-20. https://doi.org/10.1128/CMR .00072-20. Copyright \u00a9 2020 American Society for Microbiology. All Rights Reserved. Address correspondence to Daniele Focosi, daniele.focosi@gmail.com. Published 12 August 2020\ncmr.asm.org 1\n\nFocosi et al.\ndengue virus, and Zika virus) (2), chikungunya virus (3), in\ufb02uenza viruses A [e.g., A(H1N1) and A(H5N1)] (4), Ebola virus (EBOV) (5), and respiratory betacoronaviruses (SARS-CoV and Middle East respiratory syndrome-CoV [MERS-CoV]), which could put us in situations very similar to the situation with the current pandemic and which require the development of speci\ufb01c intervention protocols.\nWhile vaccination strategy is undoubtedly a viable goal, development of a vaccine requires a time frame not compatible with an emergency situation. It is also a prophylactic approach that has no use in the therapeutic setting. On the other hand, the use of antivirals is valuable for the therapeutic setting (6, 7). For the limited number of antiviral agents currently available, unless provided free of charge to developing countries, \ufb01nancial cost is an issue. Additionally, manufacturing is hard to scale up in short time frames.\nIn situations in which the new pathogen is able to induce an immune response with the production of neutralizing antibodies, passive transfusion of convalescent blood products (CBPs), in particular, convalescent plasma (CP), has proven to be a winning and logistically feasible therapeutic strategy (8). CBPs can be manufactured by collecting whole blood or apheresis plasma from a convalescent donor. This approach has been used since 1900 (9), and previous experiences have been reported elsewhere (10).\nThe main accepted mechanism of action for CBP therapy is clearance of viremia, which typically happens 10 to 14 days after infection (11). So CBP has been typically administered after the appearance of early symptoms to maximize ef\ufb01cacy. Convalescent whole blood (CWB), in addition to antibodies, provides control of hemorrhagic events, as in Ebola virus disease, if transfusion occurs within 24 h to maintain viable platelets and clotting factors. Nevertheless, CP best \ufb01ts settings where only antibodies are required.\nIn this review, we have described current technologies for CP collection, manufacturing, pathogen inactivation, and banking of CP. Then we have summarized historical settings of CBP application, with a speci\ufb01c focus on applications for COVID-19 and other future pandemics. Several articles included in this review are available as preprints which have not yet passed peer review, as indicated in the reference section.\nCP DONOR RECRUITMENT STRATEGIES Convalescent donor testing for neutralizing antibodies is mandatory in upstream\ndonor selection. Donor selection is generally based on neutralizing antibody titer, as assessed with a plaque reduction neutralization test (PRNT) (12), which requires a viable isolate, replication-competent cell lines, and skilled personnel. Since PRNT takes time to be set up and requires expensive facilities, in resource-poor settings or in time-sensitive scenarios, collection based on a retrospective PRNT or, alternatively, on an enzymelinked immunosorbent assay (ELISA) targeting the recombinant receptor binding domains (RBDs) of the viral antireceptor has often been implemented; under these circumstances, studies have suggested that ELISA ratios/indexes have good correlations with PRNT titers; e.g., the Euroimmun ELISA IgG score detected 60% of samples with PRNT titers of \u03fe1:100, with 100% speci\ufb01city using a signal/cutoff reactivity index of 9.1 (13). The current understanding of neutralization suggests that the virus-blocking effect is related to the amount of antibodies against different epitopes coating the virion, whose stoichiometry is in turn affected by antibody concentration and af\ufb01nity.\nThe donor should preferably live in the same area as the intended recipient(s) to allow consideration of mutations of the target viral antigens. SARS-CoV-2 S protein has already mutated after a few months of viral circulation (14), with one mutation outside the receptor-binding motif (23403A\u00a1G single nucleotide polymorphism, corresponding to a D614G amino acid change) currently de\ufb01ning a dominant clade (15) characterized by reduced S1 shedding and increased infectivity (16). Nevertheless, it should be considered that preferring indigenous donors could represent a drawback in areas with epidemics of other infectious diseases (e.g., malaria).\nThree approaches are theoretically available to recruit CP donors, with each having pros and cons. The least cost-effective approach is screening the general regular blood\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nClinical Microbiology Reviews cmr.asm.org 2\n\nConvalescent Plasma Therapy for COVID-19\ndonor population for the presence of anti-SARS-CoV-2 antibodies. In areas of endemicity, such a strategy provides many \ufb01t donors with the additional bene\ufb01t of seroprevalence study in the general population (80% of cases being asymptomatic) but requires a large budget.\nAlternatively, recruitment of hospital-discharged patients is highly cost-effective (patients can be easily tested before discharge and tracked), but patients who have required hospitalization are highly likely to be elderly with comorbidities and, hence, un\ufb01t to donate.\nThe intermediate approach, whenever allowed by privacy regulations, is making calls to positive cases under home-based quarantine to solicit donations; given the large numbers of such cases, some of them are likely to be regular donors, and home-based convalescence suggests that they are \ufb01t enough to donate. Nevertheless, lessons from MERS (17) and preliminary evidence with COVID-19 (18\u201320) suggest that patients with mild symptoms may develop low-titer antibodies, making antibody titration even more important in the population-wide and home-based approaches. Plasma samples collected an average of 30 days after the onset of symptoms had undetectable half-maximal neutralizing titers in 18% of donors (21).\nUnder emergency settings, it has often happened that donors are not screened for high-titer neutralizing antibodies or that low-titer donations are collected; nevertheless, as soon as the urgent requests are satis\ufb01ed and a buffer stock has been created, repeat donations should preferably focus on donors with high titers (22).\nAs recently suggested, plasmapheresis could additionally bene\ufb01t the convalescent COVID-19 donor by reducing the prothrombotic state via the citrate-based anticoagulants administered during donation and by removal of high-molecular-weight viscous components (23).\nIn addition to interventional trials, in the United States several trials have been initiated to create registries (e.g., ClinicalTrials.gov registration no. NCT04359602) or collect plasma with titers of \u03fe1:64 from immune donors for banking purposes, without immediate reinfusion (e.g., trial NCT04360278, NCT04344977, or NCT04344015). These approaches should be encouraged to better face the next waves of the COVID-19 pandemic.\nCONVALESCENT PLASMA AND PATHOGEN INACTIVATION CP should be collected by apheresis in order to ensure larger volumes than available\nwith whole-blood donations and more frequent donations and to avoid causing unnecessary anemia in the convalescent donor. Double \ufb01ltration plasmapheresis (DFPP) using fractionation \ufb01lter 2A20 is under investigation as an approach to increase IgG yield by 3 to 4 times (Table 1, trial NCT04346589 in Italy); since DFPP-derived plasma is not an ordinary blood component but, rather, a discard product, additional regulations could apply in different countries. A very exploratory approach is under investigation in a Chinese trial collecting immunoglobulins from convalescent donors by immunoadsorption (trial NCT04264858), which could be an alternative to plasma fractionation.\nTechnologies To Virally Reduce Plasma (Pathogen Inactivation) Although neither the U.S. Food and Drug Administration (FDA) (24) nor the Euro-\npean Center for Disease Control (ECDC) is recommending pathogen reduction technologies (PRT) for CP (25), several national authorities consider that, under emergency settings, donor screening and conventional viral nucleic acid testing (NAT) (i.e., HIV, hepatitis C virus [HCV], and hepatitis B virus [HBV] NAT) would not be enough to ensure CP safety (12). Under this scenario, additional virological testing and PRT approximately double the \ufb01nal cost of the therapeutic dose. Several technologies for PRT have been approved and are currently marketed.\nSolvent/detergent (S/D)-\ufb01ltered plasma provides quick inactivation of \u03fe4 logs of most enveloped viruses; although the technology was developed and is widely used for large plasma pools, small-scale reduction has been reported. The technology relies on several steps: addition of 1% tri(n-butyl) phosphate\u20131% Triton X-45, elimination of solvent and detergent via oil extraction and \ufb01ltration, and \ufb01nally sterile \ufb01ltration (26).\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nClinical Microbiology Reviews cmr.asm.org 3\n\nFocosi et al.\n\nClinical Microbiology Reviews\n\nTABLE 1 Ongoing interventional clinical trials of convalescent plasma in COVID-19 patientsa\n\nPhase(s) and indication I/II\nExposed or con\ufb01rmed children\nAll patients with COVID-19\nNon-critically ill patients\nSevere or critically ill patients\n\nTrial no.\nNCT04377672\nNCT04292340 NCT04376788\nNCT04345679 NCT04397523 NCT04356482 NCT04357106 NCT04384497\nNCT04389944 NCT04343755 NCT04360486 NCT04354831 NCT04408040 NCT04355897 NCT04332380 NCT04375098 NCT04327349 IRCT20200325046860N1 NCT04365439 NCT04374565 NCT04348877 NCT04408209 NCT04346589 NCT04333355 NCT04352751\nNCT04347681 NCT04353206 NCT04343261 NCT04388527 NCT04389710 NCT04338360 NCT04374370 NCT04358211 NCT04363034 NCT04372368 NCT04340050\n\nCountry\nUSA\nChina Egypt\nHungary North Macedonia Mexico Mexico Sweden\nSwitzerland USA\nColombia Chile Iran Iran Switzerland USA Egypt Greece Italy Mexico Pakistan\nSaudi Arabia USA\n\nStudy population (no. of participants per arm)b\n30\n15 15\n20 20 90 10 50\n15 55 EAP 131 700 100 10 30 30 200 10 29 20 60 10 20 2,000\n40 90 15 50 100 NA EAP EAP EAP up to 100 EAP up to 150 10\n\nSchedule (vs control arm)c\n5 ml/kg, equivalent to 1\u20132 U (200\u2013250 ml/U)\nNA Exchange transfusion by venesection\nof 500 ml of blood replacement by 1 U of PRBC \u03e9 1 mg/kg methylene blue i.v. over 30 min \u03e9 200 ml of CP 1 U of CP (200 ml) NA Different amounts of CP 1 U of CP (200 ml) Up to 7 infusions (200 ml each), dose-\ufb01nding study 2 U of CP (200 ml each) NA NA 1\u20132 U of CP (\u03fd7 ml/kg adjusted IBW) 200\u2013425 ml of CP 500 ml 2 U of CP (250 ml each)/24 h 200 ml of CP on days 1 and 2 NA NA NA 2 U of CP (200 ml each) in 1\u20132 days 1 400-ml unit of CP 3 doses of CP DFPP-collected CP 1\u20132 U of CP (250 ml/24 h) Children \u03fd35 kg, 15 ml/kg over 4\u2013 6 h; adults, \u03fd450\u2013500 ml over 4\u20136 h 10\u201315 ml of CP/kg body wt 1\u20132 U of CP on days 0 and 6 2 U of CP 2 U of CP 1\u20132 U of CP (200/600 ml) 1 U of CP (200/250 ml) 1\u20132 U (200\u2013400 ml/U), not to exceed 550 ml total\n1 U of CP (300 ml)\n\nDonor titerd\n\u03fe1:320\nNA NA\n\u03fe1:320 \u03fe5 AU/ml NA NA NA\nNA \u03fe1:64 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA\nNA NA NA NA NA NA NA \u03fe160 NA NA NA\n\nIII Exposed within 96 h of enrollment and 120 h of receipt of plasma\n\nNCT04323800 NCT04390503\n\nAll patients with COVID-19\n\nNCT04377568 ChiCTR2000030039 NCT04345289\n\nUSA\nCanada China Denmark\n\nNCT04372979\n\nFrance\n\nNCT04374487\n\nIndia\n\nNCT04346446\n\nNCT04380935 IRCT20200310046736N1 NCT04342182 NCT04366245\n\nIndonesia Iran Netherlands Spain\n\n150 (Exp, 75; Ctr, 75)\n200 (Exp, 100; Ctr, 100)\n100 90 (Exp, 30; Ctr, 60) 1,500 (6 arms)\n80\n100\n40\n60 45 426 72\n\n1 U of CP (200\u2013250 ml) vs nonconvalescent plasma\n1 U of CP (200\u2013250 ml) vs 5% albumin i.v.\n10 ml/kg, up to 500 ml, vs BSC 2 U of CP (200/500 ml/24 h) vs BSC 1 600-ml unit of CP vs sarilumab vs\nbaricitinib vs hydroxychloroquine vs injective placebo vs oral placebo 2 U of 200\u2013230 ml of CP vs nonconvalescent plasma Up to 3 200-ml doses of CP 24 h apart vs BSC 1\u20133 U (200 ml) of CP vs nonconvalescent plasma NA vs BSC CP vs PDIES 1 U of CP (250 ml) vs BSC NA vs BSC\n\n\u03fe1:64 NA\nNA NA NA\nNA \u03fe1:40 NA\nNA NA NA NA\n\n(Continued on next page)\n\nOctober 2020 Volume 33 Issue 4 e00072-20\n\ncmr.asm.org 4\n\nConvalescent Plasma Therapy for COVID-19\n\nClinical Microbiology Reviews\n\nTABLE 1 (Continued)\n\nPhase(s) and indication\n\nTrial no.\nNCT04344535 NCT04333251 NCT04355767 NCT04373460\n\nNCT04362176\n\nNCT04376034\n\nNon-critically ill patients NCT04356534\n\nNCT04348656 ChiCTR2000030702 ChiCTR2000030929 ChiCTR2000030010 NCT04332835 NCT04345991\n\nNCT04374526\n\nNCT04393727 NCT04358783 NCT04345523 NCT04364737 NCT04361253\n\nNCT04397757 NCT04359810\n\nSevere or critically ill patients\n\nChiCTR2000029850 ChiCTR2000030179 ChiCTR2000030627 NCT04346446\n\nNCT04385043 NCT04381858\n\nNCT04388410 NCT04405310\n\nCountry USA\nBahrain Canada China\nColombia France Italy Italy Mexico Spain USA\nChina\nIndia Italy Mexico\n\nStudy population (no. of participants per arm)b 500 115 206 1344 (Exp, 772; Ctr, 772)\n500 (Exp, 250; Ctr, 250)\n240\n40 (Exp, 20; Ctr, 20)\n1,200 50 (Exp, 25; Ctr, 25) 80 (Exp, 30; Ctr, 30) 100 (Exp, 50; Ctr, 50) 80 120\n182\n126 (Exp, 63; Ctr, 63) 30 (Exp, 20; Ctr, 10) 278 (Exp, 139; Ctr, 139) 300 220\n80 (Exp, 40; Ctr, NA) 105 (Exp, 70; Ctr, 35)\n20 (Exp, 10; Ctr, 10) 100 (Exp, 50; Ctr, 50) 30 (Exp, 15; Ctr, 15) 40\n400 (Exp, 200; Ctr, 200) 500 (Exp, 340: Ctr, 160)\n250 (Exp, 125; Ctr, 125) 80 (Exp, 40; Ctr, 40)\n\nSchedule (vs control arm)c\n450\u2013550 ml of CP vs BSC 1\u20132 U of CP (250 ml/24) vs BSC 1\u20132 U of CP (200\u2013600 ml) vs placebo 1 U of CP (200\u2013250 ml) vs\nnonconvalescent plasma 1 U of CP (250 ml at a rate of\n500 ml/h) vs placebo 1 (moderate) or 2 (severe) U of CP\nvs BSC 2 U of CP, 200 ml each, over 2 h in 2\nconsecutive days vs BSC 500 ml of CP within 12 h vs BSC NA vs BSC NA vs BSC NA vs BSC 2 U of CP (250 ml/24 h) vs BSC Up to 4 U of CP (200\u2013220 ml each)\nvs BSC 200 ml/day for 3 consecutive days vs\nBSC 1 U (200 ml) of CP vs BSC 1 U (200 ml) of CP vs BSC CP vs BSC 1\u20132 U (250 ml each) vs i.v. placebo 2 U of CP (250 ml each) within 24 h\nvs nonconvalescent plasma 2 U of CP vs BSC 1 U (200\u2013250 ml) of CP vs\nnonconvalescent plasma NA vs BSC NA vs BSC NA vs BSC 1\u20133 U (200 ml each) of CP vs\nnonconvalescent plasma NA vs BSC 2 U (200 ml each) of CP vs\npolyclonal IVIg at 0.3 gr/kg/day (5 doses) 2 U of CP vs masked i.v. saline 1 U of CP vs 20% albumin\n\nDonor titerd \u03fe1:320 \u03fe1:64 \u03fe1:80 \u05461:320\nNA\nNA\nNA\nNA NA NA NA NA NA\nNA\nNA NA NA NA NA\nNA NA\nNA NA NA NA\nNA NA\nNA NA\n\naTrials included are listed in the World Health Organization International Clinical Trial Registry Platform (ICTRP) databases (https://www.who.int/docs/default-source/ coronaviruse/covid-19-trials.xls; accessed 7 July 2020), NIH ClinicalTrials database (www.clinicaltrials.gov; accessed 7 July 2020), and Cytel Global Coronavirus COVID-19 Clinical Trial Tracker (www.covid-trials.org; accessed 7 July 2020). bWhen the information was available, the numbers of participants in the experimental group (Exp) and control group (Ctr) are given in parentheses. EAP, expanded access program; NA, not available. cNA, not available; PRBC, packed red blood cells; IBW, ideal body weight; BSC: best supportive care; PDIES, plasma-derived immunoglobulin-enriched solution; i.v., intravenous. dAU, arbitrary units.\n\nFiltration across 75- to 35-nm-pore-size hollow \ufb01bers could remove large viruses (such as betacoronaviruses) while preserving IgG (27), but this has not been implemented yet.\nIn recent years photoinactivation in the presence of a photosensitizer has become the standard for single-unit inactivation; approved technologies include combinations of methylene blue and visible light (28) (Thera\ufb02ex), amotosalen (S-59) and UV A (29) (Intercept), and ribo\ufb02avin and UV B (30) (Mirasol). These methods do not affect immunoglobulin activity.\nFatty acids are also an option. In 2002 it was reported that caprylic acid (31) and octanoic acid (32) were as effective as S/D at inactivating enveloped viruses.\nHeat treatment of plasma has been used in the past (33, 34) but comes with a risk of aggregation of immunoglobulins (35, 36).\nPooling Figure 1 represents how CP and intravenous immunoglobulin (IVIg) can be obtained\nunder modern fractionation procedures. As per CP collection, two approaches can be pursued.\nOctober 2020 Volume 33 Issue 4 e00072-20\n\ncmr.asm.org 5\n\nFocosi et al.\n\nClinical Microbiology Reviews\n\nFIG 1 Summary of possible convalescent blood products (CBP). (Adapted from reference 153 with permission of Elsevier.)\nLarge-pool products. Pharmaceutical-grade facilities typically pool 100 to 2,500 donors to manufacture S/D-inactivated plasma. IVIgs are similarly prepared from pools of 2,000 to 4,000 liters of plasma (or 100 to 1,000 liters in the case of hyperimmune IVIg) (37, 38). Such volumes can hardly be obtained from CP donors, and timely creation of dedicated CP production chains pose dif\ufb01cult good manufacturing practice (GMP) issues within plasma vendor plants (38).\nMPFS into immunoglobulins. In order to be economically sustainable, contract (private-run) fractionation typically requires well over 10,000 liters of plasma per year, and domestic (state-owned) fractionation typically requires over 100,000 to 200,000 liters per year in addition to starting up a fractionation facility. An \u201con-the-bench\u201d minipool fractionation scale (MPFS) process (5 to 10 liters of plasma, i.e., approximately 20 recovered plasma units) using disposable devices and based on caprylic acid precipitation has been under development in Egypt since 2003 and has proved effective at purifying coagulation factors (39) and immunoglobulins (6-fold enrichment) (40). The same disposable bag system has also been combined with S/D reduction (26).\nCP BANKING CP can be either frozen or transfused as a fresh product. Aliquots of 200 to 300 ml\ncan be easily achieved from a single unit using modern PRT kits. Banking CP at temperatures below \u03ea25\u02daC (according to European Directorate for the Quality of Medicines [EDQM] or FDA guidelines for ordinary plasma for clinical use [41]) is encouraged in order to produce CP as an off-the-shelf, ready-to-use product. Most regulatory systems require that CP be tracked informatically as a blood component\nOctober 2020 Volume 33 Issue 4 e00072-20\n\ncmr.asm.org 6\n\nConvalescent Plasma Therapy for COVID-19\ndifferent from ordinary plasma for clinical use. The \ufb01nal validation label should report that the donor has tested negative by PCR for the convalescent disorder and additional microbiological tests and should describe the inactivation method. A single cycle of freezing and thawing does not signi\ufb01cantly affect the quantity or function of immunoglobulins (42). Given that COVID-19 AB blood group recipients can receive CP units only from scarce matched blood group AB donors, to increase the pool of compatible units several authors have recommended titration of anti-A and anti-B isoagglutinins and transfusion of low-titer (\u03fd1:32) non-ABO-compatible CP units (i.e., O, A, and B) to AB recipients (22, 43).\nLESSONS FROM SARS SARS-speci\ufb01c neutralizing antibodies usually persist for 2 years (44), and a decline in\nprevalence and titers occurs in the third year (45). Convalescent anti-SARS immunoglobulins were manufactured on a small scale (8, 46). Three infected health care workers with SARS progression despite the best supportive care (BSC) survived after transfusion with 500 ml of CP; viral load dropped to zero at 1 day after transfusion (47). Soo et al. reported in a retrospective nonrandomized trial that treatment with CP (titer of \u03fe1:160) in 19 patients was associated with a shorter hospital stay and lower mortality than continuing treatment with high-dose methylprednisolone (48). Amotosalen photochemical inactivation of apheresis platelet concentrates demonstrated a \u03fe6.2 log10 mean reduction of SARS-CoV (49). Thera\ufb02ex reduces infectivity of SARS-CoV in plasma (50). Heating at 60\u00b0C for 15 to 30 min reduces SARS-CoV from plasma without cells (51), while maintaining 60\u00b0C for 10 h is required for plasma products (52). In addition, SARS-CoV was found to be sensitive to S/D (51, 53).\nLESSONS FROM MERS Antibody responses to MERS persist for less than 1 year, and the magnitude corre-\nlates with the duration of viral RNA shedding in sputum (but not with viral load). Patients with mild disease have very low antibody titers, making CP collection challenging in MERS convalescents (54). A study reported that only 2.7% (12 out of 443) exposed cases tested positive by ELISA, and only 75% of them had reactive microneutralization assay titers (17). CP with a PRNT titer of \u05461:80 provides clinical bene\ufb01t in MERS (55). A case of transfusion-related acute lung injury (TRALI) following CP transfusion in a patient with MERS was reported (56, 57). MERS-CoV load in plasma was reduced by Thera\ufb02ex (58), Intercept (59), Mirasol (60), and heating at 56\u00b0C for 25 min (61); in all cases, passaging of inactivated plasma in replication-competent cells showed no viral replication.\nCONVALESCENT PLASMA FOR COVID-19 As soon as the COVID-19 pandemic appeared (62, 63), several authors suggested CP\nas a potential therapeutic agent (64, 65). Of interest, the most critically ill patients show prolonged viremia (strongly correlated with serum interleukin-6 [IL-6] levels) (10), which makes feasible therapeutic intervention with antiviral agents and immunoglobulins even at late stages. Viral shedding in survivors can last as long as 37 days (62), mandating SARS-CoV-2 RNA screening in CP donors. Serum IgM and IgA antibodies appear in COVID-19 patients as early as 5 days after symptom onset (66), while IgG can be detected at day 14 (67). IgGs are generally detected after 20 days (68, 69). Severely ill female patients generate IgG earlier and at higher titers (70, 71); the greatest part of the neutralizing antibody response has been shown to be associated with the IgG1 and IgG3 subclasses (72, 73). Duration of anti-SARS-CoV-2 antibodies in plasma is currently unknown; while the overall antibody responses for other betacoronaviruses typically declines after 6 to 12 months (74), SARS-speci\ufb01c neutralizing antibodies usually persist for 2 years (44). So, in the vast majority of countries, a suitable donor could donate 600 ml of plasma (equivalent to 3 therapeutic doses under most current trials) every 14 days for a minimum of 6 months. Up to 7 plasma donations have been proven not to decrease antibody titers in convalescent donors (18). In contrast to SARS and MERS\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nClinical Microbiology Reviews cmr.asm.org 7\n\nFocosi et al.\npatients, most COVID-19 patients exhibit few or no symptoms and do not require hospitalization; this could suggest that the majority of convalescent donors are best sought in the general population although speci\ufb01c studies on antibody titers in mildly symptomatic patients suggest low titers (18\u201320).\nSARS-CoV-2 is reduced by \u03fe3.4 logs by Mirasol (75) (and likely by other PRTs); nevertheless, SARS-CoV-2 viral RNA (vRNA) is detectable at low viral loads in a minority of serum samples collected in acute infection but is not associated with infectious SARS-CoV-2 (76). Intercept treatment has been proven not to reduce SARS-CoV-2 neutralizing antibody titers (77).\nThe main contraindications to CP therapy are allergy to plasma protein or sodium citrate, selective IgA de\ufb01ciency (\u03fd70 mg/dl in patients 4 years old or older), leading to anaphylaxis from IgA-containing CP (78), or treatment with immunoglobulins in the last 30 days (because of a risk of developing serum sickness). As in many other trial settings, concurrent viral or bacterial infections, thrombosis, poor compliance, short life expectancy (e.g., multiple-organ failure), and pregnancy or breastfeeding are also contraindications (79).\nIn an early case series from China, \ufb01ve patients under mechanical ventilation (4 of 5 with no preexisting medical conditions) received transfusions of CP with an ELISA IgG titer of \u03fe1:1,000 and a PRNT titer of \u03fe40 at days 10 to 22 after admission. Four patients recovered from acute respiratory disease syndrome (ARDS), and three were weaned from mechanical ventilation within 2 weeks of treatment, with the remaining patients being stable (80).\nAnother Chinese pilot study (ChiCTR2000030046) of 10 critically ill patients showed that one dose of 200 ml of CP with a neutralizing antibody titer of \u03fe1:640 resulted in an undetectable viral load in 7 patients, with radiological and clinical improvement (81).\nA third series of 6 cases with COVID-19 pneumonia in Wuhan showed that a single 200-ml dose of CP (with titers of anti-S antibodies determined by chemiluminescent immunoassay [CLIA] only) administered at a late stage led to viral clearance in 2 patients and radiological resolution in 5 patients (82). Pei et al. reported successful treatment of 2 out of 3 patients with 200- to 500-ml doses of CP (83). Recovery from mechanical ventilation was also reported by Zhang et al. in a single patient after antibodies in CP were titrated with an anti-N protein ELISA (84). No improvement in mortality despite viral clearance was reported in a retrospective observational study recruiting 6 late-stage, critically ill patients treated with gold-immunochromatographytitrated CP, compared to results in 13 untreated controls (85). One case of recovery in a centenarian patient who received 2 CP units (S-RBD-speci\ufb01c IgG titer of \u03fe1:640) was also reported (86).\nMany more case reports and small case series are accumulating in the literature; successful treatment was reported in 3 cases with ARDS and mechanical ventilation using two 250-ml CP doses (titrated with ELISA only) in South Korea (43, 87), in 2 cases from Iraq (88), in 8 out 10 severe cases from Mexico (89), in 20 out of 26 severe cases from Turkey (90), in a kidney transplant recipient from China (91), in a case with severe aplastic anemia in Poland (92), in a case with X-linked agammaglobulinemia in Spain (93), and in 1 patient with marginal-zone lymphoma treated with bendamustine and rituximab in the United Kingdom (94). Centers in the United States reported successful treatment with CP in 18 out of 20 patients in a series (95), in 27 out of 31 patients with severe to life-threatening disease in another series (96), in one case with myelodysplastic syndrome (97), in a critically ill obstetric patient (in combination with remdesivir) (98), and in an allogeneic stem cell transplant recipient (99).\nIn a single-arm phase II trial (NCT04321421 [100]) run in Lombardy, 49 patients with moderate to severe disease were treated with up to 3 units of PRT-treated CP (250 to 300 ml/48 h) having neutralizing antibody titers of \u05461:160 in 96% of cases. Importantly, the viral inoculum was 50 50% tissue culture infective doses (TCID50) instead of the usual 100 TCID50. Seven-day mortality was 6% versus 16% in a historical cohort. One case of TRALI was reported (101).\nIn a large case series from Wuhan, 138 patients were transfused with 200 to 1,200 ml\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nClinical Microbiology Reviews cmr.asm.org 8\n\nConvalescent Plasma Therapy for COVID-19\nof CP at a median of 45 days after symptom onset and experienced a 50% lower intensive care unit (ICU) admission rate and mortality than the group treated with best supportive care. Responders had higher lymphocyte counts, lower neutrophil counts, and lower lactate dehydrogenase (LDH), type B natriuretic peptide (BNP), urea nitrogen, procalcitonin, glucose, and C-reactive protein (CRP) levels. Complete data on neutralizing antibody titers in COVID-19 convalescent plasma (CCP) units were not available, but responders tended to have received CP units with higher antibody levels (102).\nIn the \ufb01rst retrospective, randomized controlled trial published to date, 39 patients in New York with severe COVID-19 were transfused with 2 units of ABO-type matched CP with anti-Spike antibody titers of \u05461:320 (measured by a two-step Spike proteindirected ELISA). CP recipients were more likely than control patients to not increase their supplemental oxygen requirements by posttransfusion day 14 (odds ratio [OR], 0.86), but survival improved only for nonintubated patients (hazard ratio [HR], 0.19) (103).\nAnother prospective, multicenter randomized controlled trial from China (ChiCTR2000029757) enrolled 103 patients with severe to life-threatening COVID-19. The study was underpowered because of earlier than expected (200 cases) termination. CP (9 to 13 ml/kg from donors with S-RBD IgG titer of \u05461:640) was associated with a negative SARS-CoV-2 PCR test at 72 h in 87.2% of the CP group versus 37.5% of the BSC group, but clinical improvement at 28 days was statistically different only in patients with severe, but not in life-threatening, disease (104).\nTable 1 lists the other ongoing CP trials in COVID-19 patients collected from different web portals. The United States has developed a speci\ufb01c platform for facilitating clinical trials (https://ccpp19.org/), while the International Society of Blood Transfusion created a resource library (https://isbtweb.org/coronaoutbreak/covid-19 -convalescent-plasma-document-library/). At the same time, in the United States an expanded-access program (EAP) has been approved by the FDA and coordinated by Mayo Clinic and has led to treatment of more than 30,000 patients as of 8 July 2020 (https://www.uscovidplasma.org). A preliminary report on the \ufb01rst 20,000 patients (66% from intensive care units) con\ufb01rms safety (\u03fd1% severe adverse events and 14.9% mortality at 14 days) and suggests a bene\ufb01t compared to results with historical cohorts, especially if CP is administered before mechanical ventilation (105, 106); donor titers were not disclosed, and evidently some donations were not titrated before reinfusion. Largely similar data have been reported from a 25-patient case series from Houston, Texas, where CP has been used as an emerging investigational new drug (eIND) (107).\nTypically, 1 or 2 doses of 200 ml are administered (if 2 doses are used, they are administered at least 12 h apart), with infusion rates of 100 to 200 ml/h. The cumulative dose should be targeted according to body weight and antibody titer (22).\nSeveral authors have suggested plasma exchange with CP (i.e., high-volume therapeutic plasmapheresis followed by CP transfusion) rather than CP transfusion alone in order to clear proin\ufb02ammatory cytokines from the bloodstream (108, 109), and several successful case reports deploying nonconvalescent plasma have been reported (110\u2013 112). One randomized controlled trial (NCT04374539) is ongoing in patients with severe COVID-19, but unfortunately no trial to date is testing plasmapheresis followed by CP.\nUnfortunately, most trials in westernized countries (in contrast to ones ongoing in China) have no control arm, which will impair ef\ufb01cacy interpretation. When present, the control arm consists of the best supportive care alone (typically oxygen and hydroxychloroquine at 400 mg twice a day [b.i.d.] for 10 days) or combined with intravenous placebo or standard (nonconvalescent) plasma (eventually of pharmaceutical grade). Since other plasma components (e.g., aspeci\ufb01c immunoglobulins or isoagglutinins; see below) could contribute to clinical bene\ufb01t, the latter approach is ideal for dissecting the speci\ufb01c contribution of neutralizing antibodies although concerns could be raised by the prothrombotic nature of COVID-19 pathology (see Side Bene\ufb01ts from CP in COVID19, below). Even using a placebo control in late-stage patients (refractory to former lines) could pose some ethical concerns because it denies treatment opportunities to an unresponsive disease. Future trials should investigate combined antiviral and CP therapies.\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nClinical Microbiology Reviews cmr.asm.org 9\n\nFocosi et al.\nNotably, several plasma manufacturers are attempting to develop SARS-CoV-2speci\ufb01c hyperimmune sera (e.g., Takeda\u2019s TAK-888 merged with Biotest, BPL, LFB, Octapharma, and CSL Behring into the Convalescent Plasma Coalition [113]; Kedrion and Kamada have joint ventures [81]).\nMONITORING RESPONSE TO CP TREATMENT CP is considered an experimental therapy, and, as such, phase 3 randomized\ncontrolled trials should be encouraged. Despite this recommendation, in emergency settings phase 2 trials are usually started, hampering ef\ufb01cacy analysis. Response in published trials is generally measured clinically (PaO2/FiO2 ratio) or radiologically according to target organs. Nevertheless, surrogate endpoints can include anti-SARSCoV-2 antibody titer or absolute lymphocyte count increases in recipients, as well as decreases in recipients\u2019 SARS-CoV-2 viral load or IL-6 levels. Whenever quantitative PCR is not available, cycle threshold (CT) value increases in qualitative PCR after transfusion could be a proxy for reduced viral load.\nCONCERNS The \ufb01rst concern is transfusion-transmitted infection (TTI). Modern performance\nimprovement (PI) technologies, combined with NAT, reduce the risk for contracting additional TTIs. Most regulatory systems require additional tests (e.g., for hepatitis A virus [HAV] RNA, hepatitis E virus [HEV] RNA, or parvovirus B19 DNA) to be performed on CP for additional transfusion safety. CBP obtained from donors in the United Kingdom may be problematic for a couple of reasons. Currently, CBP obtained from individuals who lived for at least 6 months in the United Kingdom during the 1980-1996 outbreak of \u201cmad cow disease\u201d (bovine spongiform encephalopathy [BSE]) may not be acceptable in some countries (114) or by some individuals. In addition, there is a now a recognized risk of hepatitis E the within the U.K. blood donor population (115), most likely due to the consumption of poorly cooked pork products (116, 117), for which screening has only relatively recently been initiated (71). Although this does not preclude such SARS-CoV-2 convalescent plasma/serum from being used therapeutically within the United Kingdom, these other risks should be considered during larger clinical trials or with compassionate use in individual patients. Respiratory betacoronaviruses produce only a mild and transient viremia. With SARS-CoV, limited replication in lymphocytes (118) leads to signi\ufb01cant risk only for recipients of blood products with high concentrations of donor lymphocytes (peripheral blood stem cells, bone marrow, granulocyte concentrates, etc.). Preliminary reports have shown that SARS-CoV-2 viremia persists only in critically ill patients (10).\nThe second concern is TRALI, which can be life-threatening in patients who are already suffering from ALI. Male donors are usually preferred in order to avoid the risk of transfusing anti-HLA/HNA/HPA antibodies from parous women. In the case of COVID-19, where female patients have been shown to have higher IgG levels, this could be detrimental, and anti-HLA/HNA/HPA antibody screening could be implemented.\nAntibody-dependent enhancement (ADE) is also a theoretical concern related to passive or active antibodies (targeting S protein domains other than the RBD) facilitating IgG-coated virion entry into macrophages via Fc\u2425 receptors and/or complement receptors (119, 120), leading to activation of the RNA sensing Toll-like receptors (TLR) 3, 7, and 8 and \ufb01nally to elevated production of tumor necrosis factor (TNF) and IL-6 (a so-called cytokine storm). ELISAs discriminating the difference between total and RBD-binding antibodies could be useful to inspect the occurrence of ADE. Genetic polymorphisms (e.g., Fc\u2425RIIa [121]) can also contribute to ADE. To date, potential evidence supporting a role for ADE in COVID-19 include the following: (i) the correlation between disease severity and total anti-SARS-CoV-2 antibody levels (70, 122\u2013124), including neutralizing antibodies (125, 126); (ii) the low prevalence of symptoms in COVID-19 patients younger than 20 (who have likely not been primed by infection with the other common cross-reacting coronavirus 229E or OC43 or anyway have low-af\ufb01nity anti-coronavirus IgG [127, 128]); (iii) the occurrence, in SARS, of ADE at low antibody titers in vitro (129) and\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nClinical Microbiology Reviews cmr.asm.org 10\n\nConvalescent Plasma Therapy for COVID-19\ncorrelation in patients of high IgG titers and early seroconversion with disease severity (130). Overall, these \ufb01ndings raise concerns for usage of low-titer CP units (131). Other evidence is the high level of afucosylated IgG against S protein, facilitating FcR binding, that is produced in the most severely ill patients (132, 133).\nA last, COVID-19-speci\ufb01c, concern is worsening of the underlying coagulopathy (134) from clotting factors in transfused plasma (not only CP but also nonconvalescent plasma in control arms); since this has not been reported to date, it remains a theoretical concern.\nSIDE BENEFITS FROM CP IN COVID-19 Obviously, patients with humoral immune de\ufb01ciencies can bene\ufb01t from polyclonal\nantibodies contained in CP, and patients with hemorrhagic diathesis can bene\ufb01t from clotting factors.\nPlasma is also likely to contain antibodies against other common betacoronaviruses associated with the common cold, which have been shown to cross-react with SARSCoV-2 antigens in intravenous immunoglobulin (IVIg) preparations (135), likely stemming from recent infection with another human betacoronavirus (128). Accordingly, IVIg led to clinical and radiological recovery in 3 Chinese patients with severe COVID-19 (136), and the same team is now leading a randomized controlled trial (NCT04261426).\nAfter demonstration that blood group O health care workers were less likely to become infected with SARS-CoV (137), a research group proved that anti-A blood group natural isoagglutinins (which can also be found in CP plasma from blood group O and B donors) inhibit SARS-CoV entry into competent cells (138). Such binding could opsonize virions and induce complement-mediated neutralization (139). Since SARSCoV-2 uses the same receptor as SARS-CoV, anti-A isoagglutinins are expected to have similar effects against SARS-CoV-2 (140); accordingly, clusters of glycosylation sites exist proximal to the receptor-binding motif of the S protein from both SARS-CoV (141) and SARS-CoV-2 (142). Several publications showed that the odds ratio for acquiring COVID-19 is higher in blood group A than in blood group O (143\u2013147), and one showed the ABO gene polymorphism to be the most signi\ufb01cant at predicting severity of COVID-19 (147). COVID-19 has more severe clinical presentations and outcomes in the elderly and in males; intriguingly, elderly males are known to experience reductions in isoagglutinin titers (148, 149). Although alternative explanations exist (150, 151), studies are hence ongoing to evaluate correlations between isoagglutinin titers and outcomes in blood group O and B patients (152). If the correlations are con\ufb01rmed, while preserving ABO match compatibility, blood group O and B donors for CP in COVID-19 could be preferred, and their anti-A isoagglutinin titers should be tested.\nCONCLUSIONS CP manufacturing should be considered among the \ufb01rst responses during a pan-\ndemic while antivirals and vaccines are tested. 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Transfus Apher Sci 51:120 \u2013125. https://doi.org/10.1016/j.transci.2014.10.003.\n\nOctober 2020 Volume 33 Issue 4 e00072-20\n\ncmr.asm.org 16\n\nConvalescent Plasma Therapy for COVID-19\n\nClinical Microbiology Reviews\n\nDaniele Focosi is a hematologist employed as resident transfusion physician at the largest blood bank in Italy since 2009. He has been a transplant immunologist and immunogeneticist, quality assurance manager, and production manager. He has received awards from the European Federation of Immunogenetics, the European Society of Organ Transplantation, and the Italian Society of Hematology. He has a Ph.D. degree in Clinical and Fundamental Virology, and a master\u2019s degree in Clinical Trials. He has authored 124 articles indexed in PubMed, for a global h-index of 24, on topics ranging from emerging viral infections to new markers of immune competence.\nArthur O. Anderson is a physician/scientist, pathologist, and applied ethicist. He was the Director, Of\ufb01ce of Human Use, and Ethics and Research Integrity Of\ufb01cer at the U.S. Army Medical Research Institute of Infectious Diseases from 1974 to 2016. He held faculty appointments at Johns Hopkins University from 1972 to 1974 and at the University of Pennsylvania from 1980 to 1983. An active biomedical researcher for over 40 years, Dr. Anderson has nearly 100 publications on immunology, infectious diseases, and medical research ethics, including one entitled \u201cEthical Issues in the Development of Drugs and Vaccines for Biodefense.\u201d Now retired from his civilian position, Dr. Anderson serves as a member of the Board of Trustees at Hood College and Director at Hospice of Frederick County.\n\nJulian W. Tang is a hospital consultant and medical virologist, with special interests in the diagnosis, treatment, epidemiology, and infection control of in\ufb02uenza and respiratory viruses, congenital viral infections, HIV, and blood-borne viruses. He also has a Ph.D. in zoology. He has formerly been associate professor at the University of Alberta and assistant professor at the Chinese University of Hong Kong.\nMarco Tuccori is a clinical pharmacologist with special interest in pharmacovigilance and pharmacoepidemiology. He also has a Ph.D. in pharmacology and medical physiology. He is currently pharmacovigilance manager at the Unit of Adverse Drug Reactions Monitoring of the University Hospital of Pisa and coordinator of the Tuscan Regional Centre of Pharmacovigilance. He collaborates with the Agenzia Italiana del Farmaco (AIFA) as a member of the Working Group for Signal Detection Analysis on Drugs and Vaccines. He was a member of the Advisory Board (formerly Executive Committee) of the International Society of Pharmacovigilance (ISoP) from 2012 to 2019. He is the author of about 90 articles in peer-reviewed scienti\ufb01c journals and four chapters of books.\n\nOctober 2020 Volume 33 Issue 4 e00072-20\n\ncmr.asm.org 17\n\n\n",
    "authors": [
      "Daniele Focosi",
      "Arthur O Anderson",
      "Julian W Tang",
      "Marco Tuccori"
    ],
    "doi": "",
    "date": "2020",
    "item_type": "journalArticle",
    "url": ""
  },
  {
    "key": "CIL9MIU7",
    "title": "Will There Be a Cure for Ebola?",
    "abstract": "Despite the unprecedented Ebola virus outbreak response in West Africa, no Ebola medical countermeasures have been approved by the US Food and Drug Administration. However, multiple valuable lessons have been learned about the conduct of clinical research in a resource-poor, high risk\u2013pathogen setting. Numerous therapeutics were explored or developed during the outbreak, including repurposed drugs, nucleoside and nucleotide analogues (BCX4430, brincidofovir, favipiravir, and GS-5734), nucleic acid\u2013based drugs (TKM-Ebola and AVI-7537), and immunotherapeutics (convalescent plasma and ZMapp). We review Ebola therapeutics progress in the aftermath of the West Africa Ebola virus outbreak and attempt to offer a glimpse of a path forward.",
    "full_text": "PA57CH17-Cardile ARI 10 December 2016 10:54\n\nFurther ANNUAL\nREVIEWS\nClick here to view this article's online features:\n\u2022 Download \ufb01gures as PPT slides \u2022 Navigate linked references \u2022 Download citations \u2022 Explore related articles \u2022 Search keywords\n\nWill There Be a Cure for Ebola?\nAnthony P. Cardile, Travis K. Warren, Karen A. Martins, Ronald B. Reisler, and Sina Bavari\nUS Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702; email: anthony.p.cardile.mil@mail.mil\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nAnnu. Rev. Pharmacol. Toxicol. 2017. 57:329\u201348\nFirst published online as a Review in Advance on December 7, 2016\nThe Annual Review of Pharmacology and Toxicology is online at pharmtox.annualreviews.org\nThis article\u2019s doi: 10.1146/annurev-pharmtox-010716-105055\nCopyright c 2017 by Annual Reviews. All rights reserved\n\nKeywords\nBCX4430, brincidofovir, favipiravir, GS-5734, ZMapp, convalescent plasma\nAbstract\nDespite the unprecedented Ebola virus outbreak response in West Africa, no Ebola medical countermeasures have been approved by the US Food and Drug Administration. However, multiple valuable lessons have been learned about the conduct of clinical research in a resource-poor, high risk\u2013 pathogen setting. Numerous therapeutics were explored or developed during the outbreak, including repurposed drugs, nucleoside and nucleotide analogues (BCX4430, brincidofovir, favipiravir, and GS-5734), nucleic acid\u2013 based drugs (TKM-Ebola and AVI-7537), and immunotherapeutics (convalescent plasma and ZMapp). We review Ebola therapeutics progress in the aftermath of the West Africa Ebola virus outbreak and attempt to offer a glimpse of a path forward.\n\n329\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\nINTRODUCTION Despite the unprecedented response to the West Africa Ebola virus (EBOV) outbreak, there remain no US Food and Drug Administration (FDA)-approved Ebola medical countermeasures (MCMs). As a result, the treatment of EBOV infection remains limited to clinical supportive care, administration of investigational MCMs through expanded access, or enrollment into clinical treatment trials studying candidate MCMs. The following question or a similar variant was asked frequently during and following the West Africa EBOV outbreak: Will there be a cure for Ebola?\nTaken at face value, this seems to be a simple question. However, the meaning of cure can vary depending on context. The Merriam-Webster Online Dictionary de\ufb01nes a cure, in part, as (a) recovery or relief from a disease, (b) something (as a drug or treatment) that cures a disease, and (c) a complete or permanent solution or remedy (1). Identi\ufb01cation of a cure, therefore, may be approached from the perspective of prevention, treatment, or eradication. Only smallpox and rinderpest have been eradicated from nature (2, 3). The prospect of EBOV eradication is untenable at this time, given our incomplete understanding of potential reservoir hosts and subsequent initial transmission from reservoir hosts to humans. During the past two years, researchers have made tremendous strides in the development of EBOV MCMs for the prevention via vaccination or treatment of EBOV infection. EBOV vaccine development has been reviewed in detail recently by other sources (4, 5). The goal of this review is to summarize these advances, with a focus on therapeutics development and the challenges encountered during the recent West Africa EBOV outbreak, and to offer a perspective on the path forward. Tables 1 and 2 summarize relevant in vitro, nonhuman primate (NHP), and clinical studies of select MCMs and highlight data gaps that need to be \ufb01lled.\nREPURPOSED DRUGS Numerous drugs that are approved for other indications have been demonstrated to possess activity against EBOV. Researchers had thought that if already-approved drugs displayed in vitro or in vivo ef\ufb01cacy against EBOV, EBOV-infected patients could reasonably be treated with such therapies in an outbreak setting. Additionally, in the context of a highly lethal infection for which there were no known MCMs, some doctors and clinics used their best knowledge to select approved drugs in order to treat symptoms, despite a lack of data on EBOV infection. This approach was ineffective during the West Africa outbreak, however, and could have resulted in harm to patients, as certain drugs were given to patients outside the auspices of research protocols. For example, there were unsubstantiated claims that lamivudine, statins, and angiotensin receptor blockers (ARBs) were used successfully to treat EBOV-infected patients in West Africa (6). Similarly, a Liberian physician reported successful treatment of EBOV-infected patients with lamivudine (7). In subsequent studies, lamivudine was shown to have no in vitro activity against the 1995 isolate EBOV-Kikwit or EBOV-Makona (7). In Sierra Leone, local physicians reported treating EBOVinfected patients successfully at multiple sites with the statin atorvastatin (40 mg daily) and the ARB irbesartan (150 mg daily) (8\u201310). Fedson et al. (8\u201310) have suggested that statins and ARBs could treat the host response to Ebola infection owing to their potential to restore endothelial barrier integrity, but they can have adverse effects that could worsen EBOV disease (e.g., ARBs can cause acute kidney injury and hyperkalemia; statins can cause hepatotoxicity and myopathy). The use of lamivudine, atorvastatin, and irbesartan was not associated with approved clinical trial\n330 Cardile et al.\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nPA57CH17-Cardile ARI 10 December 2016 10:54 www.annualreviews.org \u2022 Cure for Ebola 331\n\nTable 1 Select in vitro and in vivo (NHP) characteristics of Ebola virus therapeutics\n\nTherapeutic name Amiodarone\nAmodiaquine\nBCX4430\n\nIn vitro EC50 (strain)\n0.25 \u03bcg/mL (Mayinga) (22)\n2.6 and 8.4 \u03bcM (pseudotyped virus) (14)\n11.8 \u03bcM (Kikwit), 3.4 \u03bcM (SUDV-Boniface) (28)\n\nIn vitro CC50 16.69 \u03bcg/mL\nNA\n>100 \u03bcM\n\nIn vitro therapeutic\nindex (CC50 /IC50 ) 67\nNA\n8 to >29\n\nNHP species NA NA Rhesus (31)\n\nBrincidofovir Favipiravir GS-5734\nsiEbola-3\n\n0.6\u20130.88 \u03bcM (Kikwit, Mayinga, Makona) (36)\n67 \u03bcM (Mayinga) (55)\n0.06\u20130.14 \u03bcM (Kikwit, Makona, Sudan, Bundibugyo) (67)\nNA\n\n>10 \u03bcM >1,000 \u03bcM 1.7 to >20 \u03bcM\nNA\n\n<1 to >15 >14 12 to >333\nNA\n\nNA NA Rhesus (67)\nRhesus (77)\n\nAVI-7537\n\n585 nM (Kikwit) (78)\n\nNA\n\nNA\n\nRhesus (80)\n\nZMapp\n\n0.015\u20130.04 \u03bcg/mL\n\nNA\n\nNA\n\nRhesus (96)\n\n(Kikwit); 0.004\u2013\n\n0.02 \u03bcg/mL (Makona)\n\n(96)\n\nInterferon-\u03b2\n\nNA\n\nNA\n\nNA\n\nRhesus (102)\n\nRoute of challenge and challenge virus\nNA\n\nDosing NA\n\nTime therapy was initiated postinfection\nNA\n\nNA\n\nNA\n\nNA\n\nIM Kikwit NA\n\nIM, twice daily at 1 h doses of 16 mg/kg or 25 mg/kg\n\nNA\n\nNA\n\nNA IM Kikwit IM Makona IM Kikwit IM Kikwit\nIM Kikwit\n\nNA 10 mg/kg IM\ndaily for 12 daysa 0.5 mg/kg IV for 7 days 40 mg/kg IV for 14 days 50 mg/kg IV\n10.5 \u03bcg/kg SQ\n\nNA 3 daysa\n3 days\n1h\n3, 6, and 9 days postinfection; 4, 7, and 10 days postinfection; or 5, 8, and 11 days postinfection\n18 h and 1, 3, 5, 7, and 9 days postinfection\n\nNHP survival in treatment arm NA\nNA\n66.7% (4 of 6) survival in the low-dose group and 100% (6 of 6) survival in the high-dose group\nNA\nNA 100% (6/6)a\n100% (3/3)\n75% (6/8)\n100% (18/18)\nNo survival bene\ufb01t; prolonged time to death 13.8 days versus 8.3 days for control (P = 0.0097)\n\nAbbreviations: CC50, 50% cytotoxic concentration; EC50, 50% effective concentration; IC50, 50% inhibitory concentration; IM, intramuscular; IV, intravenous; NA, not applicable; NHP, nonhuman primate; SQ, subcutaneous; SUDV, Sudan virus. aBest regimen of multiple regimens tested (67).\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nPA57CH17-Cardile ARI 10 December 2016 10:54 332 Cardile et al.\n\nTable 2 Clinically relevant characteristics of select EBOV therapeutics\n\nTherapeutic name(s)\nAmiodarone\n\nClinical studies\nAdministered as a compassionate therapy to approximately 65 patients hospitalized in Sierra Leone (retrospective analysis) (6, 24)\n\nRoute of administration\nIV or PO\n\nDosing\nDay 1: 5 mg/kg (\ufb01rst hour), total of 20 mg/kg (remaining 23 h) via IV\nDays 2\u20133: 20 mg/kg (24 h) via IV\nDays 4\u201310: 30 mg/kg via oral tablets given 3 times/day (97)\n\nAmodiaquine\nBCX4430 Brincidofovir\n\nRetrospective comparison\n\nPO\n\nof EBOV-infected\n\npatients who received\n\nartemether\u2013lumefantrine\n\nand artesunate\u2013\n\namodiaquine therapy for\n\nmalaria (31)\n\nPhase I clinical trials\n\nIM\n\nongoing (34)\n\nPhase II trial for Ebola\n\nPO\n\nterminated\n\n7.5\u201315.0 mg/kg for malaria (31)\nND Initial dose of 200 mg, then\n100 mg twice weekly for a total of 5 doses (97)\n\nPlasma half-life in humans\n20\u201347 days (28)\n211 h (31)\nND Dose-dependent,\nranging from 6.15 h at 0.025 mg/kg to 32.7 h at 1.5 mg/kg (41)\n\nClearance Hepatic (28)\nHepatic\nND Nonrenal; its\nmetabolite, cidofovir, was detected in the urine (41)\n\nAdverse drug reactions Infusion-related\nhypotension, sinus bradycardia, ventricular arrhythmias, hepatotoxicity, gastrointestinal problems (nausea, vomiting, anorexia, diarrhea, and constipation), and neurologic dysfunction; thyroid, ocular, cutaneous, and pulmonary toxicity from chronic exposure (25\u201328) Agranulocytosis, hepatotoxicity (31)\nND\nAbdominal pain, nausea, diarrhea,\naphthous stomatitis (41)\n\nClinical ef\ufb01cacy Reportedly reduced case\nfatality rates; however, the data are currently unavailable, and these claims should be interpreted with caution (6, 24).\nArtesunate\u2013amodiaquine group had a 31% lower risk of death than the artemether\u2013lumefantrine group (P = 0.004), with a stronger effect observed among patients without malaria (31).\nNo human ef\ufb01cacy data are available to date.\nClinical trials were terminated owing to slow enrollment and withdrawal of the drug for investigational use in Ebola patients by the company (11, 45).\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nPA57CH17-Cardile ARI 10 December 2016 10:54 www.annualreviews.org \u2022 Cure for Ebola 333\n\nFavipiravir\n\nSingle-arm,\n\nPO\n\nproof-of-concept trial for\n\nEbola (64)\n\nGS-5734\n\nPhase I clinical trials\n\nIV\n\nongoing (70, 71)\n\nLoading dose of 2,400 mg, followed by 1,200 mg every 8 h on day 1 (6,000 mg total) and a maintenance dose of 1,200 mg twice a day for 9 days (64, 66)\n\n4.5\u20135.8 h (67)\n\nND\n\nND\n\nTKM-Ebola-\n\nPhase I clinical trial\n\nIV\n\nND\n\nGuinea\n\ncompleted; Phase II trial\n\n(siEbola-3)\n\nterminated (87, 88)\n\nAVI-7537 ZMapp\n\nPhase I clinical trial completed (85)\nPhase I clinical trial ongoing; Phase II trial completed\n\nIV infusion in 150 mL normal saline over 30 min (85)\nIV\n\n4.5 mg/kg (85) 50 mg/kg\n\nND\n2\u20135 h (85) ND\n\nMetabolized by oxidases; metabolites excreted in the urine (67)\nND\nND\nMajority secreted in urine by 24 h (85)\n\nDuring in\ufb02uenza trials: mild to moderate diarrhea, asymptomatic increase of blood uric acid and transaminases, and decreases in neutrophil counts (67)\nND\nPostinfusion fever, rigors, dizziness, chest tightness, and tachycardia related to transient in\ufb02ammatory responses (beginning within 6 h of infusion and lasting up to 24 h) (67)\nHeadache; nausea; elevated AST, ALT, and amylase; uveitis (85)a\n\nPossible survival bene\ufb01t in patients with Ct values > 20, and no improvement in survival when Ct values are < 20 (64)\nCompassionate use in a case of EBOV recrudescence in a female nurse in the United Kingdom and a case of acute EBOV infection in a female infant in Guinea (72\u201373)\nPhase II trial terminated, as interim analysis indicated that continuing enrollment was not likely to demonstrate an overall therapeutic bene\ufb01t (87, 88)\nNo clinical data to date\n\nND\n\nCommon side effects\n\nNo statistically signi\ufb01cant\n\nreported from cases in the\n\nmortality bene\ufb01t, with\n\nliterature were\n\n37% mortality in the\n\ninfusion-related fever,\n\ncontrol arm and 22.2%\n\nhypotension, tachycardia,\n\nmortality in the treatment\n\nrash, and polypnea (67)\n\narm of the study.\n\nAbbreviations: Ct, cycle threshold; EBOV, Ebola virus; IV, intravenous; ND, not determined; PO, oral. aConsidered to be drug related; however, a retinal specialist suggested recurrent toxoplasmosis as the underlying cause of the uveitis based on the subject\u2019s region of origin (West Africa) and the presence of retinal scarring.\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\nprotocols and was not administered with adequate controls and oversight, and data have not been submitted for peer-reviewed publication to date. Claims of ef\ufb01cacy should be interpreted with caution until more data are available.\nPer World Health Organization (WHO) sources, preclinical data are available on azithromycin, erlotinib/sunitinib, atorvastatin, irbesartan, and sertraline, but these data were not available in the peer-reviewed literature at the time of writing (11). Other drugs with data for anti-EBOV activity include amitriptyline, amlodipine, azidothymidine, ba\ufb01lomycin A1, benztropine, bepridil, chloroquine, chlorpromazine, colchicine, cyproheptadine, despiramine, diltiazem, erythromycin, \ufb02uoxentine, heparin, imipiramine, nimodipine, nystatin, penbutolol, prochlorperazine, sertraline, and verapamil (12\u201320). Amiodarone and amodiaquine are discussed in more detail below, as they have more data available than many of the other drugs.\nAmiodarone (a multi-ion channel inhibitor and adrenoceptor antagonist) was found to inhibit EBOV cell entry in vitro (21). The proposed mechanism of action of amiodarone against EBOV is interference with the fusion of the viral envelope and endosomal membrane (22). The concentrations of amiodarone required for EBOV inhibition are within the range that is achieved in serum during antiarrhythmic therapy in humans (1.5\u20132.5 mg/mL) (21). Nonetheless, there are concerns that the high protein binding of the drug will result in ineffective serum concentrations. The drug was administered as a compassionate therapy to approximately 65 patients hospitalized in Sierra Leone and, in retrospective analysis, reportedly reduced case fatality rates; however, the data are currently unavailable, and these claims should be interpreted with caution (6, 23). Its use for treatment of EBOV patients could be problematic, given some of its serious side effects and drug interactions. Amiodarone can cause life-threatening ventricular arrhythmias, and in the setting of the severe electrolyte abnormalities observed during the recent outbreak, this side effect could be ampli\ufb01ed (24, 25). Amiodarone can also cause signi\ufb01cant gastrointestinal side effects (nausea, vomiting, and diarrhea) and hepatotoxicity, which could potentially exacerbate the EBOV disease process (26, 27).\nAmodiaquine is a 4-aminoquinoline antimalarial that has generated interest for the treatment of EBOV. Fifty percent inhibitory concentration (IC50) values for amodiaquine in vitro for EBOV entry and replication were lower than those for the similar antimalarial chloroquine (2.6 and 8.4 \u03bcM versus 4.7 and 16 \u03bcM, respectively) (14). At an Ebola treatment center in Liberia, the supply of artemether\u2013lumefantrine, the \ufb01rst-line antimalarial, ran out for a 12-day period in August 2014, and during that time patients received artesunate\u2013amodiaquine (28). After the in vitro EBOV activity of amodiaquine was published, a comparison of patients who received artemether-lumefantrine and artesunate-amodiaquine therapy for malaria was conducted. At admission, 194 patients were prescribed artemether\u2013lumefantrine and 71 were prescribed artesunate\u2013amodiaquine, and baseline characteristics of patients were similar between both groups and in a no-antimalarial group. A total of 125 of the 194 patients in the artemether\u2013lumefantrine group (64.4%) died, as compared with 36 of the 71 patients in the artesunate\u2013amodiaquine group (50.7%). In adjusted analyses, the artesunate\u2013amodiaquine group had a 31% lower risk of death than the artemether\u2013lumefantrine group (P = 0.004), with a stronger effect observed among patients without malaria. This information is compelling, but it is not known if this observed effect is de\ufb01nitively related to the ef\ufb01cacy of amodiaquine against EBOV, a lumefantrine-induced increased mortality in EBOV patients, or unmeasured patient characteristics that altered mortality risk (6, 28). This study is also inherently limited by its retrospective nature, and the medical record lacked information on completion of antimalarial courses of therapy (28). However, further research of amodiaquine as a therapeutic for EBOV may be warranted based on these observations.\n334 Cardile et al.\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\nDIRECT-ACTING ANTIVIRALS\nNucleoside and Nucleotide Analogues\nAntiviral nucleoside and nucleotide analogues are prodrugs that require activation by several successive phosphorylation steps catalyzed by different kinases that are present in the host cell or encoded by some of the viruses (29). These compounds can exert antiviral effects via inhibition of viral polymerases, other enzymes such as kinases, and/or incorporation into viral nucleic acids (30).\nBCX4430. BCX4430 is a novel nucleoside analogue that was synthesized as part of a smallmolecule library of inhibitors of viral RNA polymerase activity (31). BCX4430 was designed to inhibit viral RNA polymerase activity indirectly via RNA chain termination and is dependent on conversion of the parent compound to BCX4430-triphosphate (31). BCX4430 has displayed a broad antiviral spectrum in vitro, with activity against negative-sense RNA viruses (Filoviridae, Arenaviridae, Bunyaviridae, Orthomyxoviridae, Picornaviridae, and Paramyxoviridae) and positive-sense RNA viruses (Flaviviridae and Coronaviridae) (31). Of all the virus families, BCX4430 displayed the most potent in vitro activity against \ufb01loviruses (Marburg-Musoke, -Ci67, and -Angola; EBOVKikwit; and Sudan-Boniface) with 50% effective concentration (EC50) values ranging from 3.4 to 11.8 \u03bcM (31).\nIn a mouse model of EBOV disease, BCX4430 protected mice against an otherwise lethal challenge of mouse-adapted EBOV administered via intramuscular (IM) or oral routes at a dose of 150 mg/kg (the ef\ufb01cacies of lower doses were not evaluated) (31). In an as yet unpublished study to evaluate the ef\ufb01cacy of BCX4430 in a NHP EBOV disease model, BCX4430 was administered to rhesus monkeys by an IM route twice daily at doses of 16 or 25 mg/kg, beginning approximately 1 h after virus exposure (32). At both dose levels, BCX4430 conferred a statistically signi\ufb01cant survival bene\ufb01t compared to animals treated with vehicle alone, with 66.7% (4 of 6) survival in the low-dose group and 100% (6 of 6) survival in the high-dose group (32). Administration of BCX4430 also reduced plasma viral concentration in rhesus monkeys by nearly 3 log10 in both BCX4430 groups on day 8 during the most acute phase of disease (32). Finally, a two-part, doseranging study to evaluate the safety, tolerability, and pharmacokinetics of BCX4430 administered via IM injection in healthy subjects is currently recruiting patients (33). In part one, study subjects will receive a single dose of BCX4430, and in part two, subjects will receive BCX4430 for 7 days.\nBrincidofovir. Brincidofovir is a conjugate comprised of a lipid (1-0-hexadecyl-oxypropyl) covalently linked to the acyclic nucleotide analogue cidofovir (34, 35). This enables brincidofovir to be orally bioavailable and more active and reduces nephrotoxicity compared to cidofovir (34, 35). Brincidofovir has broad activity against DNA viruses, including poxviruses, herpesviruses, adenoviruses, and polyomaviruses (34).\nBrincidofovir was \ufb01rst considered as a possible EBOV therapy when in vitro data against EBOV were \ufb01rst presented in October 2014 (36). The FDA then made brincidofovir available for the treatment of EBOV patients through an Emergency Use Investigational New Drug (IND) application (37). Use of brincidofovir during the EBOV outbreak was rationalized on the basis that it had already been through Phase I studies and was in Phase II and III studies for other viral indications (cytomegalovirus and adenovirus infections) (38\u201340). At least four EBOV-infected patients may have been treated with brincidofovir (41\u201343). An open-label, multicenter study of brincidofovir against EBOV was set to commence, but in January 2015, clinical trials for\nwww.annualreviews.org \u2022 Cure for Ebola 335\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\nbrincidofovir for EBOV were terminated owing to slow enrollment and the withdrawal of the drug for investigational use in EBOV-infected patients by the company (11, 44).\nRecently, in vitro data have been published on the anti-EBOV activity of brincidofovir against wild-type EBOV and a strain of EBOV expressing green \ufb02uorescent protein (EBOV-GFP) (37). When cells were pretreated with brincidofovir and then infected with EBOV, the following variants were inhibited (EC50 values): EBOV-Mayinga (0.76 \u03bcM), EBOV-Makona (0.6\u20130.88 \u03bcM), and EBOV-Kikwit (0.66\u20130.79 \u03bcM) (37). Mechanistic studies of brincidofovir against EBOV demonstrated a different mechanism compared to its activity against DNA viruses. The precise mechanism remains unknown but has been speculated to be related to competition for phospholipases such as acid sphingomyelinase, which are required for ef\ufb01cient infection of cells by EBOV in vitro (15, 37). As opposed to DNA viruses, anti-EBOV activity required the lipid moiety, and in vitro activity against EBOV was observed for several nucleotide conjugates (37). In mouse models, brincidofovir was ineffective, and this may be related to the dose and dosing intervals used, as cidofovir diphosphate (CDV-PP) was assumed to be the active antiviral metabolite (11). Given that the half-life of intracellular CDV-PP is much longer than the plasma half-life of brincidofovir, higher doses, more frequent doses, or both would be required to show anti-EBOV activity in vivo (37). Complicating matters, in NHPs, which are the gold standard for testing the ef\ufb01cacy of EBOV therapeutics, brincidofovir is metabolized rapidly, making evaluation dif\ufb01cult (35, 37). Taken together, substantial barriers exist that make it impractical to develop brincidofovir as an EBOV MCM.\nFavipiravir. Favipiravir (T-705 or Avigan) is a pyrazinecarboxamide derivative that was developed initially as an orally active anti-in\ufb02uenza drug (45). T-705 is converted by host cells to T-705-ribofuranosyl-5 -triphosphate. It selectively inhibits viral RNA\u2013dependent RNA polymerase or causes lethal mutagenesis upon incorporation into viral RNA (46, 47). Favipiravir has broad-spectrum activity against RNA viruses, including bunyaviruses, arenaviruses, \ufb02aviviruses, noroviruses, alphaviruses, picornaviruses, and paramyxoviruses (48\u201355). Complete (100%) protection against aerosolized EBOV (E718) in interferon (IFN)-\u03b1/\u03b2 receptor knockout, immunode\ufb01cient mice was achieved when favipiravir was administered 1 h postchallenge followed by 14 days of twice-daily dosing of 150 mg/kg orally (55). In a similar study in an immunode\ufb01cient intranasal mouse model, favipiravir (300 mg/kg/day orally) was administered at six days postinfection (EBOV-Mayinga), with rapid viral clearance, reduced biochemical parameters of disease severity, and 100% survival of the exposed animals (56). However, when treatment was delayed to day 8 (peak viremia), favipiravir did not prevent death (56).\nFavipiravir had been evaluated in Phase II and III studies of in\ufb02uenza in Japan and the United States and was approved for use in in\ufb02uenza infection in Japan (57, 58). As a result, the drug was an attractive early candidate for clinical trials during the West Africa EBOV outbreak. Four case reports of patients treated with favipiravir for EBOV have been published (59\u201362). Additionally, there is one case report in which favipiravir was used to treat a patient who had recovered from acute EBOV infection and had postinfectious uveitis [vitreous \ufb02uid being positive for EBOV by polymerase chain reaction (PCR)] (61, 62).\nThe results of the JIKI (meaning hope in Kissi, a West African language) trial, a historically controlled, multisite, single-arm clinical trial to evaluate the ef\ufb01cacy of favipiravir in EBOVinfected patients in Guinea, were published recently (63). According to the authors of the study, a randomized, placebo-controlled study design was not chosen owing to ethical concerns (64). The authors argued that in the context of widespread distrust of Ebola treatment units, using a randomized design might have led more patients to avoid seeking care. Thus, the objectives of the study were to test the feasibility and acceptability of an emergency trial in the context of\n336 Cardile et al.\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\na large EBOV outbreak and to collect safety and ef\ufb01cacy data to optimize the design of future studies. All patients received favipiravir and standard care, which included oral or intravenous (IV) rehydration, electrolyte correction, empiric antimalarial and antibacterial therapies, analgesics, and antiemetic drugs. In vitro and mouse ef\ufb01cacy data for favipiravir against EBOV were combined with pharmacokinetic data from uninfected mice and humans to calculate a recommended dosing regimen for human patients infected with EBOV (65). This regimen consisted of a loading dose of 2,400 mg, followed by 1,200 mg every 8 h on day 1 (6,000 mg total) and then a maintenance dose of 1,200 mg twice a day for 9 days (63, 65). As a result, the dose proposed to treat EBOV in adults was 50% higher than the dose for in\ufb02uenza treatment (1,800 mg twice a day on day 1, 800 mg twice a day on days 2\u20135) (65). Calculations for weight-based dosing in children were also conducted (66).\nFor the primary statistical analysis, patients with cycle threshold (Ct) values >20 at admission were compared to those with Ct values <20 after analysis of recent historical data from the same Ebola treatment units revealed a strong association with mortality (63). Ct values of 20 were found to correspond to an RNA viral load of 5 \u00d7 107 genome copies/mL of plasma (7.7 log10 copies/mL). At entry into the trial, 55 subjects were enrolled with Ct >20, and 44 subjects had Ct < 20. Those with Ct values <20 had signi\ufb01cantly greater creatinine, blood urea nitrogen, aspartate aminotransferase, alanine aminotransferase, and creatine kinase levels at the time of admission than those with Ct >20. On-trial mortality was 20.0% in patients with Ct >20, which is 33% lower than the target value (30%); it was 90.9% in patients with Ct < 20, which was 7% higher than the target value (85%). Thus, there may be a survival bene\ufb01t in patients with Ct values >20 and no improvement in survival when Ct values are <20. No grade 3 or 4 clinical events were considered to be related to the drug by the investigators, and all deaths were associated with uncontrolled EBOV viremia and disease progression. There was no signi\ufb01cant difference in terms of mortality between adults who started favipiravir within 72 h and those who started favipiravir >72 h after symptoms \ufb01rst appeared [45.2% versus 54.4% (P = 0.5)].\nThe design of the JIKI trial has been criticized heavily, as there was low statistical power and no placebo arm, and as a result, the study authors admit that favipiravir ef\ufb01cacy was not proved (63). Further complicating the design, it used historical controls, which is a signi\ufb01cant confounder owing to changing case fatality rates and changing standards of care over time. Unfortunately, in this study blood samples were not able to be collected systematically to examine pharmacokinetics. Sissoko et al. (63) plan to examine plasma trough concentrations collected to update their dosing estimation model. It would be more helpful to conduct expanded safety and pharmacokinetics studies in African populations at risk for EBOV infection to determine optimal dosing for future studies.\nSissoko et al. (63) also state that their results suggest that trials of antiviral drugs in EBOV should be strati\ufb01ed by Ct value and that trials of antiviral monotherapy should primarily target patients with Ct > 20. This proposal ignores the fact that Ct values from site to site may not be generalizable owing to different methodologies and technicians (63); establishing a quali\ufb01ed, quantitative real time\u2013PCR assay will be critical to any cross-site analyses. Moreover, the proposal to stratify the analysis based on Ct value is an oversimpli\ufb01cation of the issues of protocol design. For favipiravir, researchers have not demonstrated that the dose administered was optimal: Higher doses or an alternate formulation (e.g., IV) of the drug may in fact result in ef\ufb01cacy in patients with Ct < 20. Thus, a revised conclusion could be that favipiravir as dosed in this study should not be used as a monotherapy in patients with Ct < 20. Additionally, other drugs may be more potent and have greater ef\ufb01cacy in EBOV patients with Ct < 20.\nOptimal Ebola therapy may need to parallel the treatment of HIV infection. In HIV treatment, combination therapy is critical to successful and sustained viral suppression. Furthermore, certain antiretroviral combinations (rilpivirine-based regimens, abacavir/lamivudine with efavirenz or\nwww.annualreviews.org \u2022 Cure for Ebola 337\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\natazanavir/ritonavir, and darunavir/ritonavir plus raltegrivir) have higher rates of virological failure than others when HIV viral RNA is >100,000 copies/mL (67). As in HIV antiviral therapy, therapy of EBOV may require more than one agent for ef\ufb01cacy, especially in individuals with high viral loads and more advanced disease.\nGS-5734. GS-5734 is a novel monophosphoramidate prodrug of an adenosine analogue, and data suggest that it selectively inhibits EBOV replication by targeting its RNA-dependent RNA polymerase and inhibiting viral RNA synthesis following ef\ufb01cient intracellular conversion to an active triphosphate nucleotide (68). GS-5734 has a 1 -cyano group, which provides potency and selectivity toward viral RNA polymerases. The drug was further modi\ufb01ed with a monophosphate promoiety to enhance intracellular metabolism into the active triphosphate metabolite. To demonstrate enhanced intracellular metabolism, human monocyte\u2013derived macrophages were incubated with GS-5734, with a rapid loading of cells with up to 30-fold higher levels of the active triphosphate metabolite compared to incubation with the parent 1 -cyano-substituted adenine C-nucleoside ribose analogue. GS-5734 displayed potent in vitro activity (EC50 values of 0.06\u2013 0.14 \u03bcM) against EBOV-Kikwit, EBOV-Makona, Sudan virus, Bundibugyo virus, and Marburg virus. GS-5734 also inhibited other RNA viruses, including respiratory syncytial virus, Jun\u00b4\u0131n virus, Lassa virus, and Middle East respiratory syndrome virus.\nPharmacokinetics were determined in NHPs and in rhesus macaques following IV infusion of 10 mg/kg; the half-life was 0.39 h with rapid systemic elimination and persistent levels of the active triphosphate metabolite (68). GS-5734 was distributed rapidly into peripheral blood mononuclear cells, was converted to active triphosphate metabolite within 2 h of administration, and maintained levels necessary for >50% viral inhibition for 24 h (68). In cynomolgus macaques that received 10 mg/kg IV doses of [14C] GS-5734, drug-derived material was distributed to testes, epididymis, eyes, and brain within 4 h of administration. Levels in brain at 4 h were low relative to other tissues but remained detectable above the drug plasma levels 168 h after dosing.\nThe ef\ufb01cacy of GS-5734 in EBOV-infected rhesus monkeys was assessed in the well-established EBOV-Kikwit IM infection model [1,000 PFU of 7U (7-uridylyl stretch, poly-U site in the glycoprotein sequence) virus] (68). The most effective dosing regimen of GS-5734 was 10 mg/kg administered via IM injection daily, initiated 3 days postinfection, with 100% survival. Compared to vehicle treatment, GS-5734 signi\ufb01cantly reduced plasma viral RNA by \u22651.7 log10 on day 4, and on days 5 and 7, levels were below the lower levels of quantitation (8 \u00d7 104 RNA copies/mL), whereas levels in animals receiving the vehicle exceeded 109 copies/mL. GS-5734 was associated with reduction in EBOV clinical disease scores, and mitigation of EBOV-induced laboratory abnormalities included platelet count, creatinine, transaminases, coagulation parameters, and creatinine kinase.\nIV GS-5734 is currently undergoing human Phase I safety and pharmacokinetic studies (68, 69). GS-5734 has been used as a compassionate therapy in two cases of EBOV patients who survived infection: a case of EBOV recrudescence in a female nurse in the United Kingdom and a case of acute EBOV infection in a female infant in Guinea (70, 71). GS-5734 could be a promising candidate for EBOV treatment given its potency and for eradication of persistently replicating virus in immunologically privileged sites given its tissue penetration. In addition, GS-5734 may be an attractive therapy for postexposure prophylaxis, as its active triphosphate form is rapidly accumulated and maintained in cells of the mononuclear lineage, which are important in EBOV pathogenesis.\nNucleic Acid\u2013Based Therapeutics\nTwo classes of nucleic acid\u2013based therapeutics have been studied to determine if they can treat EBOV infections: antisense phosphorodiamidate morpholino oligomers (PMOs) and small\n338 Cardile et al.\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\ninterfering RNAs (siRNAs). Both PMOs and siRNAs provided promising ef\ufb01cacy results in NHPs against EBOV, but because corporate support for further development of these products has been withdrawn, these are discussed only brie\ufb02y. PMOs are single-stranded deoxyribonucleotide oligomers that inhibit translation via steric hindrance (72, 73). siRNAs are doublestranded oligonucleotides that cause RNA interference (RNAi), silencing gene expression via mRNA degradation (72\u201374). Both oligonucleotides and naked RNAi molecules are unstable in vivo owing to nuclease digestion, are prone to poor intracellular uptake, and as a result require chemical modi\ufb01cations or additional formulation for in vivo administration (72, 73, 75, 76). The detailed development and chemical modi\ufb01cation of siRNAs and PMOs are described in detail in other sources (72, 73, 75).\nThe \ufb01rst-generation siRNA product, TKM-Ebola, was a combination of three siRNA molecules that targeted the EBOV RNA-dependent RNA polymerase L, virion protein 24 (VP24), and virion protein 35 (VP35) (77); in subsequent generations, only siRNAs designed to target L and VP35 were included (78). A PMO product, AVI-6002, is composed of AVI-7539 and AVI7537, which target VP35 and VP24, respectively (79, 80). TKM-Ebola (2 mg/kg total siRNA per dose, IV infusion) was administered to macaques 30 min after EBOV challenge, followed by doses on days 1\u20136, resulting in 100% survival (4/4) (77). Study of AVI-6002 in NHP models would eventually reveal that VP24 (AVI-7537) targeting was responsible for the observed activity, with 75% (6/8 NHPs) surviving with AVI-7537 therapy and none surviving with AVI-7539 therapy (81).\nWhen the West Africa EBOV outbreak strain was determined to be genetically unique (termed EBOV-Makona), there were concerns about the ef\ufb01cacy of siRNA and PMOs, as they are sequence-speci\ufb01c products targeting EBOV-Zaire. Researchers did not identify mutations that would be anticipated to impact AVI-7537 if used against EBOV-Makona (82). In regards to the siRNA product, mutations were detected in the binding sites of L and in VP35 targets (82). As a result, a new siRNA cocktail, siEbola-3, was designed to correct these mismatches to enable full complementarity to EBOV-Makona sequences (78). NHPs were infected with EBOV-Makona and administered siEbola-3 (0.5 mg/kg) beginning 72 h after infection, when animals were viremic and clinically ill, and had repeat daily treatments on days 4\u20139 post-challenge (78). All treated animals survived to study endpoint, whereas untreated control animals succumbed on days 8 and 9 (78).\nA Phase I clinical trial of the second-generation TKM-Ebola was initiated in January 2014, and in May 2014, the single-ascending dose portion of the study was completed. But in July 2014, it was put on clinical hold owing to concerns by the FDA regarding cytokine release, and the clinical hold has since been released (83). A Phase I clinical trial of AVI-6002 was completed in healthy adults (84). AVI-6002 was safe and well tolerated at the doses studied, with a maximum tolerated dose not observed (84). No further information on AVI-6002 or AVI-7537 has been published to date.\nAn ef\ufb01cacy study of siEbola-3 in Sierra Leone was initiated in March of 2015 (58, 85, 86). The study was an open-label, single-arm trial and was historically controlled but was terminated, as interim analysis indicated that continuing enrollment was not likely to demonstrate an overall therapeutic bene\ufb01t (85, 86). The product sponsor, Tekmira (now Arbutus Biopharma), has suspended its EBOV antiviral therapeutic program (87).\nIMMUNE THERAPEUTICS\nConvalescent Plasma and Hyperimmune Serum\nHistorically, clinical data have been limited on the bene\ufb01ts of convalescent blood products for EBOV therapy and were restricted to the case series from the 1995 EBOV-Kikwit outbreak in the Democratic Republic of the Congo and a few case reports (88). During the West Africa EBOV outbreak, the WHO had prioritized Ebola convalescent whole-blood and convalescent plasma\nwww.annualreviews.org \u2022 Cure for Ebola 339\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\ntransfusion for evaluation, with the rationale that this could be done quickly and, if proved to be safe and effective, could be implemented rapidly (89). Three convalescent plasma trials enrolled patients during the outbreak (89).\nTo date, the results of one nonrandomized, historically controlled study that was conducted in Guinea have been published (90). The authors determined that the randomization of patients was locally unacceptable in the setting of the EBOV outbreak (90). Patients of any age, including pregnant women who had symptomatic, laboratory-con\ufb01rmed EBOV, were enrolled (90). A total of 84 EBOV-infected patients were included in the primary analysis, and they were treated with two consecutive transfusions of 200\u2013250 mL of ABO-compatible convalescent plasma; levels of neutralizing antibodies were unknown and each unit of plasma was obtained from a separate convalescent donor (90). Small adults and children weighing less than 45 kg received two transfusions of 10 mL/kg of convalescent plasma (90). The transfusions were initiated on the day of diagnosis or up to 2 days later, and the level of neutralizing antibodies against EBOV in the plasma was not determined at the time of administration (90). The study was historically controlled with 418 patients who had been treated at the same center during the previous 5 months (90). From day 3 to day 16 after diagnosis, 26 of 84 patients (31%) in the convalescent-plasma group died, and 158 of 418 patients (38%) died in the control group. After adjustment for age and Ct values, mortality remained lower in the convalescent-plasma group, but the difference was not signi\ufb01cant (90). Eight patients (8%) had an adverse reaction during or soon after the transfusion with elevated temperature predominating, followed by pruritis or skin rash (90).\nThis trial had study design \ufb02aws similar to those in the JIKI trial that were dictated by the circumstances of the EBOV outbreak. The authors of this study admit that the inability to determine the level of neutralizing antibodies in the donor plasma was a limitation of the study (90). They state that the inability to conduct EBOV plaque-neutralization assays in a biosafety level 4 laboratory or to ship blood out of country was the limiting factor (90). Possibly, titers could have been conducted via ELISA or another method at the study site. If the use of plaque-neutralization titers is deemed necessary, then an assay based on an Ebola GP-pseudotyped virus or other test systems could be developed. Several in vivo studies have had varied results regarding the use of passive antibody therapy (91\u201394). Important themes that have emerged from these studies are that antibody titers in human convalescent plasma tend to be low, frequent dosing is needed to maintain antibody titers, survival is correlated in some studies with anti-EBOV IgG titers, and there is batch-to-batch titer variability in different plasma pools (91\u201394). Thus, in this clinical trial, the lack of a mortality bene\ufb01t may be related to the fact that the antibody titers were low in the transfused plasma and that plasma was not transfused frequently enough.\nThese issues could be overcome, as illustrated in an in vivo study with hyperimmune plasma. Polyclonal IgG was puri\ufb01ed from a large preparation of convalescent serum, which had been pooled from vaccinated macaques; these donor macaques subsequently survived challenge with a lethal dose of EBOV, suggesting that their antibody titers were adequate for protection (95). The fractionated IgG was evaluated and shown to have virus-neutralizing activity, and recipient NHPs were treated with 70\u2013100 mg/kg of fractionated IgG (95). Recipient NHPs were then infected with EBOV, and treatments were initiated 48 h postinfection, with additional treatments on days 4 and 8 postexposure (95). This regimen protected 100% of EBOV-challenged NHPs (95). The success with IgG treatments in this study was attributed to the polyclonal nature of the exogenous antibodies controlling viral infection and to the multiple treatments, which were thought to maintain suf\ufb01ciently high levels of IgG to permit the host\u2019s adaptive immune response to help clear the viral infection (95). However, the production of large quantities of hyperimmune plasma adequate to respond during an outbreak would be dif\ufb01cult, given that it would require a large supply of convalescent plasma or production in animals.\n340 Cardile et al.\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\nZMapp\nDuring the West Africa EBOV outbreak, ZMapp was lauded as a potential game changer, but it was available in exceedingly limited supply. The two US health-care workers who acquired Ebola in Liberia were treated with ZMapp, the \ufb01rst on days 9, 12, and 15 of illness and the second on days 10, 13, and 16, both without adverse effects (96). Both patients reported subjective improvement to varying degrees after receiving ZMapp, and both survived the infection (96). In these cases, it is not clear if ZMapp was one of the primary factors contributing to survival. Both patients were aggressively \ufb02uid resuscitated and had whole-blood transfusions in Liberia, including (in the case of Patient 1) blood from an Ebola survivor (96).\nZMapp is a mixture of MB-003 and ZMAb (97). ZMAb components were produced in Nicotiana benthamiana using the large-scale, current good manufacturing practice\u2013compatible rapid antibody production platform that was used for MB-003 (97). After a lethal IM challenge with EBOV, rhesus macaques were treated with ZMapp (50 mg/kg) at 3, 6, and 9 days postinfection; 4, 7, and 10 days postinfection; or 5, 8, and 11 days postinfection. All animals treated with ZMapp survived infection (97). Because ZMapp components were developed against EBOV-Kikwit, there were concerns that it would be ineffective or less effective during the West Africa outbreak. EBOV-Makona was evaluated via indirect comparison of published amino acid sequences, and the epitopes targeted by ZMapp were not mutated between the two virus variants (97).\nIn March 2015, a Phase I open-label study was launched to evaluate the safety and pharmacokinetics of a single dose of 50 mg/kg ZMapp in healthy adult volunteers (98). Recently, the long-awaited results of the multisite, randomized controlled trial of ZMapp in EBOV infection (PREVAILII) were presented at the Conference on Retroviruses and Opportunistic Infections (CROI) (99). Patients were randomized 1:1 to receive either the optimized standard of care (de\ufb01ned minimally as IV \ufb02uid resuscitation plus electrolyte monitoring) or the optimized standard of care plus three IV infusions of 50 mg/kg ZMapp three days apart (99). Seventy-two patients were strati\ufb01ed by baseline PCR Ct values (\u226422 versus >22) and by treatment site [United States versus Liberia or Sierra Leone versus Guinea (where favipiravir was part of the optimized standard of care)] (99). ZMapp did not confer a statistically signi\ufb01cant mortality bene\ufb01t, with 37% mortality in the control arm and 22.2% mortality in the treatment arm of the study (99). Even though the study was strati\ufb01ed, there are signi\ufb01cant confounders that may affect study results. The most concerning was that the 12 patients treated with ZMapp in Guinea were also treated with favipiravir, and it is questionable if these patients should be included in the analysis. One could easily argue that the patient treated in the United States should also be excluded from analysis given that the supportive care was likely signi\ufb01cantly different to that provided in Ebola treatment units.\nOther monoclonal antibodies and cocktails are under development, with most being in preclinical development (100\u2013104). MIL77 is the most advanced and is a collaboration between the Public Health Agency of Canada, Mapp Biopharmaceutical, and Beijing Mabworks (104). This drug is composed of ZMapp-like monoclonal antibodies in modi\ufb01ed Chinese hamster ovary cells and was administered to four patients during the West Africa outbreak (104). In a recent study, MIL77 was shown to be at least comparable to treatments using a similar formulation of the ZMapp antibodies in NHP models of EBOV-Makona disease (104).\nInterferon-\u03b2\nEBOV infection is associated with signi\ufb01cant IFN-\u03b1 production but little IFN-\u03b2 production; plasma concentrations of IFN-\u03b1 greatly exceed those seen in other viral infections (105). Early postexposure treatment with IFN-\u03b2 signi\ufb01cantly increased survival times of rhesus macaques infected with a lethal dose of EBOV, although it failed to alter mortality (105). Infected macaques\nwww.annualreviews.org \u2022 Cure for Ebola 341\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\nwere then treated with human recombinant IFN-\u03b2 (10.5 \u03bcg/kg) 18 h and 1, 3, 5, 7, and 9 days after EBOV infection, which signi\ufb01cantly prolonged the mean time to death (105). A historically controlled trial of IFN-\u03b2 was attempted in Guinea, with nine patients enrolling, but results are not currently available in the peer-reviewed literature (43).\nTHE PATH FORWARD\nThe net results of medical research that occurred during the West Africa EBOV outbreak have been described as a \u201cthin harvest\u201d in a recent article (43). Overall, the global health community was unprepared for a large EBOV outbreak and lacked the ability to initiate clinical trials rapidly in that setting. As a result of delays in implementation, many of the clinical trials discussed above were not initiated while cases existed in adequate numbers. In the future, deployable, mobile capabilities need to be developed to facilitate rapid conduct of clinical trials during an outbreak. This would require the following conditions be present in a country at risk for an outbreak: support of hostnation governments; prepositioned health care and clinical research infrastructure, including welltrained personnel, functioning laboratory support, logistical systems, human subjects research and regulatory oversight, and continuously exercised system function; trained and ready research teams; \ufb01led INDs; \ufb02exible and preapproved clinical research protocols; an established logistical tail, including available therapeutic products; and functional integration into host-nation and international response plans.\nIn addition, there were signi\ufb01cant ethical concerns about conducting blinded, randomized, placebo-controlled clinical trials in an outbreak setting with a virus with high mortality and transmission rates (106). Clinical researchers who support randomized, placebo-controlled trial designs in Ebola research argued that this design is the most ef\ufb01cient and powerful method for assessing the safety and effectiveness of available experimental interventions and that alternate trial designs are likely to lead to invalid results (107). Those against the use of placebo-controlled trials have argued that randomizing individuals in a treatment trial to the placebo-controlled arm, in which they receive supportive care only, denies them at least the possibility of bene\ufb01t that might result from an experimental treatment (107). In addition, patients that could potentially be enrolled in trials in an outbreak setting with high mortality could be considered a vulnerable population that cannot truly provide informed consent. There were also signi\ufb01cant concerns that in the context of widespread distrust of Ebola treatment units, using a randomized design could have led more patients to avoid seeking care. As a result of these concerns, only the ZMapp trial was conducted with a randomized, controlled design, whereas the other three trials (favipiravir, convalescent plasma, and TKM-Ebola) used an alternate design with historical controls. None of these trials showed ef\ufb01cacy of a product to support licensure at this time. Given that historically controlled trials were ineffective, randomized controlled trials need to be designed to mitigate ethical concerns. In addition, minimal standards for optimized supportive care and therapy for coinfections need to be adopted at all clinical trial treatment sites. One possible trial design that could facilitate this goal would be an adaptive trial, similar to that of the ZMapp trial (108). This design has been described in a recent publication and involved early, frequent interim analyses and a \u201cbarely Bayesian design\u201d (108, p. 6).\nIn conclusion, we return to the initial question posed: Will there be a cure for Ebola? At this time, despite signi\ufb01cant investments in clinical research during the West Africa EBOV outbreak, we are still a long way from a licensed MCM and thus a cure. In addition, researchers have learned that even in individuals considered to have been cured from Ebola, there can be recrudescence of the virus, and that a post-Ebola syndrome with chronic symptoms and viral persistence in immunologically privileged sites such as the eyes and gonads may occur more frequently than\n342 Cardile et al.\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 09:43:13\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\npreviously thought. As a result, future therapeutics development should take into account a need for adequate tissue penetration into sites such as the eyes, gonads, and central nervous system. Multiple valuable lessons have been learned about the conduct of clinical research in a resource-poor, high risk\u2013pathogen setting. However, the timing and number of cases that will be associated with the next outbreak in which clinical candidates can be tested are unknown and cannot be predicted with accuracy. Until the next outbreak occurs with suf\ufb01cient patient numbers, further development of EBOV antiviral therapies and vaccines will be forced to rely on ef\ufb01cacy characterizations in animal models of the disease.\nDISCLOSURE STATEMENT The authors are not aware of any af\ufb01liations, memberships, funding, or \ufb01nancial holdings that might be perceived as affecting the objectivity of this review. The views expressed herein are those of the authors and do not re\ufb02ect the of\ufb01cial policy or position of the US Army Medical Research Institute of Infectious Diseases, US Army Medical Department, US Army Of\ufb01ce of the Surgeon General, Department of the Army, Department of Defense, or US Government. The authors are employees of the US government. This work was prepared as part of their of\ufb01cial duties. Mention of trade names, commercial products, or organizations does not imply endorsement by the US Government.\nACKNOWLEDGMENTS The authors would like to acknowledge funding from Medical Countermeasures Systems and the US Defense Threat Reduction Agency.\nLITERATURE CITED 1. Merriam-Webster.com. 2016. Cure. Accessed on Mar. 1. http://www.merriam-webster.com/ dictionary/cure 2. Greenwood B. 2014. The contribution of vaccination to global health: past, present and future. Philos. Trans. R. Soc. B 369:20130433 3. Mariner JC, House JA, Mebus CA, Sollod AE, Chibeu D, et al. 2012. Rinderpest eradication: appropriate technology and social innovations. Science 337:1309\u201312 4. Ohimain EI. 2016. Recent advances in the development of vaccines for Ebola virus disease. Virus Res. 211:174\u201385 5. Sridhar S. 2015. Clinical development of Ebola vaccines. Ther. Adv. Vaccines 3:125\u201338 6. WHO (World Health Organ.). 2015. Table of drug clinical trials. WHO, Geneva. Accessed on July 23. http://www.who.int/medicines/ebola-treatment/ebola_drug_clinicaltrials/en/ 7. Hensley LE, Dyall J, Olinger GG Jr., Jahrling PB. 2015. Lack of effect of lamivudine on Ebola virus replication. Emerg. Infect. Dis. 21:550\u201352 8. Fedson DS. 2015. Immunomodulatory adjunctive treatment options for Ebola virus disease patients: another view. Intensive Care Med. 41:1383 9. Fedson DS, Jacobson JR, Rordam OM, Opal SM. 2015. 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Ethics and Ebola: Public Health Planning and Response. Rep., Pres. Comm. Study Bioethical Issues, Washington, DC. Accessed on Mar. 20, 2016. http://bioethics. gov/sites/default/\ufb01les/Ethics-and-Ebola_PCSBI_508.pdf\n108. Dodd LE, Proschan MA, Neuhaus J, Koopmeiners JS, Neaton J, et al. 2016. Design of a randomized controlled trial for Ebola virus disease medical countermeasures: PREVAIL II, the Ebola MCM study. J. Infect. Dis. 213:1906\u201313\n348 Cardile et al.\n\n\n",
    "authors": [
      "Anthony P. Cardile",
      "Travis K. Warren",
      "Karen A. Martins",
      "Ronald B. Reisler",
      "Sina Bavari"
    ],
    "doi": "10.1146/annurev-pharmtox-010716-105055",
    "date": "2017-01-06",
    "item_type": "journalArticle",
    "url": "https://www.annualreviews.org/doi/10.1146/annurev-pharmtox-010716-105055"
  },
  {
    "key": "C8KACUV7",
    "title": "Clinical Evaluation of Ebola Virus Disease Therapeutics",
    "abstract": "Ebola virus disease (EVD) was first described over 40 years ago, but no treatment has been approved for humans. The 2013\u20132016 EVD outbreak in West Africa has expedited the clinical evaluation of several candidate therapeutics that act through different mechanisms, but with mixed results. Nevertheless, these studies are important because the accumulation of clinical data and valuable experience in conducting efficacy trials under emergency circumstances will lead to better implementation of similar studies in the future. Here, we summarize the results of EVD clinical trials, focus on the discussion of factors that may have potentially impeded the effectiveness of existing candidate therapeutics, and highlight considerations that may help meet the challenges ahead in the quest to develop clinically-approved drug(s).",
    "full_text": "Author Manuscript\n\nAuthor Manuscript\n\nHHS Public Access\nAuthor manuscript\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\nPublished in final edited form as: Trends Mol Med. 2017 September ; 23(9): 820\u2013830. doi:10.1016/j.molmed.2017.07.002.\nClinical Evaluation of Ebola Virus Disease Therapeutics\nGuodong Liu1,2, Gary Wong1,2,3,4, Shuo Su5, Yuhai Bi3,4, George F Gao3,4, Gary Kobinger2,6, and Xiangguo Qiu1,2,* 1Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada 2Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Manitoba, Canada 3Shenzhen Key Laboratory of Pathogen and Immunity, State Key Discipline of Infectious Disease, Shenzhen Third People\u2019s Hospital, Shenzhen, China 4CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China 5Jiangsu Engineering Laboratory of Animal Immunology, Institute of Immunology and College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China 6D\u00e9partement de microbiologie-infectiologie et d\u2019immunologie, Universit\u00e9 Laval, Qu\u00e9bec, Canada\nAbstract\nEbola virus disease (EVD) was first described over 40 years ago, but no treatment has been approved for humans. The 2013\u20132016 EVD outbreak in West Africa has expedited the clinical evaluation of several candidate therapeutics that act through different mechanisms, but with mixed results. Nevertheless, these studies are important because the accumulation of clinical data and valuable experience in conducting efficacy trials under emergency circumstances will lead to better implementation of similar studies in the future. Here, we summarize the results of EVD clinical trials, focus on the discussion of factors that may have potentially impeded the effectiveness of existing candidate therapeutics, and highlight considerations that may help meet the challenges ahead in the quest to develop clinically-approved drug(s).\nKeywords Ebola virus; therapeutics; small molecule inhibitor; convalescent plasma; monoclonal antibody\n*Correspondence: Xiangguo.Qiu@phac-aspc.gc.ca (X Qiu). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The authors have no conflicts of interest to declare.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 2\n\nEbola Virus Disease Therapeutics Are Urgently Needed\nThe outbreak of Ebola virus (EBOV) in West Africa from December 2013 to March 2016 was the largest ever reported to date, with 28,616 cases and 11,310 deaths (http:// apps.who.int/iris/bitstream/10665/208883/1/ebolasitrep_10Jun2016_eng.pdf?ua=1).\nEBOV belongs to the genus Ebolavirus, which causes EBOV disease (EVD) \u2013 clinically manifested by a spectrum of symptoms including fever, fatigue, muscle pain, vomiting, diarrhea, anorexia, rash, bleeding and multi-organ failure [1, 2]. Disease fatality rate can be up to 90% (http://www.who.int/mediacentre/factsheets/fs103/en/). The re-emergence of EBOV in the future cannot be ruled out because it can cause sporadic infections from unknown natural reservoirs (see Glossary) and potential transmission from EVD survivors, such as those that shed virus through bodily fluids including semen and breast milk [3, 4]. Due to high fatality rates, poorly-defined natural reservoirs and transmission mechanisms, in addition to the potential for weaponization, EBOV constitutes a major public health concern.\nEBOV pathogenesis is currently only partially understood. EBOV is known to evade the Type I interferon (IFN) response through viral proteins VP30, VP35 and VP24 [5, 6], which contribute to initial viral replication and pathogenicity. Studies in non-human primates (NHPs) showed that early cellular targets of EBOV comprise macrophages and dendritic cells [7], which are currently recognized as two key players in pathogenesis [8, 9]. Dendritic cell maturation can be suppressed by EBOV, as evidenced by the failure of these cells to secrete proinflammatory cytokines and by the absence of upregulated co-stimulatory molecules, leading to impairment in antigen presentation to T-cells [10, 11]. Indeed, dysfunctional macrophages and dendritic cells likely cause deregulated innate immunity through the excessive production of proinflammatory cytokines and chemokines, as well as by suppression of adaptive immune responses against EBOV due to compromised presentation of antigen to lymphocytes and inadequate expression of co-stimulatory factors [12]. While there have been studies in mice [13] and humans [14] showing substantial involvement of adaptive immunity in advanced EVD, particularly the activation CD8+ Tcells, the systemic dissemination and robust viral replication stemming from an inability to control the infection at early disease stages eventually leads to multi-organ failure [1]. Therefore, strategies to develop targeted therapies against EVD are mostly focused on blocking viral entry and inhibiting viral replication [15].\nSubstantial efforts have been devoted to the development of EVD therapeutics in animal models over the past two decades, but remain untested in humans. The outbreak in West Africa greatly expedited the clinical evaluation of several promising therapeutics. Current candidate therapeutics mainly fall into 2 major categories: i) small molecule inhibitors, including licensed drugs to be repurposed for EVD treatment and newly developed nucleic acid-based products; and ii) immune-based therapeutics, including IFNs, plasma transfusion and monoclonal antibodies (mAbs). Full, or interim results of clinical trials for a number of experimental therapeutics have recently been reported. In this review, we summarize findings from those clinical trials that have been completed (Table 1, Key Table) and discuss the limitations that need to be overcome for the successful development of EVD-targeting therapies.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 3\n\nSmall Molecule Inhibitors: Direct Intracellular Inhibition of EBOV\nA popular approach in the search for effective therapeutics is the identification and characterization of small molecules that might inhibit EBOV, presumably through different mechanisms, including suppression of viral transcription and replication. Many small molecules, such as brincidofovir, BCX4430, favipiravir, GS-5734 and AVI-6002, have been shown to be protective in cultured cells, or in animal models such as mice and NHPs [15, 16], but remain to be assessed against EVD in humans. Recently, several small molecule drugs licensed for the treatment of other viral diseases, such as influenza and yellow fever [17, 18], or that have been newly developed against EVD, have been evaluated for their efficacy and effectiveness against EBOV infection in non-randomized clinical trials. These drugs include small compounds such as nucleotide analogs and small interfering RNAs (siRNAs) that target specific EBOV viral proteins.\n\nNucleotide Analogs: The Potential of Favipiravir in EVD Patients with Low Viral Loads\nFavipiravir (6-fluoro-3-hydroxy-2-pyrazinecarboxamide), or T-705, is a pyrazine derivative discovered from a screen of chemical compounds against influenza virus A/PR/8/34 (H1N1); it is modified intracellularly to form a purine nucleotide analog with inhibitory activity against viral RNA-dependent RNA polymerase (RdRP), but exhibits low or no inhibition of canine DNA and RNA polymerase, or human DNA polymerase [19, 20]. Favipiravir has shown inhibitory effects against a wide range of RNA viruses [17, 18, 20\u2013 25]. Recent studies in mouse models demonstrated post-exposure protection against EBOV through oral administration of favipiravir [26, 27]. In addition, favipiravir was shown to be well-tolerated in healthy or ill adults with uncomplicated influenza in phase 1\u20133 clinical trials [28].\nIn mid-November 2014, favipiravir was given to 39 patients with severe EVD admitted to the Sierra Leone-China Friendship Hospital [29]. Patients (17\u201339 years old) received oral favipiravir at doses of 800 mg bid on day 1, and 600 mg bid on day 2, based on recommendations for use in influenza infections [29]. Patients also received supportive treatments in the following days until recovery, hospital transfer or death. Survival rate and viremia in the favipiravir cohort were compared to control patients who were admitted to the same center earlier and treated with only supportive treatments. Results from the subsets with all endpoint observations available from the 2 groups (n=17 for the favipiravir group, n=18 for the control group) showed a higher survival rate in the favipiravir group (64.8% vs. 27.8%) [29]. Improvement of disease symptoms was observable in the favipiravir group, combined with a significant reduction in viral RNA load (>100 fold) determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). These results indicated that favipiravir might be able to confer a survival benefit to EVD in humans [29].\nIn December 2014, a non-randomized, single-arm proof-of-concept clinical trial (the JIKI trial) was conducted to evaluate the safety and effectiveness of favipiravir at 4 treatment centers in Guinea (ClinicalTrials.gov identifier: NCT02329054) [28]. Among the EVD patients, 111 patients (99 aged 13 and older, 12 aged 6 and younger) received no other experimental therapies and completed the trial, and were thus included in the final analyses [28]. The primary outcome (Box 1) was mortality within a period of 14 days. Doses in\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 4\nadults were determined based on results from mouse studies [30], as well as on pharmacokinetic simulation and dosage tests in humans [28].\nAdult patients were given oral favipiravir at doses of 2400 mg, 2400 mg and 1200 mg every 8 h on day 0, and 1200 mg bid for the following 9 days (target time weighted average plasma concentration was 52 \u03bcg/ml) [31]. Dosages for children were adjusted based on body weight to reach similar drug concentrations as those in adults. Since age and viral load are associated with risk of EVD death [32\u201334], the patients were grouped according to age and baseline viral loads (determined as cycle threshold (Ct) by qRT-PCR) for analysis. The patients aged \u2265 13 years were divided into two subgroups: Group A of Ct \u2265 20 (Ct = 20 is 5 \u00d7 107 genome copies/ml) with lower viral load (n=55) and Group A of Ct < 20 with higher viral load (n=44). Twelve young children (\u2264 6 years old) were included in Group YC [28].\nBy the conclusion of the trial, 59 deaths had occurred within 10 days after the first dose, and 1 death at day 17. Mortality rates were 20% in Group A of Ct \u2265 20 (11 of 55, all with Ct < 25), 90.9% in Group A of Ct < 20 (40 of 44) and 75% in Group YC (9 of 12), all meeting the predefined target mortality (30% for Group A of Ct \u2265 20, 85% for Group A of Ct < 20, and 70% for Group YC). The fatality rates in Group A were consistent with a previously observed correlation between higher viral RNA load (Ct < 20) and higher patient mortality [32, 34]. The high mortality in young children was also consistent with previous observations from two of the four treatment centers [28]. Good tolerance to favipiravir was observed during treatment, whereas continuous monitoring of viremia showed reduction in viral loads in survivors but not in non-survivors [28]. Results of available biochemical tests showed more frequent elevation of creatinine, aspartate aminotransferase and creatine phosphokinase with death in Group A Ct < 20, suggesting high levels of renal and muscular damage [28, 35, 36]. Viral clearance in the three surviving children before discharge was also observed; however, the correlations between the secondary outcomes of this study and treatment (Box 1) were not as apparent as those observed in adults and adolescents. Overall, this study indicated that high doses of favipiravir could be tolerated in EVD patients with Ct \u2265 20, and furthermore, lower fatality rates observed in Group A Ct \u2265 20 suggested that favipiravir might be more beneficial during earlier stages of EVD relative to later stages. We posit that an important consideration will now be to compare patient data of Ct \u2265 20 from treatment centers with historical data, aiming to see if there is any survival advantage in such patients. This will indicate whether the enhanced survival is solely due to favipiravir treatment. If previous data is unavailable, the efficacy of favipiravir should be tested and compared in NHPs at Ct < 20 and Ct \u2265 20.\nOf note, a follow-up study reported that favipiravir plasma concentrations in 66 patients from the trial did not reach the the predefined target level 2 days after treatment initiation, the target decreasing to a median level of ~40% of the level that had actually been predicted by a pharmacokinetic model 4 days after treatment initiation [37]. In addition, no significant correlation was observed between plasma EBOV load reduction or mortality (20/66 died) and drug concentrations. This suggests that the study may have used insufficient favipiravir concentrations for the patients, and consequently, further dose studies will be needed.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 5\n\nNucleic Acid-Based Therapeutics Need Optimization\nNucleic acid-based compounds represent another category of small molecule therapeutics for EVD. Two classes of nucleic acid-based systems have been reported, including antisense phosphorodiamidate morpholino oligomers and siRNAs. Using short oligonucleotides[15, 16], both strategies focus on targeting either viral components responsible for transcription and replication of the viral genome such as EBOV RdRp (L polymerase), or targeting antigens involved with immune suppression, such as by VP35 and VP24 [5, 6]. However, only one siRNA-based treatment has been clinically investigated.\n\nTKM-100802 is a lyophilized nanoparticle siRNA formulation consisting of three siRNAs targeting EBOV VP24, VP35 and the L polymerase responsible for viral RNA transcription and replication [38]. In the NHP model, all four animals receiving seven doses of TKM-100802 via intravenous infusion survived challenge with a lethal dose of EBOV [39]. Following NHP studies, observations from a terminated trial in healthy adults identified an optimal dose of 0.3 mg/kg/day of TKM-100802 for safety and protective efficacy [40]. During the outbreak, it was administered to five EVD patients on compassionate grounds, but no safety and efficacy assessments could be made independently because the patients simultaneously received other treatments [41, 42].\n\nBecause the EBOV outbreak in West Africa was caused by the Makona variant of EBOV, which is distinct from the Mayinga and Kikwit variants in Central Africa, the existing product was reformulated to produce TKM-130803, with sequences specifically targeting this EBOV variant. This formulation demonstrated 100% survival (three of three) in NHPs when administered 72 h after challenge with EBOV Makona [43]. In response to the urgent need for EVD therapeutics, TKM-130803 was applied to a phase 2 trial through the Rapid Assessment of Potential Interventions and Drugs for Ebola (RAPIDE) clinical trial platform (Pan African Clinical Trials Registry PACTR201501000997429) [40]. In this nonrandomized, historically controlled trial, 17 EVD patients of 18 years or older were enrolled, with 3 participants in the observational cohort and the other 14 infused intravenously with TKM-130803 at 0.3 mg/kg/day for up to 7 days. During or following infusions, no obvious cytokine release-related adverse events were observed and no termination or infusion rate change was required [40]. One patient presented exacerbated tachypnoea 48 h after the second dose, but the association with infusion was unclear [40]. Overall, TKM-130803 infusion was well-tolerated and survival at day 14 following drug administration was the primary outcome. Amongst the 14 drug-treated patients, 11 died, with two deaths within 48 hours after admission, and only three patients who had received 7 doses of TKM-130803 survived [40]. In the observational cohort, two of the three participants died 3 days after admission. The endpoint survival probability was 0.27 (95% confidence interval 0.06\u20130.58), failing to reach the pre-specified threshold of 0.55, which indicates no improvement in patient survival compared to the control cohort [40]. A possible explanation could be disease severity, as TKM-130803-treated patients all exhibited high baseline viral RNA loads (\u2265 109 copies/ml plasma for the 11 who died) associated with fatality rates > 90% [32, 44]. In addition, 50% of the patients presented symptoms related to high fatality, including bleeding and diarrhea [45]. This suggested that there was an insufficient amount of time for the drug to take full effectiveness at the given dose and/or there was an insufficient drug\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 6\nconcentration in serum, even with extensive standards of care. It is possible that the potency of TKM-130803 might be improved if given at an earlier stage in EVD, but this has not been tested. Furthermore, the selected dose (0.3 mg/kg/d) may be sub-optimal for protection considering that 0.5 mg/kg/d for 7 days could only provide up to 67% protection against EBOV Makona in NHPs [40]. Moreover, the lipid formulation used in the trial was different from the one used in previous NHP studies, which may have negatively affected the efficacy of this drug in the trial. It is also not clear if siRNA uptake efficiency could have been affected due to damaged liver and/or renal functions and vascular leakage during advanced EVD [46, 47]. Of note, a study has shown that EBOV proteins VP30, VP35 and VP40 can inhibit siRNA function, possibly through interaction with the RNAi machinery and possibly blocking siRNA assembly [48]. However, whether the effect of TKM-130803 can be impeded by these viral proteins through the above mechanisms is currently unknown.\n\nEVD Immunotherapy\nIFN-\u03b2, a Proof-of-Concept Immunomodulation Trial\nIFNs are important components of innate immunity against viral infection and have been used as broad-spectrum antiviral therapies. Protective effects of IFN-\u03b1, -\u03b2 or -\u03b3 against EBOV have been tested in different animal models including mice [49, 50], guinea pigs [51], and NHPs [52, 53]. In one EVD patient, IFNs prepared from Sendai-virus-stimulated peripheral lymphocytes were administered intramuscularly in combination with\nconvalescent serum, and this patient survived [54]. However, IFN administration in combination with other experimental therapeutics has made it difficult to assess the effect of IFNs alone. Among the few available studies on IFN monotherapy, murine IFN-\u03b3 provided up to 100% protection against a recombinant vesicular stomatitis virus expressing EBOV glycoprotein (GP) in IFN-\u03b1/\u03b2 receptor-deficient mice [50]. In another study, six doses of human IFN-\u03b2 (10.5 ug/kg) administered subcutaneously (SC) extended the survival time of NHPs challenged with EBOV or Marburg virus, but did not improve the survival rate [53], suggesting that IFN treatments might be beneficial, but likely not fully protective by themselves. Based on the in vitro observation that IFN-\u03b2 could inhibit the replication of recombinant EBOV in HEK293 cells more strongly than IFN-\u03b1 could [55], a historically controlled clinical trial tested the efficacy of IFN-\u03b2-1a in nine EVD patients in Guinea [56]. Within 2 days following qRT-PCR-mediated confirmation of EVD, IFN-\u03b2-1a (30 ug/ day) was administered SC to patients daily, until patients were tested negative for EBOV, or perished [56]. Six of the patients survived in a 21-day observation window, with a survival rate of 67%, which was 2.5-fold higher than that of a control cohort treated with supportive care in the same time period at a treatment center nearby [56]. Comparison with another historical control cohort of matched age and baseline viremia showed slightly less than 2fold higher survival in the IFN group [56], suggesting potential treatment benefit. Rapid viral clearance and improvement of certain clinical symptoms including physical strength and gastrointestinal dysfunctions were observed with IFN-\u03b2-1a treatment [56], which again, suggested a potential treatment benefit.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 7\n\nInconclusive Results for Convalescent Plasma Therapy\nConvalescent whole blood (CWB) or plasma (CP) is taken from patients who have recovered from EBOV infection and carry specific anti-EBOV antibodies, which has been used as prophylactic and/or therapeutic against EVD [57, 58]. Furthermore, efforts have been made to collect blood donations from convalescent EVD patients since the first EBOV outbreak in Zaire (now Democratic Republic of the Congo) in 1976, but studies on the therapeutic effects of convalescent blood or plasma on EVD are very limited. In 1977, a researcher who was accidentally infected with EBOV received human IFNs, in conjunction with two infusions of convalescent serum and eventually recovered from the infection [54]. During the 1995 EBOV outbreak in Zaire, eight EVD patients received whole blood transfusion donated by surviving patients [57]. Each patient was given one blood transfusion of 150\u2013450 ml, 4 to 15 days following EVD onset and seven survived [57]. However, due to the combined use of other therapeutics [54], in addition to suggestions of virus attenuation late in an outbreak (which may have exaggerated any potential advantages gained from the treatment), the therapeutic benefit of convalescent blood has not been well-investigated so far and remains unclear.\nDuring the 2013\u201316 outbreak, the World Health Organization prioritized the use of CWB and CP to treat EVD patients. In February 2015, a non-randomized clinical trial was launched in Guinea to evaluate the safety and efficacy of CP (ClinicalTrials.gov identifier: NCT02342171) [58]. Ninety-nine patients received 2 transfusions of ABO blood groupcompatible CP (200\u2013250 ml/transfusion, or 10ml/kg body weight) with a 15-min interval [58]. The source of the CP for the two blood transfusions was from separate donors [58]. Eighty-four patients who met the screening criteria were included in the final analysis of mortality and other outcomes in comparison to a historical control group of 418 patients treated with supportive care in the 5 months prior to the trial [58]. Fourteen days after treatment, 26 patients in the CP group (31%) and 158 in the control group (38%) died, and these fatality rates did not reach a pre-determined 20% difference to achieve clinical relevance, even after statistical adjustments were made based on multiple factors such as age and Ct values [58]. However, serious adverse events were not observed among the 99 patients who received CP [58]. Nevertheless, due to the unavailability of on-site methods for determining the levels of specific antibodies in CP, the quality of each transfusion (and thus efficacy) was unknown during the trial [58]. Follow-up data from this study indicated that > 90% of the CP samples contained total anti-EBOV IgG titers > 1:1000, determined by ELISA; however, only 4% contained neutralizing antibody titers of 1:160 and 75% contained a titer < 1:40 [59]. Analysis based on age and baseline Ct values revealed lower mortality in patients receiving the highest IgG doses, but also higher mortality with higher doses of neutralizing antibodies. However, neither correlation between antibody doses and mortality was significant. Thus, the effectiveness of CWB or CP-based products against EVD remains inconclusive based on the currently available data.\n\nMAb-Based Therapeutics: A Potential for ZMapp\u2122\nOver 500 mAbs against EBOV have now been isolated from recovered patients [60\u201364] or developed from animal models, such as mice and NHPs [65\u201369]. Most antibodies are neutralizing in vitro and protective in vivo resulting in survival, but most have not yet been\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 8\ntested in clinical trials. ZMapp\u2122, a cocktail composed of three humanized mAbs targeting different sites on the surface GP of EBOV, has so far been the only one tested in clinical trials [70]. This antibody blend consists of an optimized combination of two mAb cocktails, ZMAb and MB-003, previously shown to be protective in NHPs, resulting in full survival when given at 24 h after infection, or partial survival even after the appearance of viremia [52, 71]. In a landmark study, ZMapp\u2122 was shown to reverse advanced EVD and provide 100% protection for rhesus macaques when given up to 5 days after challenge [72]. In March 2015, a phase 1a open-label trial was launched to evaluate the safety and pharmacokinetics of ZMapp\u2122 in healthy human adults (ClinicalTrials.gov identifier: NCT02389192). During the 2013\u201316 outbreak, ZMAb and ZMapp\u2122 were separately given to 25 patients on a compassionate basis [73]. Twenty-two patients survived without showing serious adverse events after receiving three doses of each antibody cocktail (50 mg/kg of body weight) [73]. However, the effectiveness of the cocktails could not be accessed because the patients had also received other treatments, including CP transfusion and intensive standards of supportive care [73].\nIn February 2015, a randomized and controlled phase 1/2 clinical trial, the Partnership for Research on Ebola Virus in Liberia II (PREVAIL II), was initiated to evaluate the efficacy and effectiveness of ZMapp\u2122 (ClinicalTrials.gov identifier: NCT02363322) [74]. The trial enrolled 72 patients (200 patients in the initial plan) from Liberia, Sierra Leone, Guinea, and the US, due to the substantial decline of EVD cases at the late stages of the outbreak [74]. The patients were randomized into either a control group receiving optimized standard of care only (oSOC, with aggressive fluid resuscitation, hemodynamic support, and other interventions available in an optimized care setting), or a treatment group receiving ZMapp\u2122 plus oSOC (n=36 per group). The time from onset of symptoms to treatment in all patients represented 4 to 7 days. After assignment, patients received the first intravenous infusion of ZMapp\u2122 (50 mg/kg of body weight) within 12\u201324 h, followed by two identical doses at every third day. The primary outcome was mortality at day 28 and data from 71 patients was included in the final analysis [74].\nThe fatality rates were 37% (13 of 35) in the control group and 22% (8 of 36) in the ZMapp\u2122 group, leading to a 91.2% posterior probability of superior protection from ZMapp\u2122, which did not reach the preset threshold of \u2265 97.5% [74]. Therefore, ZMapp\u2122 in combination with oSOC did not show a statistically significant decrease in fatality rate over oSOC, even though mortality was 40% lower in the ZMapp\u2122 group relative to the control group. Measurement of secondary outcomes revealed a shorter recovery period among subjects from the ZMapp\u2122 group and the absence in most of the patients, of major safety concerns associated with antibody infusions, such as headache, myalgia, fever and blood pressure changes. These findings have suggested potential safety and therapeutic benefit.\nHowever, for ZMapp\u2122, an insufficient number of available EVD patients may have affected the precision of statistical analyses. In addition, the therapeutic benefit of ZMapp\u2122 is likely underestimated, since seven of eight deaths in the ZMapp\u2122 group occurred before the second dose of antibodies was received, suggesting that these patients may have been near, or at the terminal stage of EVD at the start of the treatment. The fatality rate (1/29) in the subgroup of patients who had finished all three doses of ZMapp\u2122 was nearly eight times\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 9\nlower than among those who survived for > 3 days since admission into the control group [74]. This suggests that additional studies are clearly needed to properly evaluate the efficacy of ZMapp\u2122. Moreover, it is not clear whether sequence differences amongst the GPs of EBOV variants may have had any impact on the efficacy of the antibodies because of potential alterations of GP epitopes. Indeed, ZMapp\u2122 antibodies were developed against the GP of the Mayinga EBOV variant, and comparison of the genomic sequences of the Mayinga and the Makona variants has revealed considerable genetic variations [75], including a non-synonymous mutation in the binding site for mAb 13C6, one component in the formulation of ZMapp\u2122. Consequently, such variations might have affected the virus neutralizing effect of ZMapp\u2122 and thus the effectiveness of this therapeutic treatment. Nevertheless, ZMapp\u2122 might still be able to provide a survival benefit in EVD patients, but its clinical implementation warrants further investigation.\n\nConcluding Remarks\nDespite challenges of testing candidate therapeutics in the midst of EVD outbreaks, valuable experience has been gained in the design and conduct of expedited clinical trials. A common problem with the trials discussed here has been the relatively low enrollment of patients because trials may have been initiated late during an outbreak, rendering it difficult to conclude whether a specific treatment protocol presented any statistically significant benefits to patients. Non-randomized single-arm studies were conducted in most trials, where all EVD patients received experimental therapies. While ethically advantageous given that patients can receive a drug which might play a beneficial role in survival, the interpretation of perceived effects can be confounded by multiple factors, including the selection of historical controls, potential placebo-like effects, and spontaneous recovery (see Outstanding Questions and Box 1). To the best of our knowledge, the ZMapp\u2122 trial may be the only randomized and controlled EVD clinical study that has allowed testing of this compound independently from the current standard of care protocol [76]. The flexibility in its design might enable a promising therapeutic for patients before a trial ends. While the control treatment group did not receive a drug that could be effective, patients still received standard medical care, adding a safeguard mechanism, should any unforeseen negative effects stem from the administration of therapeutics. Nevertheless, there is currently a clear lack of harmonization between trial designs for the different candidates, which makes it difficult to compare the outcomes between treatments. It will be important for future trials to have similar design in order to allow better comparisons.\nAlthough no therapies from the trials discussed here have demonstrated statistical superiority over supportive care, it is worth noting that several EVD treatment candidates, such as favipiravir and ZMapp\u2122, have shown a beneficial trend; this effect may reach statistical significance with further enrollment of EVD patients, or perhaps considering the elimination of patients who died early following treatment, as in the case of the favipiravir study. We posit that the evaluation of a candidate therapeutic should be adjusted based on the different stages of EVD (as determined by Ct value), since the putative compounds may have higher efficacy rates earlier in EVD, although this has not yet been directly tested (see Outstanding Questions). Alternatively, combination therapy with these single agent\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 10\ntreatments may be more effective, but the efficacy should be evidently tested in animal models prior to clinical evaluation.\nMoreover, researchers will need to focus on how evolutionary changes in EBOV structure may affect efficacy of candidate targeting compounds. Indeed, genomic variations among EBOV variants from different outbreaks have been observed [70, 75], and genomic alterations in EBOV Makona GP and L genes have been shown to enhance viral transcription and replication [77], as demonstrated in luciferase reporter assays in which mutant Makona GP and L polymerase were shown to induce stronger activities in human Huh-7 cells, as well as procure a growth advantage over wild-type Makona in both Huh-7 and monkey Vero-E6 cells [77]. Furthermore, it has been proposed that EBOV genomic alterations may be associated with elevated pathogenicity and viral shedding in NHPs because the West African isolates causing the recent outbreak induced higher mortality, higher viremia level and more severe tissue injury compared to other isolates [78] (see Outstanding Questions). Consequently, surveillance of EBOV genetic variations and their impact on the efficacy of relevant therapies will be an important consideration to ensure optimized and successful therapeutic regimens for EVD patients in the future.\nOf clinical relevance, recently, mAbs isolated from human survivors or immunized animals, including mouse and monkey, have shown cross-recognition of and broad protection against multiple members of Ebolavirus in cell lines and animal models [61, 64, 69, 79\u201382]. Indeed, crossreactive mAbs represent a better choice for putative therapeutics since treatments against other members of Ebolavirus are less developed and currently, a large range of pretherapeutic candidates exist only for EBOV. Undoubtedly, the unpredictable nature of filovirus outbreaks highlights the importance of developing successful cross-reactive but efficacious therapeutic reagents to prevent and treat such fatal diseases associated with highly pathogenic viruses.\n\nAcknowledgments\nThis study was supported by the Public Health Agency of Canada, and partially supported by a NIH grant (U19 AI109762-1) to Gary Kobinger and Xiangguo Qiu, and the National Science and Technology Major Project (2016ZX10004222) to George F. Gao, Yuhai Bi and Gary Wong. The authors have no conflicts of interest to declare. Gary Wong, Gary Kobinger, and Xiangguo Qiu were involved in the development and characterization of ZMapp discussed in this review. Yuhai Bi is supported by the Youth Innovation Promotion Association of the Chinese Academy of Sciences (CAS) (2017122). Gary Wong is supported by a grant from the National Natural Science Foundation of China International Cooperation and Exchange Program (8161101193).\nGlossary\nConvalescent serum/plasma collected from convalescent patients who presumably carry specific antibodies against the pathogen causing the disease. Convalescent plasma, serum, or whole blood can be used as therapies for infectious diseases, particularly under circumstances of limited medical resources.\nCt cycle threshold. In real-time quantitative PCR reaction, Ct refers to the cycle at which fluorescent signals from PCR amplification exceed background signals. It is a measurement\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 11\nof the amount of PCR amplicon. The numerical value of Ct is inversely related to the amount of amplicons in a reaction; that is, the lower the Ct value, the higher the number of amplicons.\nEscape mutant a variant of a microorganism, such as a virus, arising through changes in genotype in response to an outside force, such as a host immune response or the effect of therapeutics.\nGP glycoprotein. For Ebola and Marburg viruses, GP is the only surface transmembrane (envelope) protein. The GP gene of Ebola virus is transcribed into two mRNAs, producing two soluble GPs (ssGP and sGP) and one full-length GP which is cleaved into structural GP1 and GP2 by cellular proteins. The Marburg virus GP gene encodes only a single GP protein. The surface GP for these two viruses play a central role in viral entry and fusion. The Ebola GP has been reported to contribute to viral pathogenesis.\nHistorically controlled clinical trial a type of clinical trial in which a treated group of patients is compared to a control group treated from a past outbreak, instead of a concurrent, independent group.\nL gene the gene encoding the RNA-dependent RNA polymerase of filoviruses including Ebola and Marburg viruses. The L polymerase is approx. 220\u2013250 kDa and is responsible for transcription and replication of the viral genome.\nMarburg virus member of the Filoviridae family; genus Marburgvirus. Similar to Ebola virus, Marburg virus is a highly infectious and fatal human pathogen. The virus was first identified in Germany in 1967 and has caused over 10 outbreaks since then. The fatality rate of Marburg virus disease can be up to 90%.\nPhosphorodiamidate morpholino oligomer (PMO) synthetic analogs of nucleic acids, approximately18\u201330 subunits long. PMOs can bind to complementary RNA and block processing, and thus, are used for inhibition of gene expression.\nPrimary outcome a variable that is monitored in a clinical study. Considered the most important or relevant variables to be examined in a clinical trial.\nReservoir hosts natural hosts of an infectious pathogen; can carry the pathogen with little to no disease symptoms.\nSecondary outcome additional variable that is related to a clinical study question, but is less important than primary outcome.\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 12\nSingle-arm clinical study contains only one group of participants receiving the same treatment.\nsiRNA small interfering RNA: short double-stranded RNA molecule (usually 21\u201323 nucleotides in length) produced by RNase III-cleavage and processing of long double-stranded RNA. siRNA is assembled into a protein-RNA complex, binds to homologous sequences in mRNA and guides sequence-specific cleavage and degradation of mRNA. Some siRNAs can mediate methylation of genomic DNA and histones at loci complementary to siRNA, leading to silencing of gene expression.\nSmall molecule inhibitors small chemical compounds or synthetic oligonucleotides with antiviral effects, through different mechanisms, such as interfering with the functions of viral proteins responsible for transcription and replication of a virus.\nSupportive treatment applied to manage symptoms of a disease, aiming to prevent, control or relieve symptoms or side-effects related to the treatment without targeting the underlying cause.\nTime weighted average plasma concentration the average concentration of a drug in plasma over a period of time.\nTachypnoea or tachypnea, refers to abnormally rapid breathing. It may be a sign of more severe or advanced EBOV infection.\n\nReferences\n1. Baseler L, et al. The Pathogenesis of Ebola Virus Disease. Annual review of pathology. 2017; 12:387\u2013418.\n2. West TE, von Saint Andre-von Arnim A. 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Clinical Features of and Risk Factors for Fatal Ebola Virus Disease, Moyamba District, Sierra Leone, December 2014-February 2015. Emerging infectious diseases. 2016; 22:1537\u20131544. [PubMed: 27268303]\n46. Wolf T, et al. Severe Ebola virus disease with vascular leakage and multiorgan failure: treatment of a patient in intensive care. Lancet. 2015; 385:1428\u20131435. [PubMed: 25534190]\n47. Wittrup A, Lieberman J. Knocking down disease: a progress report on siRNA therapeutics. Nature reviews. Genetics. 2015; 16:543\u2013552.\n48. Fabozzi G, et al. Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the mammalian RNA interference pathway. Journal of virology. 2011; 85:2512\u2013 2523. [PubMed: 21228243]\n49. Richardson JS, et al. Evaluation of Different Strategies for Post-Exposure Treatment of Ebola Virus Infection in Rodents. Journal of bioterrorism & biodefense. 2011\n50. Rhein BA, et al. Interferon-gamma Inhibits Ebola Virus Infection. PLoS pathogens. 2015; 11:e1005263. [PubMed: 26562011]\n51. Qiu X, et al. Monoclonal antibodies combined with adenovirus-vectored interferon significantly extend the treatment window in Ebola virus-infected guinea pigs. Journal of virology. 2013; 87:7754\u20137757. [PubMed: 23616649]\n52. Qiu X, et al. mAbs and Ad-vectored IFN-alpha therapy rescue Ebola-infected nonhuman primates when administered after the detection of viremia and symptoms. Science translational medicine. 2013; 5:207ra143.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 15\n53. Smith LM, et al. Interferon-beta therapy prolongs survival in rhesus macaque models of Ebola and Marburg hemorrhagic fever. The Journal of infectious diseases. 2013; 208:310\u2013318. [PubMed: 23255566]\n54. Emond RT, et al. A case of Ebola virus infection. British medical journal. 1977; 2:541\u2013544. [PubMed: 890413]\n55. McCarthy SD, et al. A Rapid Screening Assay Identifies Monotherapy with Interferon-\u03b2 and Combination Therapies with Nucleoside Analogs as Effective Inhibitors of Ebola Virus. PLoS neglected tropical diseases. 2016; 10:e0004364. [PubMed: 26752302]\n56. Konde MK, et al. Interferon beta-1a for the treatment of Ebola virus disease: A historically controlled, single-arm proof-of-concept trial. PloS one. 2017; 12:e0169255. [PubMed: 28225767]\n57. Mupapa K, et al. Treatment of Ebola hemorrhagic fever with blood transfusions from convalescent patients. International Scientific and Technical Committee. The Journal of infectious diseases. 1999; 179(Suppl 1):S18\u201323. [PubMed: 9988160]\n58. van Griensven J, et al. Evaluation of Convalescent Plasma for Ebola Virus Disease in Guinea. The New England journal of medicine. 2016; 374:33\u201342. [PubMed: 26735992]\n59. van Griensven J, et al. Efficacy of Convalescent Plasma in Relation to Dose of Ebola Virus Antibodies. The New England journal of medicine. 2016; 375:2307\u20132309.\n60. Maruyama T, et al. Ebola virus can be effectively neutralized by antibody produced in natural human infection. Journal of virology. 1999; 73:6024\u20136030. [PubMed: 10364354]\n61. Flyak AI, et al. Cross-Reactive and Potent Neutralizing Antibody Responses in Human Survivors of Natural Ebolavirus Infection. Cell. 2016; 164:392\u2013405. [PubMed: 26806128]\n62. Bornholdt ZA, et al. Isolation of potent neutralizing antibodies from a survivor of the 2014 Ebola virus outbreak. Science. 2016; 351:1078\u20131083. [PubMed: 26912366]\n63. Corti D, et al. Protective monotherapy against lethal Ebola virus infection by a potently neutralizing antibody. Science. 2016; 351:1339\u20131342. [PubMed: 26917593]\n64. Wec AZ, et al. Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses. Cell. 2017; 169:878\u2013890 e815. [PubMed: 28525755]\n65. Wilson JA, et al. Epitopes involved in antibody-mediated protection from Ebola virus. Science. 2000; 287:1664\u20131666. [PubMed: 10698744]\n66. Takada A, et al. Identification of protective epitopes on ebola virus glycoprotein at the single amino acid level by using recombinant vesicular stomatitis viruses. Journal of virology. 2003; 77:1069\u2013 1074. [PubMed: 12502822]\n67. Qiu X, et al. Characterization of Zaire ebolavirus glycoprotein-specific monoclonal antibodies. Clinical immunology. 2011; 141:218\u2013227. [PubMed: 21925951]\n68. Keck ZY, et al. Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein. Journal of virology. 2015; 90:279\u2013291. [PubMed: 26468532]\n69. Zhao X, et al. Immunization-Elicited Broadly Protective Antibody Reveals Ebolavirus Fusion Loop as a Site of Vulnerability. Cell. 2017; 169:891\u2013904 e815. [PubMed: 28525756]\n70. Davidson E, et al. Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies. Journal of virology. 2015; 89:10982\u201310992. [PubMed: 26311869]\n71. Pettitt J, et al. Therapeutic intervention of Ebola virus infection in rhesus macaques with the MB-003 monoclonal antibody cocktail. Science translational medicine. 2013; 5:199ra113.\n72. Qiu X, et al. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature. 2014; 514:47\u201353. [PubMed: 25171469]\n73. Mendoza EJ, et al. Progression of Ebola Therapeutics During the 2014\u20132015 Outbreak. Trends in molecular medicine. 2016; 22:164\u2013173. [PubMed: 26774636]\n74. Group, P.I.W. et al. A Randomized, Controlled Trial of ZMapp for Ebola Virus Infection. The New England journal of medicine. 2016; 375:1448\u20131456. [PubMed: 27732819]\n75. Kugelman JR, et al. Evaluation of the potential impact of Ebola virus genomic drift on the efficacy of sequence-based candidate therapeutics. mBio. 2015; 6\n76. Dodd LE, et al. Design of a Randomized Controlled Trial for Ebola Virus Disease Medical Countermeasures: PREVAIL II, the Ebola MCM Study. The Journal of infectious diseases. 2016; 213:1906\u20131913. [PubMed: 26908739]\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 16\n77. Dietzel E, et al. Functional Characterization of Adaptive Mutations during the West African Ebola Virus Outbreak. Journal of virology. 2017; 91\n78. Wong G, et al. Pathogenicity Comparison Between the Kikwit and Makona Ebola Virus Variants in Rhesus Macaques. The Journal of infectious diseases. 2016; 214:S281\u2013S289. [PubMed: 27651412]\n79. Howell KA, et al. Antibody Treatment of Ebola and Sudan Virus Infection via a Uniquely Exposed Epitope within the Glycoprotein Receptor-Binding Site. Cell reports. 2016; 15:1514\u20131526. [PubMed: 27160900]\n80. Hernandez H, et al. Development and Characterization of Broadly Cross-reactive Monoclonal Antibodies Against All Known Ebolavirus Species. The Journal of infectious diseases. 2015; 212(Suppl 2):S410\u2013413. [PubMed: 25999057]\n81. Holtsberg FW, et al. Pan-ebolavirus and Pan-filovirus Mouse Monoclonal Antibodies: Protection against Ebola and Sudan Viruses. Journal of virology. 2015; 90:266\u2013278. [PubMed: 26468533]\n82. Furuyama W, et al. Discovery of an antibody for pan-ebolavirus therapy. Scientific reports. 2016; 6:20514. [PubMed: 26861827]\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 17\n\nBox 1 \u2022 \u2022 \u2022 \u2022\n\nClinician\u2019s Corner\nSingle-arm trials are advantageous in terms of ethics since all patients receive the potentially life-saving drug, but are disadvantageous scientifically since the exact impact of the drug will be unknown without a proper control group.\nRandomized, controlled trials are advantageous scientifically since a control group exists to compare the efficacy of the treatment group, but are disadvantageous ethically since not all patients receive the experimental drug.\nThe primary outcome of a clinical trial for filovirus therapeutics will always be survival, since it is only possible to test efficacy on infected patients (i.e., during an outbreak).\nTwo key secondary outcomes of a clinical trial for filovirus therapeutics will be the consideration of changes in viremia (RNA and live virus level) following treatment, as well as adverse effects.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 18\nTRENDS BOX\n\u2022 Ebola virus disease (EVD) causes severe hemorrhagic fever in humans with high fatality rates, with no approved drugs for treatment. Several candidate therapeutics were clinically assessed during the recent 2013\u20132016 outbreak in West Africa.\n\u2022 Two small molecule inhibitors of viral replication and transcription, a nucleotide analog (favipiravir) and short interfering RNA, did not yield a survival benefit in clinical trials, though administration of favipiravir appeared to be more beneficial for patients with lower viral loads (i.e. in the earlier stages of EVD).\n\u2022 The survival benefit was inconclusive in clinical trials with immune productbased therapies, including interferon, convalescent plasma and a monoclonal antibody (mAb) cocktail. The data shows that the mAb-cocktail, ZMapp\u2122, may have the best potential for a substantial therapeutic benefit.\n\u2022 Further clinical investigations that could be rapidly initiated early during an outbreak will help conclusively evaluate the true effectiveness of available candidate therapeutics.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 19\nOUTSTANDING QUESTIONS BOX\n\u2022 Have we gained sufficient knowledge from current clinical investigations into candidate therapeutics to save lives in the next outbreak?\n\u2022 Is there an optimal design to better balance study strength vs. ethical concerns for testing Ebola virus therapeutics?\n\u2022 Can more than one type of therapeutic be evaluated side-by-side in patients from the same trial during an outbreak?\n\u2022 Given that Ebola virus infections are aggressive and the timeframe for treatment is limited, can we develop better/more sensitive diagnostic systems for the early and rapid identification of EVD?\n\u2022 Can we identify markers that correlate with EVD severity so that therapeutic strategies can be personalized for more efficient treatments?\n\u2022 Are we sufficiently prepared for potential threats from other filovirus species, such as Sudan, Bundibugyo and Marburg viruses?\n\u2022 Do we have alternative methods for the treatment of Ebola virus escape mutants?\n\u2022 Is cross-protection possible across all filoviruses (especially between Marburg and Ebola)?\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nClinical Trials for Candidate EVD Therapeutics\n\nTable 1\n\nLiu et al.\n\neutics vir 30803 a scent plasma (CP) \u2122\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nClinical trial\nNCT023 29054 Nonrandomized, multicenter,\nproof-ofconcept phase 2 trial\n[28]\nPACTR2 0150100 0997429\nNonrandomized phase 2 trial\n[40]\nISRCTN 1741494 6\nNonrandomized,\nproof-ofconcept phase 1/2 trial [56]\nNonrandomized phase 2/3\ntrial [58]\nNCT023 42171 Randomized, controlled, multicen ter\n\nTreated patients 111 14 9 84 36\n\nReference treatment window 6 d in mice [26] 3 d in NHPs [43] N/A N/A 5 d in NHPs [72]\n\nAverage time from symptom onset to admission\n1.5 d (\u2264 6 y), 3\u20134 d (\u2265 13 y)\n2 d\n2\u20133 d\nNR\n4\u20137 d\n\nMedian/a verage start\npoint of treatment\n4 d from symptom\nonset\n23 hrs post admission\n1 d upon baseline Ct determination\n48 hrs upon EVD\nconfirmation\n12 to 24 hrs following randomization\n\nRoute Oral Intravenous infusion Subcutaneous injection Transfusion Intravenous infusion\n\nDose and course\n6,000 mg on d 0; 2,400 mg/d, d 1~9\n0.3 mg/kg/d for 7 d\n30 ug/d\n2 transfusion s of 200\u2013250 ml of CP or 10 ml of CP/kg, 15-min interval\n3 doses of 50 mg/kg, 3-d\ninterval\n\nPrimary outcome\nMortality on-trial\nSurvival at d 14 Survival at d 21\nMortality from 3 to 16 d after diagnosis\nMortality at d 28\n\nEfficacy (# deaths and\n% mortality)\nand effectiveness\n60 and 54%, no difference\nfrom historical controls (58%); potential benefit to \u2265 13 y and Ct \u2265 20 (20%);\n9/12 and 75%;, no benefit\n3 and 33% vs 17 and 81% in control cohort; potentially beneficial\n26 and 31%, no difference\nfrom historical controls (38%); potential benefit to < 5-y (20%) or pregnancy\n(25%)\n8 (7 before the 2nd dose) and 22% vs 37% in the control arm;\n\nAdverse reactions\nNo severe adverse reactions\nOne case of worsened tachypnoea\nMainly flu-like symptoms\nNo severe adverse reactions\nOne case of hypertension\n\nPage 20\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\neutics te\n\nClinical trial\n\nTreated patients\n\nReference treatment window\n\nAverage time from symptom onset to admission\n\nMedian/a verage start\npoint of treatment\n\nphase 1/2 trial [74]\n\nRoute\n\nDose and course\n\nPrimary outcome\n\nEfficacy (# deaths and\n% mortality)\nand effectiveness\npotentially beneficial\n\nAdverse reactions\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\npplicable ported\n\nPage 21\n\n\n",
    "authors": [
      "Guodong Liu",
      "Gary Wong",
      "Shuo Su",
      "Yuhai Bi",
      "Frank Plummer",
      "George F. Gao",
      "Gary Kobinger",
      "Xiangguo Qiu"
    ],
    "doi": "10.1016/j.molmed.2017.07.002",
    "date": "09/2017",
    "item_type": "journalArticle",
    "url": "https://linkinghub.elsevier.com/retrieve/pii/S1471491417301090"
  },
  {
    "key": "TA4ZQVDA",
    "title": "Neutralizing monoclonal antibodies for treatment of COVID-19",
    "abstract": "Several neutralizing monoclonal antibodies (mAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-C oV-2) have been developed and are now under evaluation in clinical trials. With the US Food and Drug Administration recently granting emergency use authorizations for neutralizing mAbs in non-h ospitalized patients with mild-t o-moderate COVID-19, there is an urgent need to discuss the broader potential of these novel therapies and to develop strategies to deploy them effectively in clinical practice, given limited initial availability. Here, we review the precedent for passive immunization and lessons learned from using antibody therapies for viral infections such as respiratory syncytial virus, Ebola virus and SARS-CoV infections. We then focus on the deployment of convalescent plasma and neutralizing mAbs for treatment of SARS-CoV-2. We review specific clinical questions, including the rationale for stratification of patients, potential biomarkers, known risk factors and temporal considerations for optimal clinical use. To answer these questions, there is a need to understand factors such as the kinetics of viral load and its correlation with clinical outcomes, endogenous antibody responses, pharmacokinetic properties of neutralizing mAbs and the potential benefit of combining antibodies to defend against emerging viral variants.",
    "full_text": "Reviews\n\nNeutralizing monoclonal antibodies for treatment of COVID-19\nPeter C. Taylor\u200a \u200a1\u2009\u2709, Andrew C. Adams2, Matthew M. Hufford2, Inmaculada de la Torre2, Kevin Winthrop3 and Robert L. Gottlieb\u200a \u200a4,5\nAbstract | Several neutralizing monoclonal antibodies (mAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-C\u200b oV-2) have been developed and are now under evaluation in clinical trials. With the US Food and Drug Administration recently granting emergency use authorizations for neutralizing mAbs in non-h\u200b ospitalized patients with mild-t\u200b o-\u200bmoderate COVID-19, there is an urgent need to discuss the broader potential of these novel therapies and to develop strategies to deploy them effectively in clinical practice, given limited initial availability. Here, we review the precedent for passive immunization and lessons learned from using antibody therapies for viral infections such as respiratory syncytial virus, Ebola virus and SARS-CoV infections. We then focus on the deployment of convalescent plasma and neutralizing mAbs for treatment of SARS-\u200bCoV-2. We review specific clinical questions, including the rationale for stratification of patients, potential biomarkers, known risk factors and temporal considerations for optimal clinical use. To answer these questions, there is a need to understand factors such as the kinetics of viral load and its correlation with clinical outcomes, endogenous antibody responses, pharmacokinetic properties of neutralizing mAbs and the potential benefit of combining antibodies to defend against emerging viral variants.\n\nMonoclonal antibodies (mAbs). Clonal antibodies recognizing a single epitope on an antigen. Generally used in reference to recombinant sources.\n1Botnar Research Centre, University of Oxford, Oxford, UK. 2Eli Lilly and Company, Indianapolis, IN, USA. 3Oregon Health & Science University, Portland, OR, USA. 4Baylor University Medical Center, Dallas, TX, USA. 5Baylor Scott & White Research Institute, Dallas, TX, USA. \u2709e-\u200bmail: peter.taylor@ kennedy.ox.ac.uk https://doi.org/10.1038/ s41577-021-00542-\u200bx\n\nIn the midst of the current COVID-19 pandemic, a variety of prophylactic and therapeutic treatments are being developed or repurposed to combat COVID-19. Monoclonal antibodies (mAbs) that can bind to and \u2018neutralize\u2019 the virus in infected patients are a novel class of antiviral intervention1,2. Neutralizing mAbs are recombinant proteins that can be derived from the B cells of convalescent patients or humanized mice (Fig. 1). High-\u200bthroughput screening of these B cells permits the identification of antibodies with the necessary specificity and affinity to bind to a virus and block entry of the virus, therefore abrogating pathology associated with productive infection. These mAbs are termed \u2018neutralizing\u2019 and can ultimately be used as a type of passive immunotherapy (detailed later) to minimize virulence. In this Review, we highlight the relative value that neutralizing mAbs can provide for patients and physicians, and go on to examine the role of these agents among the spectrum of potential treatments for COVID-19.\nIn the United States, three anti-\u200bsevere acute respiratory syndrome coronavirus 2 (SARS-C\u200b oV-2) mAb therapies have been granted emergency use authorization (EUA) for treatment of non-\u200bhospitalized patients with mild-\u200bto-\u200bmoderate COVID-19 \u2014 these are bamlanivimab as a monotherapy, and bamlanivimab together with etesevimab or casirivimab with imdevimab as a\n\ncombination therapy3\u20135. Therefore, several questions need to be addressed about the potential clinical use of neutralizing SARS-C\u200b oV-2 mAbs: who should get them; what is the best dose and frequency; when in the course of the infection will they be most effective; what is the duration of the protection they provide; and what is their associated benefit-\u200bto-\u200brisk ratio? In addition, neutralizing mAbs may have a prophylactic role in individuals deemed to be at high risk of severe COVID-19. Indeed, preliminary non-\u200bpeer-\u200breviewed preprint data suggest that mAbs prevent COVID-19 in high-\u200brisk individuals potentially exposed to SARS-\u200bCoV-2 in nursing homes or within households6,7.\nWhile vaccines remain the best strategy to prevent COVID-19, mAbs could potentially benefit certain vulnerable populations before or after exposure to SARS-C\u200b oV-2, such as the unvaccinated or recently vaccinated high-\u200brisk patients. The antiviral activity seen with neutralizing mAb treatment emphasizes the importance of early intervention to help counter the devastating impact the virus has had in such vulnerable populations and in other high-r\u200b isk patients. However, mAbs are complicated to produce and may be limited in initial supply. Furthermore, any protection offered would be temporary, and the duration of effective protection remains to be determined. Answers to these questions will allow\n\n382 | June 2021 | volume 21\n\n0123456789();:\n\nwww.nature.com/nri\n\nProcess Source material\nConvalescent patient\nHumanized mouse that received target antigen\n\nReviews\n\nSearch Screening for RBD-speci\ufb01c single B cells\n\nSequence and identity Analyse\n\nSelect\n\nCloning and expression Validate and characterize\n\nHigh-con\ufb01dence sequences clustered by sequence identity\n\nBinding validation\n\nMultiplexed bead-based assay\nLive cell-based assay\nFluorescence-activated single-cell sorting\n\nClonal families\n\nFunctional validation Stability\nOrganization Af\ufb01nity\n\nFig. 1 | Neutralizing monoclonal antibodies: identification, selection and production. The neutralizing monoclonal antibodies (mAbs) given emergency use authorization for treatment of COVID-19 were derived from either convalescent patients or humanized mice exposed to severe acute respiratory syndrome coronavirus 2 (SARS-C\u200b oV-2) antigens. However, mAbs can be generated by multiple methods, including from vaccinated individuals (not depicted here). The pathways of mAb generation depicted here converge in the process of selection and production. RBD, receptor-b\u200b inding domain.\n\nPassive immunotherapy The introduction of monoclonal or polyclonal antibodies derived from non-\u200bhuman or human blood products to provide protection against infection or envenomation.\nEmergency use authorization (EUA). A mechanism to facilitate the availability and use of medical countermeasures during a public health emergency. US Food and Drug Administration issuance of an EUA permits the use of unapproved medical products or unapproved uses of approved medical products when no adequate alternatives are available.\nConvalescent plasma therapy (CPT). The administration of donated plasma from an individual who has had an illness and recovered from it (for example, previously infected with severe acute respiratory syndrome coronavirus 2 (SARS-\u200bCoV-2) but has now recovered), to an infected individual. The recovered patient\u2019s plasma contains antibodies, which when administered to other patients is thought to boost the ability to fight disease.\n\nthe most efficacious use of these novel and potentially life-\u200bsaving treatments, as we discuss herein.\nPassive immunization\nMore than 125 years ago, the first major success in modern immunological intervention was developed: a therapeutic serum from animals actively immunized against diphtheria toxin8,9. Paul Ehrlich later produced a seminal article tying the curative antiserum to neutralizing antibodies10. Today, passive immunization involves infusion of antigen-\u200bspecific mAbs or polyclonal antibodies derived from non-\u200bhuman or human blood products. While polyclonal antibodies collected from immunized animals are the primary source of antisera, there is a risk of \u2018serum sickness\u2019, especially after repeated exposures, as the recipient may generate an immune response against antibodies of non-h\u200b uman origin. These risks are mitigated with the use of convalescent plasma from human patients. With careful screening (for example, to assess for the presence of infectious agents and to establish antibody titre and neutralizing capacity), convalescent plasma therapy (CPT) can be effective with minimal safety risks.\nBefore the current pandemic, CPT was used to treat infections with influenza virus11,12, respiratory syncytial virus (RSV)13, Ebola virus14 and other coronaviruses12,15\u201317. CPT appears most efficacious when used early after the onset of symptoms, rather than during severe or prolonged infection12,15,18. It also has the potential to provide protection for the immunocompromised or unvaccinated high-\u200brisk individuals recently exposed to infection13,15. Administration of plasma with higher titres of neutralizing antibodies is associated with improved clinical outcomes17; however, the antibody titres of convalescent plasma differ considerably19. CPT can\n\nbe convenient and adaptable for use in resource-\u200bpoor settings14 and can be rapidly deployed to combat novel virus outbreaks.\nThe antipathogen antibodies from convalescent plasma can mitigate infection by two main mechanisms: namely, antibody effector activity and pathogen neutralization. However, in rare cases, pathogen-s\u200b pecific antibodies can augment virulence in a process termed \u2018antibody-\u200bdependent enhancement\u2019 (ADE) (Fig. 2). ADE can occur via two distinct mechanisms. First, pathogen-\u200bspecific antibodies could increase infection by promoting virus uptake and replication in Fc\u03b3 receptor-\u200bexpressing immune cells (for example, as is seen in dengue haemorrhagic virus infection of macrophages). With SARS-\u200bCoV and SARS-\u200bCoV-2 (ref.20), in vitro evidence amassed to date indicate that these non-\u200blymphotropic coronaviruses are unable to productively replicate within haematopoietic cells21. Alternatively, ADE can be mediated via increased immune activation by Fc-\u200bmediated effector functions or immune complex formation22. In the case of respiratory virus infections, the resulting immune cascade can contribute to lung disease. While the hallmarks of severe COVID-19 have features that overlap with this type of ADE, there is currently no definitive evidence to show ADE occurs with SARS-C\u200b oV-2 infection22.\nNonetheless, steps may be considered to mitigate the potential risk of ADE. When feasible (as with neutralizing mAb therapy), the Fc region of the antibody can be modified to render it incapable of engaging effector immune responses. In the case of CPT, the potential risk of ADE can be reduced by administrating high amounts of pathogen-\u200bspecific antibodies and using plasma with high-\u200baffinity neutralizing antibodies20. However, these strategies must be balanced with the\n\nnature Reviews | IMMuNology\n\n0123456789();:\n\nvolume 21 | June 2021 | 383\n\nReviews\n\nAntibody-d\u200b ependent enhancement (ADE). The promotion of viral uptake into cells owing to the presence of suboptimal antibodies. ADE can result in enhanced viral replication and/or aberrant inflammation.\nRandomized controlled trials (RCTs). Studies that randomly assign participants into an experimental group or a control group to measure the effectiveness of a new intervention or treatment.\n\npotential loss of efficacy from effector-\u200bmediated activity. As shown in a non-\u200bprimate model of SARS-\u200bCoV, neutralizing anti-\u200breceptor-\u200bbinding domain (RBD) or anti-\u200bheptad repeat 2 antibodies provided protective immunity, whereas antibodies specific for other S protein epitopes could trigger ADE23. Furthermore, in\n\nrandomized controlled trials (RCTs), passive immunization with anti-\u200bS protein-\u200bneutralizing mAbs did not provide clinical evidence of ADE in non-\u200bhospitalized patients with COVID-19 (refs3,24,25).\nThe shortage of large RCTs of CPT has limited our understanding of the relative benefit-\u200bto-\u200brisk profile of\n\na Antibody-dependent\ncellular phagocytosis\n\nMacrophage\n\nComplement-dependent cytotoxicity\nC1q Antibody\n\nNeutralization\n\nPhagocytosis Fc\u03b3R\n\nInfected cell\n\nAntigen\nMAC Lysis\n\nE\ufb00ector cell\n\nRelease of granzyme and perforin-mediated cell apoptosis\n\nAntibody-dependent cellular cytotoxicity\n\nOpsonization\n\nb\nVirus\n\nAntibodies can enhance uptake of virions\n\nIncreased immune activation\n\nIncreased cytokine production\n\nIncreased immune cell activation and in\ufb01ltration\n\nProductive viral replication and release\n\nMacrophage\n\nIncreased infection\n\nComplement cascade activation\n\nIn\ufb02ammation\n\nFig. 2 | Mechanism of action of monoclonal antibodies for viral infection and antibody-dependent enhancement. a | Monoclonal antibodies can directly interfere with viral pathogenesis in multiple ways. First, binding of a neutralizing antibody to the virion can prevent target cell binding and/or fusion. Furthermore, antibody binding opsonizes the virions or infected cells for phagocytic uptake. If viral proteins are intercalated into target cell membranes during viral egress, monoclonal antibodies can facilitate target cell death via complement fixation and membrane attack complex (MAC) activation or antibody-\u200bdependent cytotoxicity. These mechanisms may result in apoptosis or necrosis of the infected cell. b | In some instances, opsonization of a virion can facilitate viral pathogenesis in a process termed \u2018antibody-d\u200b ependent enhancement\u2019 (ADE). ADE can occur via two distinct mechanisms. First, pathogen-s\u200b pecific antibodies could increase infection via viral uptake and replication in Fc\u03b3 receptor (Fc\u03b3R)-\u200bexpressing immune cells. Secondly, ADE can be mediated via increased immune activation by Fc-m\u200b ediated effector functions or immune complex formation. The process of ADE and its potential impact during severe acute respiratory syndrome coronavirus 2 (SARS-C\u200b oV-2) infection is expertly reviewed by Lee et al.22.\n\n384 | June 2021 | volume 21\n\n0123456789();:\n\nwww.nature.com/nri\n\nReviews\n\nImmune globulin A sterilized solution made from plasma and containing antibodies.\nViral variants Mutations arising in viruses resulting in genetic variation and the emergence of different versions of the virus.\nSpike (S) protein Protein found on the coronavirus cell surface, responsible for binding of the virus to host cells and subsequent entry of the virus. The receptor-\u200bbinding domain of the S protein is the preferred target of neutralizing monoclonal antibody therapies for severe acute respiratory syndrome coronavirus 2 (SARS-\u200bCoV-2) infection.\n\nthis treatment option. Furthermore, logistical difficulties can complicate the application of CPT. According to the EUA from the US Food and Drug Administration for CPT in patients with COVID-19, convalescent patients should be symptom-\u200bfree for a minimum of 2 weeks and have high titres of anti-\u200bSARS-\u200bCoV-2 antibodies; low-\u200btitre donations could be used for therapy following careful consideration by the health-\u200bcare provider26,27. Thus, widespread use of CPT is dependent on a readily available pool of recovering patients with high antibody titres who are willing to donate plasma, on sufficient local facilities to ensure adequate processing, screening and administration of the therapy, and on governmental coordination to regulate effective implementation.\nAdvantages of monoclonal antibodies\nThere is an increasing focus on replacing CPT with neutralizing mAbs, where dosing to ensure appropriate neutralizing capacity of the antibodies can be more precise. Today, the process to mass-\u200bproduce recombinant mAbs has become scalable to meet demand and is cost-\u200bcompetitive with other treatments. Neutralizing mAbs overcome limitations intrinsic to CPT (for example, the risk of blood-\u200bborne diseases, time to development of detectable high-\u200baffinity antibodies and risk of low antibody titres, as well as variable epitope specificity28). Furthermore, a high titre of neutralizing antibodies \u2014 which current evidence indicates is necessary for the efficacy of CPT \u2014 is inherent with neutralizing mAbs. As of March 2021, at least 20 neutralizing mAb therapies were being tested in late-\u200bstage clinical trials or had already been approved for use in nine infectious diseases, including RSV infection and Ebola29,30 (ClinicalTrials.gov).\nPalivizumab, a neutralizing mAb to the fusion protein of RSV, was initially approved in 1998 as a prophylaxis for severe RSV infection in high-r\u200b isk infants31\u201333. Previously, the standard of care for prophylaxis in these patients was monthly infusions of RSV immune globulin13,31. When administered via monthly intramuscular injections, palivizumab reduced the frequency of hospitalization and severity of RSV disease relative to placebo and was well tolerated31,32. However, palivizumab was not demonstrated in RCTs to improve clinically meaningful outcomes in infants with severe RSV infection in advanced disease stages34\u201336. Furthermore, monthly administration is required to maintain detectable levels of neutralizing mAbs, and as many as five doses may be needed to prevent severe or deadly infection37. A newer medication with a longer half-\u200blife (MEDI8897) is currently in phase II/III trials33.\nDuring the Ebola virus disease outbreak in the Democratic Republic of the Congo in 2018, an openlabel RCT (PALM) investigated four intravenously administered treatments in 681 patients actively infected with Ebola virus: the antiviral remdesivir, the triple mAb cocktail ZMapp, the single mAb MAb114, and the triple mAb combination REGN-\u200bEB3 (ref.38). After an interim analysis, the first two treatments were discontinued as MAb114 monotherapy and REGN-\u200bEB3 were superior with respect to the primary outcome, patient mortality38. One potential factor the PALM study team proposed to\n\nexplain the distinction between the therapeutics was that the full treatments for MAb114 and REGN-\u200bEB3 were administered as a single dose, thereby facilitating a rapid response, while ZMapp was given as three infusions. Indeed, patients treated with MAb114 and REGN-\u200bEB3 had faster rates of viral clearance, lending credence to this hypothesis. Overall, survival was higher in those who were treated early during symptom onset and had lower baseline viral loads. The relatively low efficacy of the ZMapp triple cocktail also serves as a reminder that the number of mAbs is not necessarily a predictor of efficacy per se, and that specific epitopes may also matter.\nTwo main uncertainties persist with passive immunization, spanning both neutralizing mAbs and CPT. First, does their use as a prophylactic or treatment potentially affect natural long-\u200bterm immunity? Considering the large doses used and the relative half-\u200blife of antibodies (~3 weeks for IgG molecules), there is a pertinent consideration whether the presence of circulating neutralizing mAbs could impact active immunity, whether through memory from infection or vaccination. In RSV infection, rodent and primate infection models indicate that the passive transfer of antibodies does diminish the development of humoral immunity in the recipient; however, long-\u200bterm memory was sufficient to protect the hosts from reinfection, largely owing to an intact T cell memory compartment39,40. Considering the limitations in translating data from animal models (where RSV replication is attenuated relative to that in its human host), additional data, particularly from clinical trials, will provide critical insight with regard to this potential challenge.\nSecond, could resistant viral variants emerge that limit the effectiveness of the therapies? The polyclonal nature of CPT, in which a spectrum of differentiated antibodies target multiple epitopes of the pathogen, may help to reduce this risk. Nevertheless, emerging preclinical data suggest SARS-\u200bCoV-2 spike (S) protein mutations escape from polyclonal serum41, and convalescent plasma has reduced neutralizing activity against some viral variants42. For mAbs, however, depending on the infectious agent and the epitope targeted, combinations of mAbs may be necessary to maintain efficacy and prevent treatment failure. Experience with mAbs targeting human immunodeficiency virus (HIV), which has a very high mutation rate, suggests that it may be more effective and durable to use multiple neutralizing antibodies (that is, combinational mAb therapy) rather than a single one43\u201346. These particular mAbs to HIV also need to be broadly neutralizing and target epitopes generally conserved among viral variants. On the other hand, infections involving pathogens with lower mutation rates and/or accessible broadly conserved epitopes may not require combinational mAb therapy; for example, MAb114 monotherapy, which targets a broadly conserved epitope on the Ebola virus\u2019s RBD, was as effective as the combination therapy REGN-E\u200b B3 (ref.38) and more effective than the ZMapp triple cocktail. It is important to note though that the global nature of the COVID-19 pandemic presents a larger risk of escape variants emerging than during the Ebola outbreak, owing to the sheer number of infections and high levels of circulating virus among populations.\n\nnature Reviews | IMMuNology\n\n0123456789();:\n\nvolume 21 | June 2021 | 385\n\nReviews\n\nNucleocapsid (N) protein Protein that encloses and protects a viral genome, such as the severe acute respiratory syndrome coronavirus 2 (SARS-C\u200b oV-2) genomic RNA.\n\nFrom the collective clinical data with MAb114, REGN-\u200bEB3 and palivizumab, the general benefits and risks associated with neutralizing mAbs are similar to those observed with traditional passive immunization against infectious agents. The agents themselves are relatively tolerable for patients, efficacious during the early onset of disease symptoms and in certain cases as a prophylactic, but with limited efficacy once infections are severe. The distinctions between these therapies are largely logistical; CPT is more rapidly implemented during an emerging pandemic when few therapeutic options are yet available, while neutralizing mAbs take time to discover and it takes time for regulatory approval for their use to be obtained as well as to scale up manufacturing capacity. The use and promise of passive immunization during the coronavirus outbreaks of the twenty-\u200bfirst century (that is, with SARS-\u200bCoV, Middle East respiratory syndrome-\u200brelated coronavirus and SARS-\u200bCoV-2) have re-\u200bemphasized these past lessons while also highlighting additional insights, as we discuss next.\nPassive immunization for coronaviruses\nDuring the SARS epidemic in 2003, immune system kinetics following SARS-\u200bCoV infection were different in patients who recovered compared with those who finally succumbed to the viral infection and sequelae. In patients with fatal outcomes, the levels of endogenous neutralizing antibodies peaked at 15 days from symptom onset and then decreased drastically until the time of death. By contrast, in patients who went on to recover, peak neutralizing antibody responses were observed at 20 days from symptom onset47,48. Those who recovered tended to develop antibody responses with diverse isotypes (IgM, IgG and IgA) against two proteins on the virus, the nucleocapsid (N) protein and the S protein, while patients with fatal outcomes had restricted antibody responses to the N protein only. In a serological survey of confirmed convalescent serum samples, 88% had anti-\u200bSARS-\u200bCoV antibodies 31\u2013180 days after the onset of symptoms; the geometric mean of the neutralizing antibody titre was 1:61 (ref.19).\nOwing to the brief duration of the SARS epidemic in 2003, few observational trials examining CPT were conducted. The largest involved 80 patients and was conducted at the Prince of Wales Hospital in Hong Kong15. Those given CPT before day 14 following onset of symptoms had a better outcome than those given CPT after day 14 (P\u2009<\u20090.001); mortality was also lower in the former group (6.3% versus 21.9%). Patients who tested positive for SARS-\u200bCoV by PCR had better outcomes if they were seronegative when given CPT than those who were already seropositive (66.7% versus 20%; P\u2009<\u20090.001). A summary of eight observational studies using CPT during the SARS 2003 outbreak (including the aforementioned Prince of Wales Hospital study) suggested CPT was associated with reduced mortality, shorter hospital stays and reduced overall viral loads in the respiratory tract12. The treatments were considered tolerable12; however, information on minor complications may have been under-\u200breported. Importantly, robust studies on the effectiveness and safety of CPT were not completed,\n\nand thus these results must be interpreted with caution, particularly as patients treated at later times with CPT may demonstrate selection bias for an already refractory pathophysiology.\nIn SARS-\u200bCoV-2 infection, plasma collected from 175 patients who had recovered from mild COVID-19 demonstrated neutralizing antibody and S-\u200bbinding antibody titres that correlated with increased age, greater inflammation (that is, higher C-\u200breactive protein levels) and lower lymphocyte counts; the vast majority of the SARS-\u200bCoV-2 neutralizing antibodies were not cross-\u200breactive with SARS-\u200bCoV49. Several reports indicate convalescent patients can maintain high titres of neutralizing antibodies several weeks after infection49\u201351.\nObservational studies have reported that CPT has been associated with improved outcomes in COVID-19 (ref.52). For example, in a small case series in China, five critically ill patients with acute respiratory distress syndrome showed improved clinical status following CPT. Thirty-\u200bseven days after transfusion, three patients had been discharged and two were in a stable condition53. In a cohort analysis of 35,222 patients hospitalized with COVID-19 from the United States Convalescent Plasma Expanded Access Program, reduced mortality was associated with earlier time to transfusion (after diagnosis) and convalescent plasma with higher antibody levels18. Because these studies were observational, there was limited procedural control including standardization of the level of neutralizing antibodies.\nIn an open-\u200blabel RCT of CPT for patients (n\u2009=\u2009103) with severe or life-\u200bthreatening COVID-19 in China, donors were required to have high levels of antibodies specific to the RBD of the S protein54. However, the study was terminated early; the hazard ratio for the time to clinical improvement within 28 days in the CPT group versus the standard treatment control group was 1.4 (favouring CPT) but was not statistically significant. The proportion of patients with severe disease who achieved the primary end point was significantly higher in the CPT group (21 of 23 patients) versus the standard treatment group (15 of 22 patients; P\u2009=\u20090.03), but no distinction was noted in patients with life-\u200bthreatening COVID-19 (ref.53). In a blinded RCT in Argentina, CPT (with a median titre of 1:3,200 anti-\u200bSARS-\u200bCoV-2 antibodies) also failed to demonstrate benefit in patients with COVID-19-\u200bassociated severe pneumonia55. Furthermore, in an open-\u200blabel RCT in India (PLACID) in hospitalized patients with hypoxaemia (generally comparable with the definition of severe COVID-19 from other trials), CPT did not demonstrate benefit in terms of patient mortality or transition to worsening disease56. Similarly, preliminary data from the RECOVERY RCT among 10,406 hospitalized patients showed no proof of mortality benefit in the primary end point of 28-\u200bday mortality in the CPT group versus the standard treatment group57,58.\nDespite inconsistent clinical efficacy, there is evidence that CPT was associated with greater viral clearance than standard-\u200bof-\u200bcare treatment54,56; collectively, this indicates increased viral clearance alone was not sufficient to clearly improve the clinical outcomes in patients with established COVID-19 (that is, in patients\n\n386 | June 2021 | volume 21\n\n0123456789();:\n\nwww.nature.com/nri\n\nReviews\n\nTime-w\u200b eighted average An average that takes both the numerical level and the time of a particular variable into consideration.\nMedically attended visits Medical visits such as telemedicine visits, in-p\u200b erson outpatient visits to or from a medical provider, urgent care or emergency department visits, or hospitalization.\n\nhospitalized with COVID-19). Because the currently available data on CPT are derived predominantly from inpatient (severe or critical) COVID-19 RCTs, the suitability of CPT as prophylaxis or treatment at the onset of COVID-19 symptoms remains to be determined by appropriately controlled clinical trials.\nMonoclonal antibodies for COVID-19 The primary antigenic epitope on SARS-\u200bCoV and SARS-\u200bCoV-2 is the S protein, which facilitates target cell binding and fusion upon engaging the cell-\u200bsurface angiotensin-c\u200b onverting enzyme 2 (ACE2) receptor, which is found on cells in the respiratory system, gastro\u00adintestinal tract and endothelium59\u201363. Thus, antibodies directed\n\nSARS-CoV-2\n\nImdevimab Casirivimab\nRBD\n\nS protein RBD\n\nAntibody prevents viral binding and/or fusion with host cell\n\nBamlanivimab Etesevimab\n\nACE2\n\nFig. 3 | Inhibition of SARS-CoV-2 target cell engagement by neutralizing monoclonal antibodies. Neutralizing monoclonal antibodies (mAbs) being developed to combat COVID-19 are generated against the receptor-b\u200b inding domain (RBD) of the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-\u200bCoV-2). The anti-R\u200b BD mAbs prevent binding of the S protein to its cognate receptor, angiotensin-c\u200b onverting enzyme 2 (ACE2), on target host cells. Three neutralizing mAb regimens have been given emergency use authorization for treatment of COVID-19. (1) Casirivimab and imdevimab bind distinct epitopes on the RBD with dissociation constants KD of 46 and 47 pM, respectively. Imdevimab binds the S protein RBD from the front or lower-l\u200beft side, while casirivimab targets the spike-\u200blike loop from the top direction (overlapping with the ACE2-\u200bbinding site3,68). (2) Bamlanivimab binds an epitope on the RBD in both its open confirmation and its closed confirmation with dissociation constant KD\u2009=\u200971pM, covering 7 of the approximately 25 side chains observed to form contact with ACE2 (ref.4). (3) Bamlanivimab and etesevimab bind to distinct, but overlapping, epitopes within the RBD of the S protein of SARS-\u200bCoV-2. Etesevimab binds the up/active conformation of the RBD with dissociation constant KD\u2009=\u20096.45 nM (ref.5); it contains the LALA mutation in the Fc region, resulting in null effector function.\n\nto the S protein can neutralize the ability of the virus to bind and fuse with the target host cell. Humanized murine technology or convalescent plasma from recovered patients has been used to derive neutralizing mAbs targeted to the RBD of the S protein64\u201366 (Fig. 3). To date, most advanced research efforts for therapeutic use of neutralizing mAbs are focusing on a handful of pro\u00ad ducts in clinical development, some of which are already authorized on the basis of phase I/II and phase II data for emergency use (Table 1).\nREGN-\u200bCOV2 therapy. REGN-\u200bCOV2 is a combination of two potent neutralizing mAbs \u2014 namely, casirivimab and imdevimab, which are IgG1 mAbs with unmodified Fc regions. These two mAbs were chosen from a pool of more than 200 neutralizing mAbs present in the initial isolation of thousands of antibodies and were derived from parallel efforts using humanized mice and the sera of patients recovering from COVID-19 (refs67,68). The antibodies bind two distinct and non-\u200boverlapping sites on the RBD3,67. The rationale for this antibody combination is that it is unlikely that a mutation in the S protein of SAR-\u200bCoV-2 will simultaneously render both antibodies ineffective. In extensive in vitro testing, this combination retained its ability to neutralize all known S protein mutations67. Further, casirivimab and imdevimab combination therapy initiated antibody-\u200b mediated cytotoxicity and cellular phagocytosis in virally infected cells in vitro3. This product was tested in rhesus macaques and golden hamsters infected with SARS-C\u200b oV-2, which serve as models for mild and severe disease, respectively69. In both models, prophylactic and therapeutic treatment with casirivimab and imdevimab not only resulted in a reduction in viral load but also diminished the incidence and severity of lung disease relative to a placebo.\nAn ongoing phase I/II/III placebo-\u200bcontrolled trial (NCT04425629) is investigating the safety and efficacy of a single infusion of casirivimab and imdevimab \u2014 2,400\u2009mg (n\u2009=\u2009266, interim), 8,000\u2009mg (n\u2009=\u2009267, interim) or matching placebo (n\u2009=\u2009266) \u2014 for symptomatic adults who have not previously been hospitalized within 3 days of a positive active SARS-C\u200b OV-2 diagnosis (and within 7 days of the first symptoms)3. In the modified full analysis set for the phase I/II analysis, the median age was 42 years (7% aged 65 years or older), 85% of patients were white, 9% were Black and 34% were considered at high risk (for example, they were elderly, had obesity or had underlying chronic medical conditions). Pooled treatment achieved the primary end point of time-\u200b weighted average change from the baseline in viral load (log10 copies per millilitre), collected from a nasopharyngeal swab, in patients with a positive baseline for viral RNA (n\u2009=\u2009665). The difference in time-\u200bweighted average from day 1 through day 7 for the pooled doses of casirivimab and imdevimab compared with placebo was \u22120.36 log10 copies per millilitre (P\u2009<\u20090.0001). The combination was reported to reduce viral load particularly in patients with higher viral loads who were seronegative at the baseline3,70. On a key clinical end point, a lower proportion of patients treated with casirivimab and imdevimab had COVID-19-\u200brelated medically attended visits\n\nnature Reviews | IMMuNology\n\n0123456789();:\n\nvolume 21 | June 2021 | 387\n\nReviews\n\nTable 1 | Neutralizing monoclonal antibodies for SARS-C\u200b oV-2 currently in development up to 11 December 2020\n\nSponsors\n\nDrug code/International Status proprietary name\n\nTrial ID\n\nActual starta Estimated primary completiona\n\nJunshi Biosciences and Eli JS016, etesevimab Lilly and Company\n\nEUA when used in combination with bamlanivimabb\n\nNCT04441918 NCT04441931 NCT04427501\n\n5 Jun. 2020 19 Jun. 2020 17 Jun. 2020\n\n11 Dec. 2020 2 Oct. 2020c 20 Sep. 2020c\n\nTychan Pte Ltd\n\nTY027\n\nPhase I; phase III pending\n\nNCT04429529 NCT04649515\n\n9 Jun. 2020 4 Dec. 2020d\n\n19 Nov. 2020c 31 Aug. 2021\n\nBrii Biosciences\n\nBRII-196\n\nPhase I\n\nNCT04479631\n\n12 Jul. 2020 Mar. 2021\n\nBrii Biosciences\n\nBRII-198\n\nPhase I\n\nNCT04479644\n\n13 Jul. 2020 Mar. 2021\n\nAbbVie\n\nABBV-47D11\n\nPhase I pending\n\nNCT04644120\n\n10 Dec. 2020 5 Sep. 2021\n\nSorrento Therapeutics Inc. COVI-\u200bGUARD (STI-1499) Phase I\n\nNCT04454398\n\nSep. 2020d\n\nJan. 2021\n\nMabwell (Shanghai) Bioscience Co. Ltd\n\nMW33\n\nPhase I\n\nNCT04533048\n\n7 Aug. 2020 16 Nov. 2020c\n\nHiFiBiO Therapeutics\n\nHFB30132A\n\nPhase I\n\nNCT04590430\n\n20 Oct. 2020 Apr. 2021\n\nOlogy Bioservices\n\nADM03820\n\nPhase I pending\n\nNCT04592549\n\n4 Dec. 2020 30 Sep. 2021\n\nHengenix Biotech Inc\n\nHLX70\n\nPhase I pending\n\nNCT04561076\n\n9 Dec. 2020d 6 Sep. 2021\n\nUniversity of Cologne and DZIF-10c Boehringer Ingelheim\n\nPhase I/II pending\n\nNCT04631705 NCT04631666\n\n14 Dec. 2020 8 Dec. 2020\n\n31 Jul. 2021 31 Jul. 2021\n\nSorrento Therapeutics Inc. COVI-\u200bAMG (STI-2020)\n\nPhase I/II pending NCT04584697\n\nDec. 2020c\n\nApr. 2021\n\nBeigene\n\nBGB DXP593\n\nPhase I; phase II pending\n\nNCT04532294 (phase I)\nNCT04551898 (phase II pending)\n\n8 Sep. 2020 2 Dec. 2020\n\n19 Feb. 2021 25 Jan. 2021c\n\nSinocelltech Ltd\n\nSCTA01\n\nPhase I; phase II/III NCT04483375\n\npending\n\nNCT04644185\n\n24 Jul. 2020 10 Feb. 2021d\n\n17 Nov. 2020c 10 May 2021\n\nAstraZeneca\n\nAZD7442 (AZD8895 and AZD1061)\n\nPhase I; phase III pending\n\nNCT04507256 NCT04625725\n\n18 Aug. 2020 21 Nov. 2020\n\n25 Oct. 2021 21 Apr. 2021\n\nNCT04625972\n\n2 Dec. 2020 21 Jan. 2022\n\nCelltrion\n\nCT-\u200bP59\n\nPhase I; phase II/III NCT04525079\n\n18 Jul. 2020 31 Aug. 2020\n\nNCT04593641\n\n4 Sep. 2020 22 Oct. 2020\n\nNCT04602000\n\n25 Sep. 2020 Dec. 2020\n\nVir Biotechnology Inc and VIR-7831/GSK4182136 GlaxoSmithKline\n\nPhase II/III\n\nNCT04545060\n\n27 Aug. 2020 Mar. 2021\n\nAbCellera and Eli Lilly and Company\n\nBamlanivimab; combination of bamlanivimab and etesevimab\n\nEUAb\n\nNCT04411628 (phase I) NCT04427501 (phase II) NCT04497987 (phase III)\n\n28 May 2020 17 Jun. 2020 2 Aug. 2020\n\n26 Aug. 2020c 20 Sep. 2020c 8 Mar. 2021\n\nNCT04501978 (phase III) 4 Aug. 2020 Jul. 2022\n\nNCT04518410 (phase II/III) 19 Aug. 2020 May 2023\n\nRegeneron\n\nREGN-\u200bCOV2 (casirivimab EUAb and imdevimab)\n\nNCT04425629 (phase I/II) 16 Jun. 2020 NCT04426695 (phase I/II) 11 Jun. 2020\n\n10 Apr. 2021 16 Apr. 2021\n\nNCT04452318 (phase III) 13 Jul. 2020 15 Jun. 2021\n\nA complete list can be found at COVID-19 Biologics Tracker. EUA, emergency use authorization; SARS-C\u200b oV-2, severe acute respiratory syndrome coronavirus 2. aDates as of 7 April 2021. bHave recieved EUA in the United States. cActual primary completion date. dEstimated start date.\n\nAbsolute risk reduction The difference between the risk of an event in the control group and the risk of an event in the treated group.\n\n(2.8% for pooled doses versus 6.5% for placebo). In post hoc analyses, a lower proportion of patients treated with casirivimab and imdevimab had COVID-19-r\u200b elated hospitalizations or emergency department visits compared with patients who received placebo (2% versus 4%). The absolute risk reduction for casirivimab and imdevimab compared with placebo was greater for patients at high risk of progression to severe COVID-19 and/or hospitalization (3% versus 9%). Collectively, these results supported the EUA of Regeneron\u2019s casirivimab and\n\nimdevimab cocktail in the United States in November 2020 (ref.71).\nBamlanivimab monotherapy. Bamlanivimab is a potent neutralizing mAb (IgG1 with an unmodified Fc region) to the S protein that was derived from the convalescent plasma of a patient who had COVID-19 (refs24,66). Bamlanivimab binds the S protein\u2019s RBD, engaging its cognate epitope in both up and down conformations, which makes this antibody potentially useful as a\n\n388 | June 2021 | volume 21\n\n0123456789();:\n\nwww.nature.com/nri\n\nReviews\n\nPersistent high viral load The continued presence of a high viral load in patients, which is associated with increased risk of hospitalization.\n\nmonotherapy. There have been historical precedents for the effectiveness of neutralizing mAbs as a monotherapy (for example, MAb114 for Ebola)38. To assess theoretical risk of ADE, bamlanivimab was studied in primary human macrophages and immune cell lines exposed to SARS-\u200bCoV-2 at concentrations down to 100-\u200bfold below the effective concentration for half-\u200bmaximum response, and in these studies, it did not demonstrate productive viral infection4. Prophylactic efficacy was tested in rhesus macaques given bamlanivimab 24\u2009hours before a virus challenge66. The symptoms in this model were mild overall, but the treatment significantly decreased viral load and replication in the respiratory tract following inoculation, supporting its antiviral efficacy.\nIn the phase II portion of the ongoing phase II/III BLAZE-1 trial (NCT04427501), ambulatory adults with mild-\u200bto-\u200bmoderate symptoms of COVID-19 within 3 days of a first-\u200bpositive nasopharyngeal swab positive for SARS-\u200bCoV-2 received a single infusion of one of three doses of bamlanivimab (700, 2,800 or 7,000\u2009mg) or placebo in an outpatient setting24. A pre-\u200bplanned interim analysis was conducted of 452 patients who had reached day 11 following infusion (median age 45\u201346 years (12% aged 65 years or older), 88% white, 6% Black and 68% at high risk (for example, they were elderly, had obesity or had underlying chronic medical conditions))24. The study revealed the viral clearance time course via the intrinsic immune response and the enhanced clearance with neutralizing mAb infusion, concomitant with improved clinical response. Viral loads were assayed from serial nasopharyngeal swabs, with postinfusion measurements enabled by a novel partnership with home-\u200bhealth research. Following infusion, the log viral load had begun decreasing relative to the baseline as early as the first postinfusion assessment on day 3 (\u22120.85 for placebo versus \u22121.35 for pooled bamlanivimab doses), continued to decrease on day 7 (\u22122.56 for placebo versus \u22122.90 for pooled bamlanivimab doses) and further decreased by day 11 (\u22123.47 for placebo versus \u22123.70 for pooled bamlanivimab doses). In a post hoc analysis, patients with early persistent high viral load (described as log viral load of 5.27 or greater at trial day 7) had a higher risk of hospitalization, and the risk was further increased for elderly patients and patients with obesity. Clinical evidence demonstrating the efficacy of bamlanivimab came from two predefined secondary end points. First, at day 29, the percentage of patients who were hospitalized with COVID-19 was 6.3% for the placebo group and reduced to 1.6% for the group with pooled bamlanivimab doses. In post hoc analyses, hospitalization among elderly patients (65 years or older) or patients with obesity (body mass index 35\u2009kg\u2009m\u22122 or greater) was 15% for the placebo group and 4% for the group who received pooled bamlanivimab doses. Absolute risk reduction for hospitalizations was more evident for patients with risk factors. Second, amelioration of baseline symptoms was greater for the pooled bamlanivimab doses than for placebo from day 2 through to day 11. Collectively, these results supported the EUA of bamlanivimab monotherapy in the United States and Canada in November 2020 (ref.4).\n\nBamlanivimab and etesevimab. Other treatment arms of the BLAZE-1 trial studied bamlanivimab together with etesevimab (an S protein-\u200bbinding IgG1 with a modi\u00adfied Fc region, resulting in null effector function)25,72. Bamlanivimab and etesevimab together significantly decreased viral load (mean changes from the baseline and percentage of patients with persistent high viral load) compared with placebo at day 3 to day 11 (ref.25). Bamlanivimab- and etesevimab-\u200btreated patients had fewer COVID-19-\u200brelated hospitalizations relative to the placebo group (5.8% for placebo reduced to 0.9% for bamlanivimab together with etesevimab). Recently released placebo-\u200bcontrolled phase III data from 1,035 patients randomized 1:1 to receive bamlanivimab together with etesevimab versus placebo demonstrated that in high-\u200brisk ambulatory patients (including patients aged 12\u201317 years with specific risk factors and patients aged 18 years or older with specific adult risk factors) treatment with bamlanivimab and etesevimab together was associated with a 70% reduction in COVID-19-\u200brelated hospitalizations and deaths relative to placebo treatment (7.0% for placebo reduced to 2.1% for bamlanivimab together with etesevimab)25,73. On the basis of these data, an additional EUA of bamlanivimab together with etesevimab has been issued5.\nMonoclonal antibody therapies in severe COVID-19. There are concurrent studies investigating neutralizing mAbs for patients hospitalized with severe COVID-19. The REGN-\u200bCOV2 trial in hospitalized patients enrols patients with or without supplemental oxygen and is ongoing74,75. In prospectively designed analysis of REGN-\u200bCOV2, there may be clinical benefit in patients treated with casirivimab and imdevimab and who were seronegative at the time of treatment76. In the ACTIV-3 RCT (n\u2009=\u2009326, 1:1 randomization), bamlanivimab added to standard of care (typically including remdesivir) did not demonstrate additional clinical benefit in hospital\u00ad ized patients77. In line with similar studies investi\u00ad gating CPT or neutralizing mAbs for patients with severe viral disease (including COVID-19)15,18,34\u201336,54,56, the evidence indicates that rapid viral clearance, in itself, is insufficient. Rather, additional factors, such as an excessive immune response, are the primary drivers for continued disease in this particular patient population. Thus, early disease seen in outpatients is likely virally driven, whereas the pathophysiology for inpatient advanced disease is predominantly a postviral or periviral phenomenon, with clinical status uncoupled from viral load.\nAdverse events associated with monoclonal antibody therapies. In terms of risk associated with mAb treatment of COVID-19, treatment-a\u200b ssociated adverse events were comparable to those with placebo. The most frequent side effects observed in RCTs include nausea, diarrhoea, dizziness, headache and vomiting24,25,78. One per cent of patients receiving casirivimab and imdevimab reported a grade 2 or higher infusion-r\u200b elated reaction within 4 days of administration (comparable to 1% reported for placebo treatment)78. In the phase II portion of BLAZE-1, nine patients reported an infusion-\u200brelated reaction\n\nnature Reviews | IMMuNology\n\n0123456789();:\n\nvolume 21 | June 2021 | 389\n\nReviews\n\n(1.9% (6/309) with bamlanivimab monotherapy, 1.8% (2/112) with bamlanivimab and etesevimab together, and 0.6% (1/156) with placebo). Most reactions occurred during infusion; these were mild in severity and were not dose related25. Regarding evidence of ADE, in vitro data indicate neutralizing mAbs do not enhance productive infection of immune cells with SARS-\u200bCoV-2 (refs3,4). From the clinical data available to date, there is no clear evidence these therapies result in enhanced immune responses consistent with ADE24,25,78. Furthermore, the safety profiles of modified and modified plus unmodified mAbs to treat SARS-\u200bCoV-2 infection are similar, suggesting that ADE may not play a role in clinical outcomes25.\nEmergence of drug-\u200bresistant SARS-\u200bCoV-2 strains. For patients with COVID-19 who receive neutralizing mAbs, there is potential for the development of drug-\u200bresistant variants, which become more obvious when selective pressure is applied in the setting of drug treatment78,79. For bamlanivimab, non-\u200bclinical studies using serial passage of SARS-\u200bCoV-2 and directed evolution of the SARS-\u200bCoV-2 S protein identified viral variants (E484D/K/Q, F490S, Q493R and S494P, amino acid substitutions in the S protein RBD) that had increased resistance to this drug4.\nIn clinical trials of bamlanivimab, genotypic and phenotypic testing are monitoring SARS-C\u200b oV-2 strains for potential S protein variations that are associated with bamlanivimab resistance. In clinical trials of bamlanivimab, viral sequencing is being performed for all patients, regardless of treatment status/progression. In other studies where only treatment failures are sampled, the selective pressure exerted by the antiviral activity cannot be assessed. In the BLAZE-1 RCT, which was limited to US investigative sites, known bamlanivimab-\u200bresistant variants at the baseline were observed at a frequency of 0.27% to date4. In the same trial, treatment-\u200bemergent variants were detected at S protein amino acid positions E484, F490 and S494 (including E484A/D/G/K/Q/V, F490L/S/V and S494L/P); considering all variants at these positions, 9.2% and 6.1% of participants in the 700-\u200bmg bamlanivimab arm (the EUA dose) harboured such a variant after the baseline at allele fractions of 15% or greater and 50% or greater, respectively, compared with 8.2% and 4.1%, respectively, of participants in the placebo arm. Most of these variants were first detected on day 7 following infusion, and were detected at only a single time point. The clinical impact of these variants is currently unknown4.\nAs with bamlanivimab, casirivimab and imdevimab therapy has the potential to lead to the development of resistant viral variants. In non-\u200bclinical studies, serial passage of vesicular stomatitis virus (VSV) encoding the SARS-C\u200b oV-2 S protein in the presence of the drugs identified escape variants with reduced susceptibility to casirivimab (K417E/N/R, Y453F, L455F, E484K, F486V and Q493K) or imdevimab (K444N/Q/T and V445A)3. Each viral variant showing reduced susceptibility to one mAb remained susceptible to the other mAb; all identified variants retained susceptibility to the combination. In a separate experiment, neutralization assays\n\nwere performed with VSV pseudotyped with 39 variants of the S protein identified in circulating SARS-\u200bCoV-2. The G476S, S494P and Q409E variants had reduced susceptibility (5-\u200bfold, 5-\u200bfold, and 4-f\u200bold, respectively) to casirivimab, and the N439K variant had reduced susceptibility (463-\u200bfold) to imdevimab. The casirivimab and imdevimab combination was active against all individual variants tested3. It has been reported that the combination of mutants at residues 417 and 439 may abrogate the effectiveness of the casirivimab and imdevimab combination80.\nIn the casirivimab and imdevimab RCT NCT04425629, interim data indicated only one variant (G446V) detected in 4.5% of participants at an allele fraction of 15% or greater, each detected at a single time point3. The clinical impact is unknown. In a VSV pseudoparticle neutralization assay, the G446V variant had reduced susceptibility to imdevimab (135-f\u200bold) but retained susceptibility to both casirivimab alone and the casirivimab and imdevimab combination.\nHowever, not all variants must be considered clinically relevant mutations associated with resistance to treatment. During the Ebola outbreak in 2018, a genomic assessment of 48 viral genomes determined that this outbreak was due to a distinct viral variant. The sequence information allowed researchers to evaluate the relevance of the distinct mutations to the available vaccine and therapeutics and to conclude that the neutralizing antibodies MAb114 and ZMapp would likely be effective against the currently circulating variant81. A similar practice for SARS-\u200bCoV-2 surveillance may be prudent to determine whether emergent S protein variants pose a threat to the efficacy of neutralizing mAb therapies.\nIndeed, three SARS-\u200bCoV-2 variants of particular interest have been identified and are circulating globally. In the United Kingdom, a variant called \u2018B.1.1.7\u2019 with a large number of mutations was identified in the autumn of 2020. In South Africa, a variant called \u2018B.1.351 was identified. Originally detected in early October 2020, B.1.351 shares some mutations with B.1.1.7. In Brazil, a variant called \u2018P.1\u2019 was identified that contains a set of additional mutations that may affect its ability to be recognized by first-\u200bgeneration neutralizing mAbs and by the immune responses generated by first-g\u200b eneration vaccines. Although these variants have been detected in the United States, according to real-\u200btime data accessed via the GISAID COVID-19 variant tracker82 these COVID-19 variants do not currently represent a significant proportion of COVID-19 infections in the United States82,83, while recent California (B.1.427/B.1.429) and New York (B.1.526) variants do. To date, the effect of these variants on the neutralizing capacity of vaccines and mAbs is unknown. A recent preprint suggests that the variants identified in the United Kingdom and South Africa are more resistant to CPT and vaccine sera42.\nBamlanivimab and imdevimab maintain full neutralization activity against the primary SARS-\u200bCoV-2 receptor-\u200bbinding site variants (69-70del and N501Y) implicated in the strain originating in the United Kingdom, suggesting that these mAbs should maintain full activity against the new strain originating in the United Kingdom42,84. From what is known about the\n\n390 | June 2021 | volume 21\n\n0123456789();:\n\nwww.nature.com/nri\n\nReviews\n\nstrains that were first identified in South Africa, Brazil as well as the ones in California and New York, it appears that some of the first generation of antibody therapies may not be as effective and it will be important for physicans to refer to the most up to date factsheet3\u20135,42.\nClinical use in COVID-19\nBamlanivimab, bamlanivimab together with etesevimab, and casirivimab with imdevimab decrease viral load when given early on in the course of SARS-\u200bCoV-2 infection and favourably impact clinical outcomes for patients with mild-\u200bto-\u200bmoderate COVID-19 (refs24,70). Although full clinical trial data are pending, top-\u200bline and interim results from multiple trials suggest that the therapies may also function as prophylaxis in at-r\u200b isk patients recently exposed to SARS-\u200bCoV-2 (refs6,7). One signal emerging from early data is that patients with persistently higher viral loads progress more frequently towards medically attended visits, emergency department visits or hospitalization, and this effect is most pronounced for patients with pre-e\u200b xisting risk factors for disease progression3,24. It remains a tenet that antivirals, whether small molecules or neutralizing mAbs, work best when deployed early. By extrapolation from early viral load data, ideally patients would receive treatment as soon as possible (that is, within hours to days following a positive test or symptom onset). In the trial setting, by day 7 to day 11 most patients either are progressing towards clearance of the virus24 or have experienced clinical decline and hospitalization, further emphasizing the need for early intervention. As the clinical trial timelines typically represent an offset of several days from initial diagnosis, corresponding to day 10\u201314 of clinical illness, the actionable message remains unchanged \u2014 treat patients as early as possible to maximize the chance of altering the disease trajectory and promote recovery.\nThe COVID-19 pandemic poses logistical and medical challenges for the distribution of neutralizing mAbs. Up to 10% of initially asymptomatic, minimally symptomatic and mild infections progressed to severe disease including respiratory distress85. While approximately 78% of patients admitted to hospital have at least one documented co-\u200bmorbidity86, there continue to be patients lacking any identified co-\u200bmorbidity who subsequently become critically ill. Thus, the absence of co-\u200bmorbidities does not completely eliminate the risk of severe disease and sequalae, and there is an urgent need for additional insight into a more personalized predictive algorithm to unlock as-\u200byet-\u200bunidentified risk factors. Contrary to the discussion in the lay media, COVID-19 can potentially claim the lives of young adults in their prime, even in the absence of any known underlying risk factors. Given that persistently high SARS-\u200bCoV-2 viral loads may be associated with severe clinical outcomes87\u201389, it is possible that early reassessment of viral loads might help guide who among the \u2018lower-r\u200b isk\u2019 population might be helped by neutralizing mAbs. RCT evidence indicates that the clinical value of neutralizing mAb therapy is more pronounced in individuals who are seronegative at diagnosis70. Collectively, measuring viral load and serology would allow strategic deployment for patients without otherwise identifiable\n\nrisk factors while targeting early supply to the high-\u200brisk population. However, this strategy would be contingent on rapid turnaround of laboratory testing. Meanwhile, it also seems reasonable to use neutralizing mAbs early on during the disease for patients with well-\u200bidentified risk factors for severe disease evolution90.\nAnother way to classify candidate patients for neutralizing mAbs would be to select patients who are expected to have poor antiviral responses (for example, elderly or immunocompromised patients) or to identify patients with poor T cell and/or B cell function via experimental techniques (such as by serology or flow cytometry). Regarding the latter, there is a lack of published evidence on humoral immune response dynamics and correlation with clinical outcomes. Furthermore, technical difficulties in stratifying patients on the basis of antibody production, lymphocyte function and/or viral load might pose a significant impediment to the timely identification of the most appropriate patients for neutralizing mAb therapy.\nFinally, antiviral and antimicrobial therapies are traditionally plagued by their promoting escape variants, and sometimes combination therapy can mitigate this risk. As a first-\u200bgeneration approach for neutralizing mAbs, monotherapies have been developed and have been demonstrated to be efficacious, but it is expected that a greater number of combination therapies will follow. For example, in phase II/III trials involving patients with COVID-19, bamlanivimab and the bamlanivimab and etesevimab combination had similarly improved magnitudes and timings of symptom relief relative to the placebo25. However, to date, the bamlanivimab and etesevimab combination does not appear to lead to the emergence of drug-\u200bresistant variants of SARS-\u200bCoV-2. This is similar to what has been observed for the other authorized neutralizing mAb combination (casirivimab and imdevimab), as already described herein. At present, there remains a role for continued use of monotherapy while transitioning towards combination therapies so as to mitigate the selection pressure for viral escape as manufacturing capacity becomes available, to ensure longevity of the therapies and to reduce the potential rate of treatment failure.\nSummary and conclusions\nThe sudden arrival and devastating spread of the COVID-19 pandemic has stimulated an accelerated programme of international research to identify effective ways to limit the spread of infection and to reduce the morbidity and mortality associated with COVID-19 (refs91,92). The first data are now emerging on vaccines designed to prevent disease93,94. In the context of active SARS-\u200bCoV-2 infection, clinical trials suggest that mortality in infected patients with hypoxia could be reduced with agents such as dexamethasone, baricitinib (in combination with remdesivir) and tocilizumab (data still under review)95\u201399. Furthermore, many trials have been conducted or are under way with various immune-\u200bmodulating medications designed to limit the tissue damage associated with the later stages of COVID-19. However, to date there have been few unequivocal successes. Neutralizing mAbs, particularly in\n\nnature Reviews | IMMuNology\n\n0123456789();:\n\nvolume 21 | June 2021 | 391\n\nReviews\n\ncombination with other medications, are an attractive approach with potential utility in both prophylactic and treatment settings. Encouraging early clinical trial data support further investigation of neutralizing mAbs to determine the optimal dosing regimen. Unanswered questions regarding this novel therapeutic approach set a pressing research agenda; we need to establish which at-r\u200b isk individuals would benefit most from prophylactic\n\nneutralizing mAbs, the duration of protection offered by these mAbs and any potential impact of mAb therapy on subsequent vaccination. It will also be important to determine the optimum timing for administration of neutralizing mAbs on the basis of viral load, serology and other potential clinical factors.\nPublished online 19 April 2021\n\n1. Renn, A., Fu, Y., Hu, X., Hall, M. D. & Simeonov, A. Fruitful neutralizing antibody pipeline brings hope to defeat SARS-\u200bCov-2. Trends Pharmacol. Sci. 41, 815\u2013829 (2020).\n2. Shanmugaraj, B., Siriwattananon, K., Wangkanont, K. & Phoolcharoen, W. Perspectives on monoclonal antibody therapy as potential therapeutic intervention for coronavirus disease-19 (COVID-19). Asian Pac. J. Allergy Immunol. 38, 10\u201318 (2020).\n3. Regeneron Pharmaceuticals Inc. Fact sheet for health care providers: emergency use authorization (EUA) of casirivimab and imdevimab. Regeneron https:// www.regeneron.com/sites/default/files/treatment-\u200b covid19-eua-f\u200bact-sheet-f\u200bor-hcp.pdf (2020).\n4. US Food and Drug Administration. Fact sheet for health care providers emergency use authorization (EUA) of bamlanivimab. FDA https://www.fda.gov/ media/143603/download (2020).\n5. US Food and Drug Administration. Fact sheet for health care providers emergency use authorization (EUA) of bamlanivimab and etesevimab. FDA https:// www.fda.gov/media/145802/download (2021).\n6. Regeneron Pharmaceuticals Inc. Regeneron reports positive interim data with REGEN-\u200bCOV\u2122 antibody cocktail used as passive vaccine to prevent COVID-19. Regeneron https://newsroom.regeneron.com/news-\u200b releases/news-\u200brelease-details/regeneron-\u200breportspositive-i\u200bnterim-data-r\u200begen-covtm-a\u200b ntibody (2021).\n7. Eli Lilly and Company. Lilly\u2019s neutralizing antibody bamlanivimab (LY-\u200bCoV555) prevented COVID-19 at nursing homes in the BLAZE-2 trial, reducing risk by up to 80 percent for residents. Eli Lilly and Company https://investor.lilly.com/news-\u200breleases/news-\u200breleasedetails/lillys-\u200bneutralizing-antibody-\u200bbamlanivimably-c\u200b ov555-prevented (2021).\n8. Llewelyn, M. B., Hawkins, R. E. & Russell, S. J. Discovery of antibodies. BMJ 305, 1269\u20131272 (1992).\n9. Behring, E. A. & Kitasato, S. Ueber das Zustandekommen der Diphtherie-\u200bimmunitat und der Tetanus-i\u200bmmunitat bei Thieren. Dtsch. Med. Wochenschr. 28, 1321\u20131332 (1890).\n10. Kaufmann, S. H. Immunology\u2019s foundation: the 100-year anniversary of the Nobel Prize to Paul Ehrlich and Elie Metchnikoff. Nat. Immunol. 9, 705\u2013712 (2008).\n11. Hung, I. F. et al. Convalescent plasma treatment reduced mortality in patients with severe pandemic influenza A (H1N1) 2009 virus infection. Clin. Infect. Dis. 52, 447\u2013456 (2011).\n12. Mair-\u200bJenkins, J. et al. The effectiveness of convalescent plasma and hyperimmune immunoglobulin for the treatment of severe acute respiratory infections of viral etiology: a systematic review and exploratory meta-\u200banalysis. J. Infect. Dis. 211, 80\u201390 (2015).\n13. Groothuis, J. R. et al. Prophylactic administration of respiratory syncytial virus immune globulin to high-risk infants and young children. N. Engl. J. Med. 329, 1524\u20131530 (1993).\n14. Sahr, F. et al. Evaluation of convalescent whole blood for treating Ebola virus disease in Freetown, Sierra Leone. J. Infect. 74, 302\u2013309 (2017).\n15. Cheng, Y. et al. Use of convalescent plasma therapy in SARS patients in Hong Kong. Eur. J. Clin. Microbiol. Infect. Dis. 24, 44\u201346 (2005).\n16. Yeh, K. M. et al. Experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a Taiwan hospital. J. Antimicrob. 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People at increased risk and other people who need to take extra precautions. CDC https://www.cdc.gov/ coronavirus/2019-ncov/need-\u200bextra-precautions/index. html?CDC_AA_refVal=https%3A%2F%2Fwww.cdc. gov%2Fcoronavirus%2F2019-ncov%2Fneed-\u200bextraprecautions%2Fpeople-\u200bat-increased-\u200brisk.html (2021).\n\n91. Wang, C., Horby, P. W., Hayden, F. G. & Gao, G. F. A novel coronavirus outbreak of global health concern. Lancet 395, 470\u2013473 (2020).\n92. Zhu, N. et al. A novel coronavirus from patients with pneumonia in China, 2019. N. Engl. J. Med. 382, 727\u2013733 (2020).\n93. Dai, L. & Gao, G. F. Viral targets for vaccines against COVID-19. Nat. Rev. Immunol. 21, 73\u201382 (2020).\n94. Krammer, F. SARS-\u200bCoV-2 vaccines in development. Nature 586, 516\u2013527 (2020).\n95. Horby, P. et al. Dexamethasone in hospitalized patients with Covid-19 - preliminary report. N. Engl. J. Med. https://doi.org/10.1056/NEJMoa2021436 (2020).\n96. Kalil, A. C. et al. Baricitinib plus remdesivir for hospitalized adults with Covid-19. N. Engl. J. Med. https://doi.org/10.1056/NEJMoa2031994 (2020).\n97. US Food and Drug Administration. Fact sheet for healthcare providers emergency use authorization (EUA) of baricitinib. FDA https://www.fda.gov/ media/143823/download (2020).\n98. The REMAP-C\u200b AP Investigators et al. Interleukin-6 receptor antagonists in critically ill patients with Covid19\u2013preliminary report. Preprint at medRxiv https://doi.org/10.1101/2021.01.07.21249390 (2021).\n99. RECOVERY Collaborative Group et al. Tocilizumab in patients admitted to hospital with COVID-19 (RECOVERY): preliminary results of a randomised, controlled, open-\u200blabel, platform trial. Preprint at medRxiv https://doi.org/10.1101/2021.02.11. 21249258 (2021).\nAcknowledgements This work was supported by Eli Lilly and Company. C. J. Antalis and H. Green (Eli Lilly and Company) provided editorial assistance.\nAuthor contributions The authors contributed equally to all aspects of the article.\nCompeting interests P.C.T. has received research grants, consultation fees and/or speaking fees from AbbVie, Biogen, Bristol-\u200bMyers Squibb, Celgene, Celltrion, Fresenius, Galapagos, Gilead, GlaxoSmithKline, Janssen, Eli Lilly and Company, Sanofi, Nordic Pharma, Pfizer, Roche and UCB. A.C.A., I.d.l.T. and M.M.H. are employees and shareholders of Eli Lilly and Company. K.W. has received research grants from BristolMyers Squibb and Pfizer and consulting fees from AbbVie, AstraZeneca, Bristol-\u200bMyers Squibb, Eli Lilly and Company, Galapagos, GlaxoSmithKline, Gilead, Novartis, Pfizer, Regeneron, Roche, Sanofi and UCB. R.L.G. reports nonfinancial support from Gilead Sciences Inc. and personal fees from Gilead Sciences Inc. outside the submitted work.\nPeer review information Nature Reviews Immunology thanks the anonymous reviewers for their contribution to the peer review of this work.\nPublisher\u2019s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\nRelated links ClinicalTrials.gov: https://clinicaltrials.gov COviD-19 Biologics Tracker: https://www.antibodysociety. org/covid-19-biologics-tracker/ GisAiD COviD-19 variant tracker: https://www.gisaid.org/\n\u00a9 The Author(s), under exclusive licence to Springer Nature Limited. 2021\n\nnature Reviews | IMMuNology\n\n0123456789();:\n\nvolume 21 | June 2021 | 393\n\n\n",
    "authors": [
      "Peter C. Taylor",
      "Andrew C. Adams",
      "Matthew M. Hufford",
      "Inmaculada De La Torre",
      "Kevin Winthrop",
      "Robert L. Gottlieb"
    ],
    "doi": "10.1038/s41577-021-00542-x",
    "date": "06/2021",
    "item_type": "journalArticle",
    "url": "https://www.nature.com/articles/s41577-021-00542-x"
  },
  {
    "key": "436NHDS3",
    "title": "Monoclonal antibodies for the treatment of Ebola virus disease",
    "abstract": "Introduction: To date, the management of patients with suspected or confirmed Ebolavirus disease (EVD) depends on quarantine, symptomatic management and supportive care, as there are no approved vaccines or treatments available for human use. However, accelerated by the recent large outbreak in West Africa, significant progress has been made towards vaccine development but also towards specific treatment with convalescent plasma and monoclonal antibodies.",
    "full_text": "Expert Opinion on Investigational Drugs\nISSN: 1354-3784 (Print) 1744-7658 (Online) Journal homepage: www.tandfonline.com/journals/ieid20\nMonoclonal antibodies for the treatment of Ebola virus disease\nA. L. Moekotte, M. A. M. Huson, A. J. van der Ende, S.T. Agnandji, E. Huizenga, A. Goorhuis & M. P. Grobusch\nTo cite this article: A. L. Moekotte, M. A. M. Huson, A. J. van der Ende, S.T. Agnandji, E. Huizenga, A. Goorhuis & M. P. Grobusch (2016) Monoclonal antibodies for the treatment of Ebola virus disease, Expert Opinion on Investigational Drugs, 25:11, 1325-1335, DOI: 10.1080/13543784.2016.1240785 To link to this article: https://doi.org/10.1080/13543784.2016.1240785\n\u00a9 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 08 Oct 2016.\nSubmit your article to this journal\nArticle views: 6735\nView related articles\nView Crossmark data\nCiting articles: 4 View citing articles\nFull Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=ieid20\n\nEXPERT OPINION ON INVESTIGATIONAL DRUGS, 2016 VOL. 25, NO. 11, 1325\u20131335 http://dx.doi.org/10.1080/13543784.2016.1240785\nREVIEW\nMonoclonal antibodies for the treatment of Ebola virus disease\nA. L. Moekottea, M. A. M. Husona, A. J. van der Endeb,c, S.T. Agnandjid,e, E. Huizengab,c, A. Goorhuisa,c and M. P. Grobuscha,c,d,e\naCenter of Tropical Medicine and Travel Medicine, Department of Infectious Diseases, Division of Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; bLion Heart Medical Center, Yele, Sierra Leone; cLion Heart Medical Research Unit, Yele, Sierra Leone; dCentre de Recherches M\u00e9dicales en Lambar\u00e9n\u00e9 (CERMEL), Lambar\u00e9n\u00e9, Gabon; eInstitute of Tropical Medicine, University of T\u00fcbingen, T\u00fcbingen, Germany\n\nABSTRACT\nIntroduction: To date, the management of patients with suspected or confirmed Ebolavirus disease (EVD) depends on quarantine, symptomatic management and supportive care, as there are no approved vaccines or treatments available for human use. However, accelerated by the recent large outbreak in West Africa, significant progress has been made towards vaccine development but also towards specific treatment with convalescent plasma and monoclonal antibodies. Areas covered: We describe recent developments in monoclonal antibody treatment for EVD, encom-\npassing mAb114 and the MB-003, ZMAb, ZMapp\u2122 and MIL-77E cocktails.\nExpert opinion: Preventive measures, are, and will remain essential to curb EVD outbreaks; even more so with vaccine development progressing. However, research for treatment options must not be neglected. Small-scale animal and individual human case studies show that monoclonal antibodies (mAbs) can be effective for EVD treatment; thus justifying exploration in clinical trials. Potential limitations are that high doses may be needed to yield clinical efficacy; epitope mutations might reduce efficacy; and constant evolution of (outbreak-specific) mAb mixtures might be required. Interim advice based on the clinical experience to date is that treatment of patients with mAbs is sensible, provided those could be made available in the necessary amounts in time.\n\nARTICLE HISTORY Received 6 June 2016 Accepted 21 September 2016 Published online 10 October 2016\nKEYWORDS Ebolavirus disease; therapy; monoclonal antibodies; MB-003; ZMAb; ZMappTM; MIL-77E; mAb114\n\n1. Introduction\n1.1. Ebola virus disease: outbreak history\nEbola virus disease (EVD) was first recorded in 1976 when two outbreaks occurred, namely in (now South) Sudan, and in the Democratic Republic of Congo, formerly Zaire [1]. Since then, around 35 outbreaks have been reported mainly from Central Africa, with case fatality rates between 25% and 90% [2]. The latest one in West Africa was caused by Zaire ebolavirus and started in the Southeast of Guinea in December 2013. It rapidly spread to Liberia and Sierra Leone to become the largest recorded outbreak in history [3,4] and only tailed off slowly [5]. With more than 28,600 cases and more than 11,300 deaths (mortality rate 39.5%), it constituted a humanitarian disaster and public health emergency of international concern.\n1.2. The Ebola viruses\nEVD is caused by viruses of the Filoviridae family, which encompasses the genus Ebolavirus (with several distinct species), Marburgvirus (with the single species Marburg marburgvirus), and Cuevavirus (with the single recently identified species Lloviu cuevavirus) [6]. Filoviruses are enveloped, nonsegmented, negative-stranded RNA viruses that derive their name from their characteristic filamentous particles [7]. The Ebolavirus genus\n\nencompasses five antigenically distinct viruses; each named after the location of the outbreak in which they were first identified. These include Zaire ebolavirus, Sudan ebolavirus, Reston ebolavirus, Ta\u00ef Forest ebolavirus (formerly known as C\u00f4te d\u2019Ivoire ebolavirus), and Bundibugyo ebolavirus [8]. Zaire ebolavirus and Sudan ebolavirus most frequently cause disease in humans. Reston virus has not been linked to disease in humans.\nThe Ebola virus structure has been described in detail by Bharat et al. [9] and Beniac et al. [10] Messaoudi et al. [11] depict the Ebola virus \u2018life cycle\u2019 in detail. Ebola viruses (EBOV) produce nonstructural small soluble glycoproteins (ssGPs) and soluble glycoproteins (sGPs) [12]; the latter are secreted in large quantities from infected cells. The only EBOV transmembrane surface protein is another glycoprotein (GP), which forms trimeric spikes consisting of glycoprotein 1 (GP1) and glycoprotein 2 (GP2). This GP structure provides the target for vaccines and monoclonal antibodies (mAbs). Murin et al. [13] described GP structure and antibody binding sites in detail.\n1.3. Clinical manifestations\nAfter an incubation period of 2\u201321 days, EBOV can cause severe disease in humans, characterized by the sudden appearance of flu-like signs and symptoms including fever, chills, malaise, and myalgia; followed by gastrointestinal manifestations such as\n\nCONTACT M. P. Grobusch m.p.grobusch@amc.uva.nl Center of Tropical Medicine and Travel Medicine, Department of Infectious Diseases, Division of Internal Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Amsterdam 1100 DD, The Netherlands\n\u00a9 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.\n\n1326\n\nA. L. MOEKOTTE ET AL.\n\nArticle highlights\n\u25cf The management of patients with suspected or confirmed Ebolavirus disease still depends on quarantine, symptomatic management and supportive care; however, accelerated by the recent large outbreak in West Africa, significant progress has been made towards vaccine development but also towards specific treatment by convalescent plasma and monoclonal antibodies.\n\u25cf To date, around 20 Ebolavirus monoclonal antibodies have been identified and characterized, of which several were found promising to progress to testing in non-human primate models.\n\u25cf The monoclonal antibodies, which emerged from testing in NHPs as promising for treatment of humans include the single antibody mAb114; the two-mAb cocktail MIL77E and the 3-MAb cocktails MB-003, ZMab and ZMappTM.\n\u25cf The individual mAbs each bind to one of three distinct regions of the Ebolavirus surface glycoprotein; its base, a glycan cap, or a mucin-like domain. MAbs binding to the GP base are neutralizing, whereas antibodies binding the glycan caps or the mucin-like domains appear predominantly non-neutralizing.\n\u25cf Available data are based on in vitro studies and studies in NHPs,\n\u2122 except for ZMAb and ZMapp : four case reports are available\ndescribing compassionate use in humans with EVD. To date, clinical trials on mAb114, MB-003, ZMAb or MIL-77E have not been con-\n\u2122 ducted. A phase I/II clinical trial on ZMapp is ongoing.\n\u25cf Challenges to further develop and to bring mAb therapy to the field are multitude; however, interim advice based on the clinical experience to date is that treatment of Ebola patients with mAbs is sensible, provided those could be made available in the necessary amounts in time.\nThis box summarizes key points contained in the article.\ndiarrhea, nausea, and vomiting [14]. Disease progression is rapid, and clinical signs such as ecchymosis, petechial bleedings, and maculopapular rash can be observed. Laboratory findings include leucopenia, thrombocytopenia, and elevated serum aminotransferase concentrations. In addition, signs and symptoms of respiratory and central nervous system infection such as chest pain, dry cough, behavioral disorders, and seizures can be present. The terminal disease phase leads to either death or a frequently prolonged convalescence period. Death by shock and multiorgan failure typically occurs within 6\u201310 days after onset of disease [14].\nThe high fatality rate of EVD, in combination with the absence of treatment and vaccination options, constitutes an important public health threat, illustrated by the classification of Ebola virus as category A pathogens of Bioterrorism by the Centers for Disease Control and prevention (CDC) [15].\n2. Overview of the market: prevention and treatment of Ebola virus disease\nTo date, there are no approved vaccines or treatments available for human use, and the current protocol for patients with suspected or confirmed EVD is quarantine, symptomatic management, and supportive care, including rehydration, restoration of electrolyte deficiencies, early treatment of secondary bacterial infections, empiric malaria treatment, and vital organ function support (blood transfusion, mechanical ventilation, and hemodialysis) in case of disease progression; mostly limited to settings\n\nwhich allow for maximal care [16]. However, during the latest outbreak in West Africa, significant progress has been made beyond these limited prevention and treatment options, despite the ethical, time-related, organizational, and technical obstacles that typically hamper the design and implementation of clinical trials in outbreak situations. To that end, the fast approval of several clinical vaccine trials was a quantum leap forward. Progress with the development of effective treatments has developed more slowly. However, several interventions prioritized by WHO [17] have reached phase II clinical trials, including favipiravir, TKM-130803, convalescent plasma, and a monoclonal antibody\ncocktail (ZMapp\u2122, Mapp Biopharmaceutical, San Diego, CA, USA).\nThe focus of this review was on monoclonal antibodies and their combination considered as promising for the treatment of EVD in humans. In order to put their development into perspective, other interventions will be briefly discussed first.\n2.1. Vaccines\nThe two most promising candidates are chimpanzee adenovirus 3 vector vaccine expressing Zaire ebolavirus GP (ChAd3-EBO-Z) and a recombinant vesicular stomatitis virus (rVSV-ZEBOV) vaccine. Both vaccines showed so far acceptable safety and tolerability profiles, and high titers of surface and neutralizing antibodies were induced after vaccination [18\u201321]. An interim analysis of the rVSV-ZEBOV phase II/III trial in Guinea demonstrated a promising 100% efficacy [22]. Phase II/III clinical trials for both vaccines are ongoing [23].\n2.2. Favipiravir\nFavipiravir, originally developed for the treatment of severe influenza, is a virus RNA polymerase inhibitor, which has shown to be effective in the treatment of EVD in mice. From a nonrandomized trial in Guinea, no conclusions could be drawn regarding efficacy [24].\n2.3. TKM-100802/TKM-130803\nTKM-100802 is a small RNA-interfering (siRNA) molecule directed against the gene products encoding for two viral proteins: L polymerase, involved in transcription and replication of EBOV, and Viral Protein-35, involved in suppression of the host immune response [25]. Survival rate was 100% in a limited number of NHPs treated [26]. TKM-100802 is currently on partial clinical hold by the Food and Drug Administration (FDA) due to concerns about the occurrence of a cytokine release syndrome, a proinflammatory reaction mediated by activated immune cells, following administration of the drug [25]. TKM-130803 is a new formulation of TKM-100802 in which the siRNA has been adapted by three nucleotide substitutions to enhance specificity to the EBOV that caused the latest outbreak. A nonrandomized, single-arm, phase II trial conducted in 2015 in Sierra Leone failed to show improved survival compared to historical survival rates [25].\n\nEXPERT OPINION ON INVESTIGATIONAL DRUGS\n\n1327\n\n\u2122 Figure 1. Monoclonal antibodies for the treatment of Ebola virus disease. ZMapp and MIL-77E are composed of mAbs included in predecessor formulations\n(MB-003 and ZMAb). Adapted from Gonzalez-Gonzalez et al. [30].\n\n2.4. Convalescent plasma (polyclonal antibody therapy)\nThe rationale behind treatment with convalescent plasma is that EVD survivors have developed a protective immune response against the virus through generation of neutralizing antibodies and/or polyclonal Ebola-specific IgG that may or may not neutralize the virus but still may exhibit other protective mechanisms. Administration of these antibodies by transfusion could support virus clearance. Furthermore, the administration of these antibodies likely prevents virus infection/replication/dissemination within the patient, rather than virus clearance.\nAn important prerequisite to prevent infection with bloodborne diseases is that safety of virus-inactivated convalescent plasma transfusions is guaranteed, which can be challenging in EVD endemic areas [27]. Clinical trials were initiated in Guinea, Sierra Leone, and Liberia; without severe adverse reactions reported to date. While no efficacy data are available yet, experience from nonrandomized studies suggests safety, acceptability, and feasibility of convalescent plasma as EVD treatment [28]. Of note, in their concise overview on antibody therapeutics for EVD authored by the major leaders in the field of monoclonal antibody cocktail development [29], Zeitlin et al. present remarkable original data of an EVD patient treated sequentially with polyclonal antibodies in the form of convalescent plasma and then subsequently with monoclonal antibodies (ZMAb: see below); with the administration of the latter resulting in a dramatic increase in anti-Ebola IgG levels and a pronounced reduction of Ebola viral load, suggesting a synergistic effect.\n\ncombined) and MB-003, ZMab and ZMappTM (three antibodies combined, respectively; Figure 1).\n3. Monoclonal antibodies in the treatment of Ebola virus disease\n3.1. Introduction of the compounds\nMAb114 is a human monoclonal antibody, recently isolated from a survivor of the 1995 EVD outbreak in the Democratic Republic of Congo [31]. MB-003 and ZMAb are monoclonal antibody cocktails isolated from immunized mice. MB-003 contains three mouse\u2013human mAbs: 13C6, 6D8, and 13F6, which are IgG antibodies consisting of mouse variable regions chimerized with human constant regions and produced in tobacco plants (Nicotiana benthamiana). The mAbs were obtained from mice injected with Venezuelan equine encephalitis virus replicons encoding Ebola virus GP [32]. ZMAb contains three mAbs: 2G4, 4G7, and 1H3, which are murine immunoglobulins G (IgG) obtained from mice immunized with vesicular stomatitis virus (VSV), with the VSV GP gene replaced with one encoding Ebola virus GP\n[33,34]. ZMapp\u2122 is a cocktail of three chimeric mouse\u2013\nhuman mAbs produced in tobacco plants. Capitalizing on the two predecessor cocktails (Figure 1), it contains the mAbs 13C6, 2G4, 4G7 [35]. MIL77E is a chimeric antibody cocktail containing two mAbs (13C6 and 2G4 from\nZMapp\u2122) and is produced in engineered Chinese hamster\novary cells [36].\n\n2.5. Monoclonal antibodies\nAs comprehensively reviewed recently by Gonzalez-Gonzalez et al. [30]; up to date, around 20 monoclonal antibodies have been identified and characterized, of which some were found promising to progress to testing in nonhuman primate models. This included KZ52, isolated from a human survivor and successful in protecting mice and guinea pigs from lethal infection, but failing to protect NHPs when administered as single antibody [13].\nThe monoclonal antibodies, which emerged from testing in NHPs as promising for treatment of humans are subject to this review and are discussed in detail below. They include the single antibody mAb114; the mAb cocktails MIL77E (two mAbs\n\n3.2. Chemistry\nAll mAbs are IgG antibodies (Figure 1): large molecules, composed of four polypeptide chains, comprising two identical light chains and two identical heavy chains, forming a flexible Y-shaped structure. The two heavy chains are linked with each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. Each chain consists of a series of similar, although not identical, sequences, each about 110 amino acids long. Each of these sequences corresponds to a discrete, compactly folded protein domain region. The light chain of the IgG molecule is made up of two domains, whereas the heavy chain contains four [37].\n\n1328\n\nA. L. MOEKOTTE ET AL.\n\nMurin et al. [13] describe the specific structure of EVDprotective antibodies in detail and discuss them in relation to the specificities of the GP target; Gonz\u00e1lez-Gonz\u00e1lez et al. summarize the up-to-date knowledge on the structure and mode of action of mABs as well as the structure of the targeted Ebola virus GP [30].\n3.3. Pharmacodynamics\nMonoclonal antibodies bind GP, the only surface protein of Ebola virus, which plays a key role in virus attachment and fusion to host membranes. The individual mAbs each binds to one of three distinct regions; the GP base (where GP1 and GP2 interface), a glycan cap, or a mucin-like domain (Figure 2). MAbs binding to the GP base (4G7 and 2G4) are neutralizing in vitro, whereas antibodies binding the glycan caps (mAb114, 1H3, and 13C6) or the mucin-like domains (13F6 and 6D8) appear non-neutralizing in vitro [13], with the exception of mAb114 [31]. 13C6 and 6D8 do neutralize in presence of complement [32]. During Ebola virus entry, mucin-like\n\ndomains and glycan caps as well as their binding mAbs are removed before receptor engagement and therefore appear non-neutralizing; whereas the GP base with its binding mAbs remains intact before entry. MB-003 does not contain mAbs binding the GP base, but only mAbs binding domains that are cleaved off GP before viral infusion. ZMAb contains one mAb against the glycan cap and two against the GP base. Two mAbs (1H3 and 13C6) also bind sGP, the soluble version of GP, which is secreted from infected cells during EVD. It has been suggested that sGP acts as a decoy for antibodies, and therefore, mAbs which bind sGP may be less effective [13].\n3.4. Pharmacokinetics and metabolism\nTo date, the half-life of monoclonal antibodies remains unknown, as pharmacokinetics and metabolism have not been studied in detail.\n3.5. Clinical efficacy\nTo date, clinical trials on mAb114, MB-003, ZMAb, or MIL-77E have not been conducted. A phase I and phase I/II clinical trial\non ZMapp\u2122 are registered (Box 1). Available data are based\non in vitro studies and studies in NHPs, except for ZMAb and\nZMapp\u2122: four case reports are available describing compas-\nsionate use in humans with EVD. Table 1 provides a synopsis of existing in vivo studies in NHPs and humans. Table 2 summarizes all published efficacy study results for the different mAbs in NHPs. Table 3 provides an overview on biochemical studies and reviews to date.\n\n3.5.1. mAb114 MAb114 has in vitro activity against recent and previous outbreak variants of Ebola and monotherapy with mAb114 fully protected three rhesus macaques when given as late as 5 days after a challenge with a lethal dose of EBOV [31]. This study suggests that a single antibody has therapeutic potential. However, although all three rhesus macaques survived EVD with only mAb114, the authors also saw that animals given mAb114 alone at 1\u20133 dpi had high viremia and showed clinical signs, whereas a combination of mAb114 and mAb100 prevented viremia and clinical signs, and thus, it is currently still preferable to administer a mAb cocktail, and\n\nFigure 2. Viral entry and the effect of neutralizing antibodies.\n(a) Ebola expresses trimers of Glycopotein 1 (GP1) and Glycoprotein 2 (GP2) on its surface. GP1 is covered by a glycan cap and mucin like domain (MLD), both highly glycosylated regions, which cover conserved sites of receptor binding sites. After binding to a target cell receptor, Ebola virus particles the cell through macropinocytosis (II) or clathrin mediated endocytosis. They are then taken up in early endosomes (IV) and fuse with the endosome membrane to release viral RNA to allow for transcription, translation of viral proteins and viral replication (not shown in this figure, the lifecycle of Ebola is reviewed in more detail in Messaoudi et al. [11]. An infected cell releases both Ebola virus particles and glycoprotein dimers and monomers, which are thought to act as decoy targets for neutralizing antibodies, thereby diminishing the number of antibodies available for viral entry interference.\n(b) Neutralizing antibodies prevent viral entry into the target cell. Different antibodies bind to different sites of the glycoprotein. 2G4 and 4G7, which are part of ZMAb and ZMApp, bind to the GP base (I), 13C6, which is part of MB-003 and ZMApp, and 1H3, which is part of ZMAb, bind to the glycan cap (II), and 6D8 and 13F6, which are part of MB-003, bind to the mucin like domain (III). Glycoprotein structure and antibody binding are described in more detail in Murin et al. [13].\n\nBox 1. Drug summary\n\nDrug/molecule name\nPhase Indication\nPharmacology description/ mechanism of action\nRoute of administration Chemical structure Pivotal trial\n\nMB-0031, ZMAb1, MIL-77E1, mAb1141, and ZMappTM2\nPreclinical1, phase I/II clinical2 EVD or postexposure prophylaxis\nfollowing EBOV exposure Binding virus glycoprotein\nIntravenous IgG mAbs Phase I and phase I/II trials ongoing for\n\u2122 ZMapp (https://clinicaltrial.gov/ct2/\nshow/NCT02389192;https://clinicaltrial. gov/ct2/show/ NCT02363322 respectively)\n\nTable 1. Monoclonal antibody treatment in vivo studies.\n\nAntibody\n\ntreatment\n\nTitle\n\nAuthor\n\nPopulation\n\nObjective\n\nConclusion\n\nYear/ reference\n\nAnimal studies\n\nMAb114 Protective monotherapy against lethal Corti et al. Rhesus macaques\n\nIsolate mAbs from human EVD survivors and Monotherapy with mAb114 shows 100% survival in\n\nEbola virus infection by a potently\n\nidentify those who show protection as a\n\nNHPs when administrated as late as 5 days after\n\nneutralizing antibody\n\nsingle or dual-combination agent\n\nchallenge with EBOV\n\nMB-003\n\nDelayed treatment of Ebola virus infection Olinger et al. Rhesus macaques\n\nEfficacy of RAMP MB-003 vs. CHO MB-003, 75% and 83% survival; MB-003 administration 1 h p.i.\n\nwith plant-derived monoclonal\n\ngiven 1, 24, and 48 h p.i.\n\nderived from CHO and RAMP, respectively\n\nantibodies provides protection in rhesus\n\n67% survival 24 and 48 h p.i. with RAMP MB-003\n\nmacaques\n\nTherapeutic interventions of Ebola virus Pettitt et al. Rhesus macaques\n\nEfficacy of MB-003 in NHP administered after 43% survival with MB-003 administration after fever +\n\ninfection on rhesus macaques with the\n\nthe onset symptoms\n\nviremia\n\nMB-003 monoclonal antibody cocktail\n\nZMAb\n\nSuccessful treatment of Ebola virus-\n\nQiu et al.\n\nCynomolgus macaques Evaluate survival and immune response in 100% (4/4) and 50% (2/4) survival with ZMAb 25 mg/\n\ninfected cynomolgus macaques with\n\nEBOV-infected NHP treated with ZMAb\n\nkg administration i.v. at D3,6,9 beginning at 24 h\n\nmonoclonal antibodies\n\nand 48 h p.i., respectively\n\nmAbs and Ad-vectored IFN-\u03b1 therapy\n\nQiu et al.\n\nCynomolgus macaques Evaluate efficacy of ZMAb given simultaneously ZMAb + Ad-IFN simultaneously 75\u2013100% survival.\n\nrescue Ebola-infected NHP when\n\nwith Ad-IFN and ZMAb administration\n\nZMAb following Ad-IFN 50% survival\n\nadministered after the detection of\n\nfollowing Ad-IFN\n\nviremia and symptoms\n\nSustained protection against Ebola virus Qiu et al.\n\nCynomolgus macaques Evaluate whether NHP EVD survivors treated 100% and 67% survival when rechallenged 10 and\n\ninfection following treatment of\n\nwith ZMAb are still protected at subsequent 13 weeks after initial exposure, respectively\n\ninfected NHP with ZMAb\n\nexposure\n\n\u2122 ZMapp\n\nReversion of advanced Ebola virus disease Qiu et al.\n\nin nonhuman primates with ZMapp\n\nRhesus macaques\n\n\u2122 Identify the optimized antibody combination The optimized combination ZMapp (13C6, 2G4, 4G7)\n\nfrom MB-003 and ZMAb components and\n\nshowed 100% survival in NHP initiated up to 5 days\n\nMIL-77E\n\nTwo-mAb cocktail protects macaques against the EBOV Makona variant\n\nQiu et al.\n\nCase reports/\n\nRhesus macaques\n\ndetermine the therapeutic window\nConfirm whether CHO-produced MIL-77E\nshows the same efficacy as plant-produced\n\u2122 ZMapp and evaluate the impact of\nremoving mAb 4G7\n\np.i.\n\u2122 MIL-77E shows a comparable efficacy with ZMapp even when used as a 2 mAb cocktail. However,\ntreatment was initiated at day 3 compared to day 5\n\u2122 for Zmapp\n\nseries in\n\nhumans\n\nZMAb\n\nEbola virus disease complicated with\n\nPetrosillo et al. 1 human\n\nDescribe the course of EVD under optimized ZMAb administrating was associated with a sharp\n\ninterstitial pneumonia\n\ncondition\n\ndecay of plasma viral loads (twice)\n\nClinical features and viral kinetics in a Schibler et al. 1 human\n\nDescribe clinical, biological, and virological FU Rapid recovery, virus half-life decrease, but in the\n\nrapidly cured patient with EVD\n\nof EVD\n\ncontext of aggressive supportive care measures\n\n\u2122 ZMapp\n\nClinical care of two patients with Ebola Lyon et al.\n\nvirus disease in the United States\n\n2 humans\n\nReport the clinical course of EVD in two health- Improvement of both patients was observed shortly\n\ncare\n\n\u2122 after receiving ZMapp ; however, this occurred in\n\nworkers optimally treated in Emory\n\nthe context of receiving other care as well\n\nUniversity\n\nHospital\n\n2016 [31] 2012 [38] 2013 [39] 2012 [40] 2013 [41] 2013 [42] 2014 [35] 2016 [36]\n2015 [43] 2015 [44] 2014 [45]\n\nEXPERT OPINION ON INVESTIGATIONAL DRUGS\n\nRAMP: rapid antibody manufacturing platform; CHO: Chinese hamster ovary; p.i.: postinfection; NHP: nonhuman primate; EBOV: Ebola virus; mAbs: monoclonal antibodies; Ad-IFN: adenovirus-vectored interferon-\u03b1; EVD: Ebola virus disease; FU: follow-up; mAb: monoclonal antibody.\n\n1329\n\n\u2122 Table 2. MB-003a, ZMAba, ZMapp , MIL-77E, and mAb114 efficacy in NHPs.\n\nTreatment MAb114 MB-003 ZMAb (\u00b1Ad-IFN)\nZMapp\u2122\nMIL-77E\n\nChallenge virus EBOV EBOV EBOV\nEBOV\nEBOV\n\nChallenge dose Lethal doseb\n690 PFU\n1067 PFU 1000 PFU\n1000 PFU 1000 PFU 1000 PFU 2512 PFU 628 PFU\n1000 PFU\n\nNHP model (all macaques) Rhesus\nRhesus\nRhesus Cynomolgus\nCynomolgus Rhesus Cynomolgus Rhesus Rhesus\nRhesus\n\nTime of first administration\n24 h 120 h 24 h 48 h 103\u2013120 h 24 h 48 h 72 h 72 h 24 h (Ad5-IFN) and 96 h (ZMAb) 72 h 72 h 96 h 120 h 72 h\n\nNHP: nonhuman primate; EBOV: Ebola virus; PFU: plaque forming unit; Ad-IFN: adenovirus-vectored interferon-\u03b1. a Data as provided by Wong et al. [54] b Not defined in article. c NCT02363322\n\nDose\n50 mg/kg 50 mg/kg 50 mg/kg 50 mg/kg 50 mg/kg 25 mg/kg 25 mg/kg 50 mg/kg + 1x10\u2079 PFU Ad-IFN 50 mg/kg + 1x10\u2079 PFU Ad-IFN 50 mg/kg + 1x10\u2079 PFU Ad-IFN 50 mg/kg 50 mg/kg 50 mg/kg 50 mg/kg 50 mg/kg\n\nNumber of doses (days of treatment)\n2 (1, 2, 3) 3 (5, 6, 7) 4 (1, 5, 8, 10) 4 (2, 6, 8, 10) 3 (every 72 h) 3 (1, 4, 7) 3 (2, 5, 8) 3 (3, 6, 9) 3 (3, 6, 9) 3 (every 72 h) 3 (3, 6, 9) 3 (3, 6, 9) 3 (4, 7, 10) 3 (5, 8, 11) 3 (3, 6, 9)\n\nSurvival\n3/3 (100%) 3/3 (100%) 2/3 (67%) 2/3 (67%) 3/7 (43%) 4/4 (100%) 2/4 (50%) 3/4 (75%) 4/4 (100%) 2/4 (50%) 6/6 (100%) 6/6 (100%) 6/6 (100%) 6/6 (100%) 3/3 (100%)\n\nClinical trial No No No\nYesc\nNo\n\nA. L. MOEKOTTE ET AL.\n\n1330\n\nEXPERT OPINION ON INVESTIGATIONAL DRUGS\n\n1331\n\nTable 3. Antibody treatment reviews and biochemical studies.\n\nYear/\n\nTitle\n\nAuthor\n\nObjective\n\nConclusion\n\nreference\n\nReviews Ebola hemorrhagic fever Antibody therapeutics for Ebola\nvirus disease\n\nZeitlin et al.\n\nPostexposure therapy of Filovirus Wong et al. infections\n\nAnti-Ebola therapies based on Gonz\u00e1lez-Gonz\u00e1lez monoclonal antibodies: current et al. state and challenges ahead\nBiochemical studies Epitopes involved in antibody- Wilson et al\nmediated protection from Ebola virus Mechanism of binding to Ebola Davidson et al. virus GP by the ZMapp, ZMAb, and MB-003 cocktail antibodies\n\nStructures of protective antibodies reveal sites of vulnerability on Ebola virus\n\nMurin et al.\n\nDescribe the historic evidence for and Whole blood or plasma infusions unlikely to have an 2016 [29]\n\nagainst the use of AB in EVD\n\nimpact; whether mAbs will be effective remains\n\nto be determined\n\nSummarize and evaluate the available mAbs most promising. Survival 43\u2013100% depending 2014 [54]\n\nand experimental postexposure\n\non mAbs, time till administration and addition of\n\ntreatment of Filovirus\n\nAd-IFN\n\nDiscuss knowledge on antibody\n\nMAbs are promising, bottlenecks are costs and\n\n2015 [30]\n\ntherapy as well as challenges in the availability\n\nproduction of mAbs\n\n2000 [32]\n\nIdentify binding epitopes and their affinity, conservation, and accessibility for ZMapp, ZMAb, and MB-003\nIdentify epitopes and their mechanisms for the mAbs in ZMapp, ZMAb, and MB-003\n\nThe six different mAbs bind nonidentical epitopes. 4G7 and 13C6 are most effective based on their affinity, complementarity, and accessibility\nC1H3 and c13C6 compete for binding at the same site on GP\n\n2015 [33] 2014 [13]\n\nEVD: Ebola virus disease; Ad-IFN: adenovirus-vectored interferon-\u03b1; p.i.: postinfection; mAbs: monoclonal antibodies; AB: antibodies; GP: glycoprotein.\n\nfollow-up studies, to the best of our knowledge, have not been conducted to date.\n3.5.2. MB-003 Olinger et al. challenged eight rhesus macaques with a lethal dose of EBOV (690 plaque-forming units [PFU]). Initiation of treatment with MB-003 (50 mg/kg) started 24 and 48 h after virus challenge, with three animals in each treatment group. Two controls were treated with phosphate-buffered saline (PBS) or a control mAb. Animals received three additional doses (at day 5, 8, and 10 for the 24 h group and day 6, 8, and 10 for the 48 h group). In both groups, two animals survived with low viremia and showed no signs of illness, whereas both controls died on day 7. Hence, survival in this small group of subjects was 67% in the 24 h and the 48 h treatment groups, respectively, versus zero in controls [38].\nTo study the use as a therapeutic option rather than a postexposure prophylaxis, the same study group investigated whether MB-003 was effective after the onset of positive viremia (through reverse transcription polymerase chain reaction [RT-PCR]) and elevated temperature. They challenged nine rhesus macaques with an EBOV dose of 1067 PFU. Three of seven (43%) animals survived following treatment after symptom onset, in contrast to none of the two controls [39].\n3.5.3. ZMAb Qiu et al. studied the efficacy of ZMAb administrated 24 and 48 h after challenging cynomolgus macaques with a lethal dose of EBOV (1000 PFU). The four animals in each group were treated intravenously with three doses of ZMAb (25 mg/kg), each dose three days apart. One control received PBS. All four animals (100%) in the 24 h group survived; two of four animals (50%) in the 48 h group survived, whereas the one control did not (0%) [40]. The same study group tried to\n\nextend the treatment window of ZMAb by adding adenovirusvectored interferon-\u03b1 (Ad-IFN) to the treatment. In the first experiment, four cynomolgus macaques received three doses of ZMAb (50 mg/kg) intravenously at a 3-day interval, beginning 3 days after a lethal dose of 1000 PFU of EBOV. The animals were supplemented with Ad-IFN (1 \u00d7 109 PFU/kg) at receipt of the first dose of ZMAb. Three of the four animals survived the challenge with mild signs of disease; one had to be euthanized after a prolonged illness period. The second experiment focused on extending the treatment window. First, Ad-IFN was administered one day after EBOV challenge. Then, the ZMAb regime was started after onset of viremia and fever at day 4 in all four animals; two of them survived infection. In a third experiment, four rhesus macaques were treated with the same regime as the cynomolgus macaques in the first experiment (Ad-IFN and ZMAb initiated at day 3). In all four animals, viremia and fever higher than 40\u00b0C were detected before treatment. All four treated animals survived, whereas two controls (treated with PBS or control mouse IgG) succumbed to infection [41]. In another study, Qiu et al. performed an experiment with macaques surviving a previous EBOV challenge by receiving ZMAb or ZMAb and Ad-IFN. The animals were rechallenged 10 or 13 weeks after the initial challenge, respectively, to evaluate whether the immunity developed following the first infection was protective without further intervention. Survival rates were 100% and 67%, respectively [42]. Two cases of compassionate use of ZMAb in humans with EVD have been described. Petrosillo et al. describe the case of a physician with EVD who was evacuated to Italy for medical help. He was treated with convalescent plasma, favipavir, and ZMAb. Having received two doses of ZMAb, a sharp and sustained decline of plasma viral load was seen, with an increase of IgM and IgG after the start of viremia decline [43]. Schibler et al. reported a case of EVD in a medical doctor who was airlifted to Switzerland for medical care. The patient\n\n1332\n\nA. L. MOEKOTTE ET AL.\n\nreceived favipiravir and two doses of ZMAb (50 mg/kg). A temporal relation was seen between the ZMAb infusion combined with favipavir and an accelerated decay of viral load [44]. Both patients compassionately treated with ZMAb survived. Since both patients received additional treatment, the significance of the effect of the mAb cocktail cannot be conclusively established.\nTo the best of our knowledge, four additional EVD patients were treated compassionately with ZMAb, of which all survived [33].\n3.5.4. ZMappTM ZMappTM is the mAb cocktail, which evolved from MB-003 and ZMAb. It has shown a 100% efficacy in NHPs when administered up to 5 days after lethal challenge with EBOV. Multiple NHPs presented with severe stage of disease, indicated by the elevated liver enzymes, mucosal hemorrhages, and rash, at the time of administration [35]. A randomized, phase I/II safety and efficacy study is currently ongoing in Guinea, Sierra Leone, and the United States. No data on efficacy are available yet [17]. Several patients have been\ncompassionately treated with ZMapp\u2122. Lyon et al. describe\ntwo American health-care workers infected with EBOV who were transported to the USA and treated at Emory University in Atlanta. Both patients\u2019 conditions improved shortly after a\nfirst dose of ZMapp\u2122. Since this improvement occurred in\nthe context of aggressive rehydration, electrolyte balancing, and others supportive care measures, the significance of the effect of the mAb cocktail cannot be conclusively established [45].\n3.5.5. MIL77E MIL77E, containing 13C6 and 2G4, showed full protection in NHPs when administered 3 days after challenge with a lethal dose of EBOV [36]. No further studies have yet been conducted with this combination of monoclonal antibodies. Of note, 13C6:2G4 was given in a 1:2 ratio, respectively. For all other antibody cocktails mentioned previously (i.e. MB-003, ZMAb, ZMapp), the ratio was 1:1:1.\n3.6. Safety and tolerability\nIn the four primary NHP studies, there was no mentioning of adverse effects observed [39\u201342]. In the case report by Schibler et al., urticaria were observed on day 17 (ZMAb was given on days 5 and 8) likely due to a food allergy, although a delayed reaction to infusion of ZMAb cannot be excluded [44]. In the case report by Petrosillo et al., no adverse reactions were seen [43]. In the case series by Lyon et al., two patients\nwere treated with ZMapp\u2122, without adverse reactions [45].\n3.7. Development of cross-reactive antibodies\nMost of the currently investigated antibody cocktails are Ebola virus specific, and cross-neutralizing activity against Ebola viruses has not been demonstrated [46]. Ebola virus is responsible for the majority of filovirus hemorrhagic fever outbreaks, including the 2014 outbreak in West Africa. However, other members of the Filoviridae have also caused\n\nhuman epidemics, including seven outbreaks of Sudan virus, two outbreaks of Bundibugyo virus [47], and 12 outbreaks of Marburg virus [48]. Therefore, broadly protective treatment options are needed. Multiple mAbs have been generated, that elicit cross-reactivity in vitro and efficacy in mouse models for different Ebola virus species [49,50] including Marburg virus [51\u201353]. Furuyama et al. generated mAb 6D6, which was found to efficiently neutralize the infectivity of vesicular stomatitis virus (VSV) pseudo-typed with GPs of all known Ebola viruses (EBOV, SUDV, TAFV, BDBV, and RESTV), but not Marburg virus (MARV). In a mouse model of EBOV infection, 6D6 was 100% effective when given a single dose 24 h after challenge. A second mouse model of SUDV infection showed severe weight loss in untreated mice. Treatment with 6D6 24 h after infection delayed the onset of the disease and significantly reduced the weight loss [51]. Keck et al. created a set of pan-ebolavirus and pan-filovirus mAbs derived from cynomolgus macaques immunized repeatedly with a mixture of engineered GPs and virus-like particles (VLPs) for three different filovirus species (EBOV, SUDV, and MARV). The most protective mAb, FVM04 effectively neutralized EBOV and SUDV in vitro and showed weak binding to MARV. In a mouse model of EBOV infection, FVM04 was fully protective when used in two doses (2 h and 3 days postchallenge). When given a single dose 3 days postchallenge, survival rate was 40% [53].\n3.8. MAbs production techniques and capacity\nAnti-Ebolavirus mAB production is highly complex, and the currently available production facilities would not suffice to facilitate a quick large-scale outbreak response. Gonz\u00e1lezGonz\u00e1lez et al. [30] thoroughly examined the currently available and potential future techniques and highlight that firstly, MAbs required for successful treatment attempts may amount to as much as 10 g/patient; and that within a massive outbreak situation, MAbs needs may rapidly run up to many kilograms. Pointing out that with the current technique to produce MAbs from transfected tobacco plants; in order to produce, as an example, 50 kg of MAbs to allow for the treatment of maybe 5000 individuals, the authors calculate that 150 tons of tobacco leaves would have to be transfected. This is a dimension exceeding current facilities by far; apart from the fact that the MAbs recovery process from tobacco biomass poses its own formidable technical challenges [30].\nAn alternative to full-length MAbs production to be explored in the future could be a stable expression in mature tobacco plants, which would, even if technically feasible, require a considerable financial ad hoc effort and time to establish (which is precisely what is lacking in outbreak situations) or expression in mammalian cells, which appears theoretically feasible but would by far exceed the current global production capacity. A future option to be still thoroughly researched could be engineered mAb fragments in place of full-length MAbs, which could make it probably more easily into mass production [30].\n\nEXPERT OPINION ON INVESTIGATIONAL DRUGS\n\n1333\n\n4. Conclusions\nTwo promising vaccines are underway; however, mass immunization in the near future is unlikely, considering the costs and the amount of people at risk of getting EVD. Therefore, development of postexposure prophylaxis options or treatment for EVD is important. Monoclonal antibodies are currently the most promising experimental postexposure and treatment options against EVD, as evidenced by consistently higher rates of survival in lethally challenged NHPs [30,54].\nMonoclonal antibodies of particular interest are mAb114\nand ZMapp\u2122 which both showed to be 100% protective in\nNHPs when given as late as 5 days after exposure [31,35]. All\nEVD patients compassionately treated with ZMAb or ZMapp\u2122\nsurvived, and a clear decrease in viral load following infusion was seen. However, considering the multiple additionally applied and different supportive treatments, it remains unclear whether this effect is due to the antibodies alone [43\u201345]. The only antibody treatment currently undergoing\nclinical trial is ZMapp\u2122.\n5. Expert opinion\nPrevention and prophylaxis strategies, including early case detection, rapid diagnostic testing, and \u2013 hopefully soon \u2013 production and mass administration of vaccines will be key to prevent or at least curb future Ebola outbreaks. However, it goes without saying that the development of specific treatment options must not be neglected. As a consequence of the unprecedented large West African outbreak, research in this area has remarkably intensified recently.\nEvidence from small-scale animal studies and some limited evidence from individual cases support the view that both suitable single mAbs and mAb cocktails are effective for the treatment of human EBOV infections following exposure; thus justifying further preclinical development and testing in clinical trials.\nThe ultimate goals in this field are a comprehensive understanding of why, how, and to what extent monoclonal antibodies play an essential role in overcoming disease; the identification of the most potent full-length mAbs, and ideally engineering highly efficient fragments thereof; the recognition of (situation-specific) optimal compositions of mAb cocktails; on an operational level, the development of a framework enabling the accomplishment of clinical studies in the challenging conditions of an ongoing outbreak; and the facilitation of large-scale production to enable timely mass deployment once needed.\nRecent research by a small number of research groups leading the field has identified a multitude of mAbs effective in animal studies including nonhuman primates to facilitate survival of Ebola virus challenge; moreover, anecdotal evidence of individually treated patients has triggered the further clinical development of at least one full-length mAb cocktail. Over the past couple of years, insight into the viral GP target of the various mAbs as well as into their composition and role within the virus\u2013host interaction has massively grown, as well that techniques to accelerate and refine the further developments of mAbs for the treatment of EVD have been developed.\n\nWhile impressive progress has been made, knowledge on viral entry into host cells and the various mechanisms of Ebola virus to evade an efficient host immune response, as well as knowledge gaps on how mAb therapy precisely works is still incomplete.\nOver the coming years, next-generation full-length antibodies and designed fragments from the most suitable candidates will be identified and progress to testing in NHPs, as well as that the understanding of the immunological complexity of non-neutralizing and neutralizing protective antibodies will grow further.\nChallenges to move on with the development of Ebola therapies will remain multitude, however; including the potentially very high doses needed to yield clinical efficacy; epitope mutations which might reduce efficacy and require a constant evolution of (possibly outbreak-specific) mAb mixtures; as well as that monoclonal antibodies work best early after exposure, before the development of clinical disease, which poses a conceptual challenge and a common problem with antiviral therapy.\nAdequately designed phase I studies are needed to pave the way for eventual, sufficiently powered phase II and III trials. This is a major obstacle, in view of the fact that the disease manifests itself in outbreaks, with associated ethical and logistical difficulties in timely conducting randomized controlled clinical trials. As well, anti-Ebolavirus mAb production is highly complex, and the currently available production facilities are not sufficient to facilitate a quick large-scale emergency response.\nTo that end, the probably biggest overall challenge today may be to develop innovative and ethically acceptable ways of facilitating appropriately designed \u2018ad hoc\u2019 clinical trials to be initiated swiftly (and seen through) in outbreaks-to-come, and to stimulate investment into adequate production techniques and facilities for rapid upscaling beforehand.\nWhile more research data are pending, and in the light of high EVD mortality, an interim advice based on the clinical experience to date is that treatment of patients with mAbs is sensible, provided those could be made available timely in the quantities required. For the time being, however, there remains a clear discrepancy between the theoretical potential of MAbs for large-scale EVD treatment and today\u2019s experimental treatment options for very few individual study patients.\nFunding\nThis paper was not funded.\nDeclaration of interest\nThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.\nReferences\nPapers of special note have been highlighted as either of interest (\u2022) or of considerable interest (\u2022\u2022) to readers.\n1. World Health Organization. Ebola haemorrhagic fever in Zaire, 1976. Bull World Health Organ. 1978;56:271\u2013293.\n\n1334\n\nA. L. MOEKOTTE ET AL.\n\n2. Centers of Disease Control and Prevention (CDC). Outbreaks Chronology: Ebola virus disease. [cited 2015 Nov 11]. Available from: http://www.cdc.gov/vhf/ebola/outbreaks/history/chronology.html\n3. Saez AM, Weiss S, Nowak K, et al. Investigating the zoonotic origin of the West African Ebola epidemic. EMBO Mol Med. 2014;7:17\u201323.\n4. WHO Ebola response team. Ebola virus disease in West Africa - The first 9 months of the epidemic and forward projections. N Engl J Med. 2014;371:1481\u20131495.\n5. WHO situation report \u2013 28 April 2016. [cited 2016 May 22]. Available from: http://www.who.int/csr/disease/ebola/situationreports/archive/en/\n6. Negredo A, Palacios G, V\u00e1zquez-Mor\u00f3n S, et al. Discovery of an ebolavirus-like filovirus in europe. PLoS Pathog. 2011;7;e1002304.\n7. Kiley MP, Bowen ET, Eddy GA, et al. Filoviridae: a taxonomic home for Marburg and Ebola viruses? Intervirol. 1982;18:24\u201332.\n8. Kuhn JH, Becker S, Ebihara H, et al. Proposal for a revised taxonomy of the family Filoviridae: classification, names of taxa and viruses, and virus abbreviations. Arch Virol. 2010;155:2083\u20132103.\n9. Bharat TA, Noda T, Riches JD, et al. Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography. Proc Natl Acad Sci USA. 2012;109:4275\u20134280.\n10. Beniac DR, Melito PL, Devarennes SL, et al. The organisation of ebola virus reveals a capacity for extensive, modular polyploidy. PLoS ONE. 2012;7:e29608.\n11. Messaoudi I, Amarasinghe GK, Basler CF. Filovirus pathogenesis and immune evasion: insights from Ebola virus and Marburg virus. Nature Rev Microbiol. 2015;13:663\u2013676.\n12. Mehedi M, Falzarano D, Seebach J, et al. A new Ebola virus nonstructural glycoprotein expressed through RNA editing. J Virol. 2011;85:5406\u20135414.\n13. Murin CD, Fusco ML, Bornholdt ZA, et al. Structures of protective antibodies reveal sites of vulnerability on Ebola virus. Proc Natl Acad Sci USA. 2014;111:17182\u201317187.\n14. Feldmann H, Geisbert TW. Ebola haemorrhagic fever. Lancet. 2011;377:849\u2013862.\n15. Centers for Disease Control and Prevention (CDC). Bioterrorism agents/diseases. [cited 2016 Apr 29]. Available from: http://emer gency.cdc.gov/agent/agentlist.asp#e\n16. Grobusch MP, Visser BJ, Boersma J, et al. Ebola virus disease: Basics the medical specialist should know. Neth J Crit Care. 2015;22:6\u201314.\n17. World Health Organization. Ebola vaccines, therapies, and diagnostics - Questions and Answers. [cited 2016 Apr 29]. Available from: http://www.who.int/medicines/emp_ebola_q_as/en/\n18. Tapia MD, Sow SO, Lyke KE, et al. Use of ChAd3-EBO-Z Ebola virus vaccine in Malian and US adults, and boosting of Malian adults with MVA-BN-Filo: a phase 1, single-blind, randomised trial, a phase 1b, open-label and double-blind, dose-escalation trial, and a nested, randomised, double-blind, placebo-controlled trial. Lancet Infect Dis. 2016;16:31\u201342.\n19. Agnandji ST, Huttner A, Zinser ME, et al. Phase 1 trials of rVSV ebola vaccine in Africa and Europe. N Engl J Med. 2016;374:1647\u20131660.\n20. Regules JA, Beigel JH, Paolino KM, et al. A recombinant vesicular stomatitis virus ebola vaccine \u2013 preliminary report. N Engl J Med. 2015 Apr 1. [Epub ahead of print]. DOI: 10.1056/NEJMoa1414216\n21. Ewer K, Rampling T, Venkatraman N, et al. A monovalent chimpanzee adenovirus ebola vaccine boosted with MVA. N Engl J Med. 2016;374:1635\u20131646.\n22. Henao-Restrepo AM, Longini IM, Egger M, et al. Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trial. Lancet. 2015;386:857\u2013866.\n23. Sridhar S. Clinical development of Ebola vaccines. Ther Adv Vaccines. 2015;3:125\u2013138.\n24. Sissoko D, Laouenan C, Folkesson E, et al. Experimental treatment with favipiravir for ebola virus disease (the JIKI Trial): a historically controlled, single-arm proof-of-concept trial in Guinea. PLoS Med. 2016;13:e1001967.\n25. Dunning J, Sahr F, Rojek A, et al. Experimental treatment of ebola virus disease with TKM-130803: A single-arm phase 2 clinical trial. PLoS Med. 2016;13:e1001997.\n\n26. Geisbert TW, Lee ACH, Robbins M, et al. Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study. Lancet. 2010;375:1896\u20131905.\n27. Kreil TR. Treatment of Ebola virus infection with antibodies from reconvalescent donors. Emerging Infect Dis. 2015;21:521\u2013523.\n28. Van Griensven J, De Weiggheleire A, Delamou A, et al. The use of ebola convalescent plasma to treat ebola virus disease in resourceconstrained settings: a perspective from the field. Clin Infect Dis. 2016;62:69\u201374.\n29. Zeitlin L, Whaley KJ, Olinger GG, et al. Antibody therapeutics for Ebola virus disease. Curr Opin Virol. 2016;17:45\u201349.\n\u2022 Very concise overview on mAb cocktail development against EVD by one of the groups leading the field.\n30. Gonz\u00e1lez-Gonz\u00e1lez E, Alvarez MM, M\u00e1rquez-Ipi\u00f1a AR, et al. AntiEbola therapies based on monoclonal antibodies: current state and challenges ahead. Crit Rev Biotechnol. 2015;26:1\u201316.\n31. Corti D, Misasi J, Mulangu S, et al. Protective monotherapy against lethal Ebola virus infection by a potently neutralizing antibody. Science. 2016;351:1339\u20131342.\n\u2022 Monotherapy with mAB114 derived from plasma of a Kikwit 1995 EVD survivor protected a limited number of macaques when administered up to 5 days postchallenge.\n32. Wilson JA, Hevey M, Bakken R, et al. Epitopes involved in antibodymediated protection from Ebola virus. Science. 2000;287:1664\u2013 1666.\n33. Davidson E, Bryan C, Fong RH, et al. Mechanism of binding to ebola virus glycoprotein by the ZMapp, ZMAb, and MB-003 cocktail antibodies. J Virol. 2015;89:10982\u201310992.\n\u2022 This paper elucidates the mechanism of action of monoclonal antibody cocktails.\n34. Qiu X, Alimonti JB, Melito PL, et al. Characterization of Zaire ebolavirus glycoprotein-specific monoclonal antibodies. Clin Immunol. 2011;141:218\u2013227.\n35. Qiu X, Wong G, Audet J, et al. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature. 2014;514:47\u201353.\n\u2022\u2022 Demonstration that ZMapp emerging from MB-003 and ZMab cocktails resulted in a 100% survival rate in a limited number of EVD-infected macaques when treated within 5 days postchallenge.\n36. Qiu X, Audet J, Lv M, et al. Two-mAb cocktail protects macaques against the Makona variant of Ebola virus. Sci Transl Med. 2016;8:329\u2013333.\n\u2022\u2022 MIL-77E fully protects NHPs when administered 3 days postchallenge with a lethal EBOV dose.\n37. Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: the immune system in health and disease. 5th ed. New York: Garland Science; 2001.\n38. Olinger GG Jr, Pettitt J, Kim D, et al. Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques. Proc Natl Acad Sci USA. 2012;109:18030\u201318035.\n\u2022 60% of NHPs versus no controls survived EBOV infection with MB-003 administered up to 48 h postinfection.\n39. Pettitt J, Zeitlin L, Kim do H, et al. Therapeutic intervention of Ebola virus infection in rhesus macaques with the MB-003 monoclonal antibody cocktail. Sci Transl Med. 2013;5:199ra113.\n\u2022\u2022 EBOV-challenged NHPs survived when given MB-003 after onset of disease.\n40. Qiu X, Audet J, Wong G, et al. Successful treatment of ebola virusinfected cynomolgus macaques with monoclonal antibodies. Sci Transl Med. 2012;4:138ra81.\n\u2022\u2022 The authors demonstrate that EBOV-infected NHPs may develop strong specific immune responses and survive following administration of ZMAb and interferon after disease onset.\n41. Qiu X, Wong G, Fernando L, et al. mAbs and Ad-vectored IFN-\u03b1 therapy rescue Ebola-infected nonhuman primates when administered after the detection of viremia and symptoms. Sci Transl Med. 2013;5:207ra143.\n42. Qiu X, Audet J, Wong G, et al. Sustained protection against Ebola virus infection following treatment of infected nonhuman primates with ZMAb. Sci Rep. 2013;3:3365.\n\n\u2022\u2022 Of NHPs which survived EBOV disease when challenged and administered ZMAb; 100% and 60%, respectively, survived when rechallenged at week 10 or 13, respectively.\n43. Petrosillo N, Nicastri E, Lanini S, et al. Ebola virus disease complicated with viral interstitial pneumonia: a case report. BMC Infect Dis. 2015;15:432.\n44. Schibler M, Vetter P, Cherpillod P, et al. Clinical features and viral kinetics in a rapidly cured patient with Ebola virus disease: a case report. Lancet Infect Dis. 2015;15:1034\u20131040.\n45. Lyon GM, Mehta AK, Varkey JB, et al. Clinical care of two patients with Ebola virus disease in the United States. N Engl J Med. 2014;371:2402\u20132409.\n46. Nakayama E, Yokoyama A, Miyamoto H, et al. Enzyme-linked immunosorbent assay for detection of filovirus species-specific antibodies. Clin Vaccine Immunol. 2010;11:1723\u20131728.\n47. Chippaux JP. Outbreaks of Ebola virus disease in Africa: the beginnings of a tragic saga. J Venom Anim Toxins Incl Trop Dis. 2014;20:44.\n\nEXPERT OPINION ON INVESTIGATIONAL DRUGS\n\n1335\n\n48. Pigott DM, Golding N, Mylne A, et al. Mapping the zoonotic niche of Marburg virus disease in Africa. Trans R Soc Trop Med Hyg. 2015;109:366\u2013378.\n49. Furuyama W, Marzi A, Nanbo A, et al. Discovery of an antibody for pan-ebolavirus therapy. Sci Rep. 2016;6:20514.\n50. Flyak AI, Shen X, Murin CD, et al. Cross-reactive and potent neutralizing antibody responses in human survivors of natural ebolavirus infection. Cell. 2016;164:392\u2013405.\n51. Holtsberg FW, Shulenin S, Vu H, et al. Pan-ebolavirus and pan-filovirus mouse monoclonal antibodies: protection against ebola and Sudan viruses. J Virol. 2015;90:266\u2013278.\n52. Flyak AI, Ilinykh PA, Murin CD, et al. Mechanism of human antibodymediated neutralization of Marburg virus. Cell. 2015;160:893\u2013903.\n53. Keck ZY, Enterlein SG, Howell KA, et al. Macaque monoclonal antibodies targeting novel conserved epitopes within filovirus glycoprotein. J Virol. 2015;90:279\u2013291.\n54. Wong G, Qiu X, Olinger GG, et al. Post-exposure therapy of filovirus infections. Trends Microbiol. 2014;22:456\u2013463.\n\n\n",
    "authors": [
      "A. L. Moekotte",
      "M. A. M. Huson",
      "A. J. Van Der Ende",
      "S.T. Agnandji",
      "E. Huizenga",
      "A. Goorhuis",
      "M. P. Grobusch"
    ],
    "doi": "10.1080/13543784.2016.1240785",
    "date": "2016-11-01",
    "item_type": "journalArticle",
    "url": "https://www.tandfonline.com/doi/full/10.1080/13543784.2016.1240785"
  },
  {
    "key": "GK68N9KY",
    "title": "Ebola Virus Disease: Therapeutic and Potential Preventative Opportunities",
    "abstract": "The 2014 Ebola virus disease (EVD) epidemic in West Africa was unprecedented in its geographical distribution, scale, and toll on public health infrastructure. Standard public health measures were rapidly overwhelmed, and many projections on outbreak progression through the region were dire. At the beginning of the outbreak there were no treatments or vaccines that had been shown to be safe and e\ufb00ective for treating or preventing EVD, limiting health care providers to o\ufb00er supportive care under extremely challenging circumstances and at great risk to themselves. Over time, however, drugs and vaccines in the development pipeline were prioritized based on all available research data and were moved forward for evaluation in clinical trials to demonstrate safety and e\ufb03cacy. The armamentarium against EVD eventually included biologics such as monoclonal antibodies, convalescent plasma, and vaccines as well as small molecule therapeutics such as small interfering RNAs and nucleoside analogs. This article provides a high-level overview of the interventions and prophylactics considered for use in the outbreak and discusses the challenges faced when attempting to deploy investigational countermeasures in the midst of an evolving epidemic.",
    "full_text": "Ebola Virus Disease: Therapeutic and Potential Preventative Opportunities\nROBERT FISHER1 and LUCIANA BORIO2\n1Food and Drug Administration, Of\ufb01ce of Counterterrorism and Emerging Threats, Silver Spring, MD 20993; 2Food and Drug Administration, Of\ufb01ce of the Chief Scientist, Silver Spring, MD 20993\n\nDownloaded from https://journals.asm.org/journal/spectrum on 20 August 2024 by 2c0f:f6d0:26:2d:8551:cc20:4633:4ac.\n\nABSTRACT The 2014 Ebola virus disease (EVD) epidemic in West Africa was unprecedented in its geographical distribution, scale, and toll on public health infrastructure. Standard public health measures were rapidly overwhelmed, and many projections on outbreak progression through the region were dire. At the beginning of the outbreak there were no treatments or vaccines that had been shown to be safe and e\ufb00ective for treating or preventing EVD, limiting health care providers to o\ufb00er supportive care under extremely challenging circumstances and at great risk to themselves. Over time, however, drugs and vaccines in the development pipeline were prioritized based on all available research data and were moved forward for evaluation in clinical trials to demonstrate safety and e\ufb03cacy. The armamentarium against EVD eventually included biologics such as monoclonal antibodies, convalescent plasma, and vaccines as well as small molecule therapeutics such as small interfering RNAs and nucleoside analogs. This article provides a high-level overview of the interventions and prophylactics considered for use in the outbreak and discusses the challenges faced when attempting to deploy investigational countermeasures in the midst of an evolving epidemic.\nINTRODUCTION\nIn mid-1976 an outbreak of hemorrhagic fever was reported in southern Sudan and northern Zaire. Patients with the disease, which appeared \ufb01rst in southern Sudan in June 1976, presented with in\ufb02uenza-like symptoms including headache, fever, and myalgias and rapidly progressed to a more severe illness characterized by diarrhea, vomiting, chest pains, and hemorrhage. The disease was associated with a high mortality rate and was transmitted between close contacts of the severely ill, resulting in a substantial number of cases being linked to a local hospital (1). An outbreak of a disease\n\nwith similar symptoms was noted in northern Zaire beginning in September 1976, and by 24 October there were 280 deaths out of a total of 318 cases, for a case fatality rate of 88% (2). When samples derived from patients affected by the Sudan and Zaire outbreaks were used to infect Vero cells in culture, guinea pigs, or mice, a \ufb01lamentous virus similar to Marburg virus was observed (3, 4). Virus particles with a similar morphology were also identi\ufb01ed in postmortem liver samples from patients in Zaire (4, 5). Antigenic comparisons of the new virus isolates and Marburg demonstrated that while there was cross-reactivity between the Sudan and Zaire viruses, they were distinct from Marburg virus, and the new isolates were designated \u201cEbola virus\u201d (EBOV) after a river near the outbreak site in Zaire (5). Unlike Marburg virus, for which a single species has been described, at least \ufb01ve different species of the Ebolavirus genus exist (6). Zaire ebolavirus and Sudan ebolavirus are the species most frequently associated with human disease (7).\nReceived: 26 January 2016, Accepted: 8 February 2016, Published: 13 May 2016 Editors: W. Michael Scheld, Department of Infectious Diseases, University of Virginia Health System, Charlottesville, VA; James M. Hughes, Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA; Richard J. Whitley, Department of Pediatrics, University of Alabama at Birmingham, Birmingham, AL Citation: Fisher R, Borio L. 2016. Ebola virus disease: therapeutic and potential preventative opportunities. Microbiol Spectrum 4(3):EI100014-2016. doi:10.1128/microbiolspec.EI10-0014-2016. Correspondence: Robert Fisher, robert.fisher@fda.hhs.gov \u00a9 2016 American Society for Microbiology. All rights reserved.\n\nASMscience.org/MicrobiolSpectrum\n\n1\n\nFisher and Borio\n\nBefore the large 2014\u20132015 epidemic in West Africa, outbreaks of Ebola virus disease (EVD) had occurred with some frequency, but they have been largely limited to Western and Central Africa including Uganda, Sudan, Cote-d\u2019Ivoire, Gabon, the Republic of the Congo, and the Democratic Republic of the Congo, and most involved only a handful of cases (8). After the initial 1976 outbreak and prior to the 2014\u20132015 epidemic, three outbreaks stood out as exceptional due to high case numbers: the emergence of Ebola virus in Kikwit, Democratic Republic of the Congo, in 1995 (9), of Sudan virus in Uganda during 2000\u20132001 (10), and again of Ebola virus in the Democratic Republic of the Congo in 2007 (11).\nMembers of the Ebolavirus genus are \ufb01lamentous, enveloped viruses containing linear, nonsegmented \u223c19-kb single-stranded RNA genomes that encode seven genes and eight proteins (12, 13); due to RNA editing, the glycoprotein (GP) gene produces separate proteins for the virion envelope GP and a secreted glycoprotein (sGP) (14). While sGP has multiple pathophysiologic and immunomodulatory roles (15), GP is critical for virus binding and fusion, and as the sole surface protein on the intact virion it is an obvious target for vaccine and monoclonal antibody development. Other structural components of the virus such as VP24, VP40, and VP35 also antagonize the innate immune response (16), activities that may be restored by blocking the interaction of the viral proteins with their cellular targets. Finally, as a single-stranded RNA virus, the Ebola virus relies on an RNA-dependentRNA-polymerase (L) to transcribe the negative strand genome into monocistronic mRNAs for protein synthesis and a full-length antigenome as a template for replication. As might be expected, the polymerase represents a virus-speci\ufb01c target that can be exploited through the use of nucleoside analogs or knockdown strategies.\nMEDICAL COUNTERMEASURE DEVELOPMENT DURING THE 2014\u20132015 EPIDEMIC\nThe successful response to any epidemic involving a disease as contagious as Ebola must focus on controlling the spread of disease through the implementation of standard public health measures, such as identifying and isolating infected persons, tracing their contacts to detect secondary infections, protecting contacts and health care workers from exposure, and ensuring the safe burial of the deceased. However, applying these\n\npublic health measures on a large scale has presented complex challenges because of the limited public health infrastructure within most countries where Ebola outbreaks have occurred, and especially in West Africa when the epidemic emerged in 2014 (17). With 28,598 cases of EVD and 11,299 deaths as of 3 November 2015 (18), the unprecedented scale and speed of the Ebola epidemic in Guinea, Liberia, and Sierra Leone underscored the need for safe, effective, and rapidly deployable medical countermeasures (MCMs). These MCMs include diagnostic tests to assist in disease surveillance and case detection, vaccines to protect health care workers and help interrupt transmission, and drugs to improve the outcomes of infected patients.\nSigni\ufb01cant dif\ufb01culties in successfully implementing risk communication strategies were encountered, and health care systems and Ebola treatment centers in the affected West African countries rapidly became overwhelmed (19), leading to extremely pessimistic projections on the potential course of the epidemic (20, 21). These projections added to the urgency of conducting properly designed clinical trials to evaluate a number of investigational MCMs still in early stages of development. The sooner one could establish whether an investigational MCM was safe and effective for the treatment or prevention of EVD, the sooner it could be incorporated into the response to the public health emergency (22, 23). One factor limiting the prompt evaluation of some investigational drugs was their availability, since most of the more promising antiviral candidates had been produced only in limited quantities for early development purposes. Convalescent blood and/or plasma was the exception to this rule, but the infrastructure to safely collect, store, and use this resource was not extant in West Africa until November 2014 (24), and the \ufb01rst clinical trials of Ebola therapeutics in West Africa were initiated in December 2014 (25).\nSeveral therapies had been investigated in vitro or in animal model systems over the years that preceded the 2014 epidemic. However, at the beginning of the West African epidemic there were no treatments or vaccines that had been shown to be safe or effective for treating or preventing EVD. Clinicaltrials.gov documents only \ufb01ve studies of \u201cEbola treatments\u201d (as compared to vaccines) prior to March 2014; these were all phase 1 safety studies (26), and of these, one was being developed for postexposure prophylaxis, not treatment. Of the four phase 1 safety studies for drugs with treatment indications, only one was completed, while the others were either withdrawn or terminated.\n\nDownloaded from https://journals.asm.org/journal/spectrum on 20 August 2024 by 2c0f:f6d0:26:2d:8551:cc20:4633:4ac.\n\n2\n\nASMscience.org/MicrobiolSpectrum\n\nEbola Virus Disease: Therapeutic and Potential Preventative Opportunities\n\nAs the epidemic progressed, a number of different compounds, most of which had at least some activity demonstrated in animal models, were selected for evaluation in clinical trials. Product classes considered for treatment of EVD included both biologics (monoclonal antibodies and convalescent plasma) and drugs (small interfering RNAs [siRNAs], nucleoside analogs, and others).\nSupportive Care\nSupportive care was key to improving patient outcomes and is expected to remain a central component of the care of acutely ill patients, even in the setting of a proven speci\ufb01c treatment. When possible, volume replacement of patients was an important component due to intravascular depletion resulting from emesis, voluminous diarrhea, and \ufb02uid shifts within the body that deplete the intravascular space. Antiemetics and antidiarrheals were administered when possible, as were empiric antibiotics because of possible bacteremia from transmigration of Gram-negative bacteria from the gut (27, 28). Antimalarials were also commonly administered because many patients admitted to Ebola treatment units had concomitant malaria infections. Diazepam for sedation and morphine for analgesia (29) were also commonly used. Even when suf\ufb01cient medication and supplies were available, patient care suffered in the initial months because demand for health care personnel greatly exceeded what was available in affected areas (30, 31). Despite these challenges, there appeared to be an increasing level of care in West Africa over the course of the epidemic (32), with ensuing improved outcomes in the later months of the epidemic.\nSupportive care delivered in developed settings, such as Europe and the United States (33, 34) also proved to be demanding. In contrast to the standard of care in West Africa, however, extensive physiological monitoring and aggressive medical interventions were available for patients admitted or evacuated to these facilities (35). A patient treated in Germany for severe EVD required blood products, ventilation, vasopressors, antibiotics and antifungals, and hemodialysis. In addition to general supportive measures, investigational interventions were provided to most patients in Europe and the United States (36), although the effects of these interventions (bene\ufb01cial or harmful) could not be assessed. Not all patients needed intensive levels of support; some patients, presumably those with less severe disease, rapidly recovered after receiving only minimal support, such as intravenous \ufb02uids (37).\n\nTherapeutics\nTwo American health care workers developed EVD while caring for patients in West Africa and were evacuated to the United States for medical care. In addition to a high level of supportive care, they also received the investigational compound ZMapp (35). Both patients survived, and although no conclusion of safety or ef\ufb01cacy could be drawn from the use of the investigational compound under these circumstances, there were widespread calls for access to early-stage investigational candidates such as ZMapp. An ethics panel advising the WHO indicated that while it was ethical to provide therapies with unproven safety and ef\ufb01cacy pro\ufb01les, \u201cinvestigators have a moral duty to evaluate these interventions (for treatment or prevention) in the best possible clinical studies that can be conducted under the circumstances of the epidemic\u201d so that effective therapies could be rapidly identi\ufb01ed (38).\nClinical trials for promising treatments were put into place as rapidly as possible, but assembling the infrastructure for conducting trials took some time. In the interim, the use of investigational candidates in the United States, Europe, and West Africa continued on a case-by-case basis under what is colloquially referred to as \u201ccompassionate use\u201d; in the United States such use is one type of expanded access that can be permitted under an investigational new drug application (39). This type of use of experimental intervention is not designed to generate conclusions about the safety or ef\ufb01cacy of the investigational drugs being used, given the lack of appropriate comparator groups. Unfortunately, even after clinical trials were established, access to investigational agents outside of clinical trials was advocated for by some organizations as a stop-gap measure, even though this delayed the gathering of interpretable data that would allow the most ef\ufb01cient identi\ufb01cation of bene\ufb01cial treatments or the rapid discontinuation of harmful therapies.\nAntibodies ZMAPP A series of monoclonal antibodies targeting EBOV GP was developed by researchers at the U.S. Army Medical Research Institute for Infectious Diseases, and several were demonstrated to be protective in mouse models (39). Three of these antibodies (13F6, 13C6, and 6D8) were modi\ufb01ed to deimmunize (13F6) and/or chimerize (all three antibodies) through the addition of a human Fc region. The mixture of these three antibodies (MB-003) was found to be effective in a mouse challenge model even when administered as late as 48 hours post-EBOV\n\nDownloaded from https://journals.asm.org/journal/spectrum on 20 August 2024 by 2c0f:f6d0:26:2d:8551:cc20:4633:4ac.\n\nASMscience.org/MicrobiolSpectrum\n\n3\n\nFisher and Borio\n\ninfection (40, 41). A parallel effort led by the Public Health Agency of Canada also evaluated mixtures of monoclonal antibodies directed against EBOV GP and demonstrated that a combination of three murine monoclonals (1H3, 2G4, and 4G7; ZMAb) were also effective as a postexposure intervention in mice and guinea pigs (42). MB-003 conferred a survival bene\ufb01t compared to placebo when dosed after the onset of fever in rhesus macaques challenged with EBOV (43), while ZMAb protected cynomolgus macaques from an EBOV challenge when treatment was initiated 48 hours postinfection (44).\nThe realization that a cooperative effort would bene\ufb01t both groups led to a collaboration that examined whether an optimized cocktail could be formulated from the individual components of ZMAb and MB-003. As a result, it was demonstrated that a mixture of c13C6 (from MB-003) and c2G4 + c4G7 (humanized versions of two ZMAb components) provided protection even when treatment was delayed to 5 days postinfection in the rhesus macaque EBOV challenge model (45). The combination of c13C6, c2G4, and c4G7 was trademarked as \u201cZMapp\u201d by MappBio and advanced as a candidate therapy for human EVD in January 2014 (http://mappbio.com/z-mapp/). ZMAb was used in at least two patients in Europe, while ZMapp was used in at least nine EVD patients (46) in the United States, Europe, and West Africa prior to the establishment of a randomized, controlled clinical trial in the United States and West Africa designed to evaluate the safety and ef\ufb01cacy of this investigational product in adults and children infected with EBOV (47). This trial, launched in February 2015 (48), has enrolled approximately 70 research participants at the time of this writing.\nAlthough preliminary results with ZMapp in animal models have been promising and led to its prioritization for evaluation in clinical trials, the plant-based production system created a bottleneck that prevented timely increases in production. Initially, the ability to rapidly move the ZMAb components from a hybridoma platform to one more suitable for preparation of clinical material was considered a major advantage (49), and the use of transgenic Nicotiana benthamiana plants improved glycosylation and, thus, antibody-dependent cytotoxicity of the antibodies (41). Unfortunately, only a handful of treatment courses were available at the onset of the West African outbreak, and these were depleted by mid-August 2014 (50). While funding was rapidly made available through the Biomedical Advanced Research and Development Authority (BARDA) (46), the throughput of the facility producing the drug substance\n\nwas extremely limited and was expected to produce only an additional 10 to 20 treatment courses by the end of 2014 (51).\nMIL-77 Produced by a Chinese company, MabWorks, MIL-77 is a cell-derived monoclonal cocktail similar to ZMapp. MIL-77 was developed using the sequence information referenced in the ZMapp patents, raising intellectual property concerns (52). It was \ufb01rst administered to a United Kingdom medic who subsequently recovered from EVD. however, the ef\ufb01cacy could not be attributed to MIL77 (53) in the absence of a properly designed trial, because some patients with EVD recover, especially in the setting of advanced supportive care. According to the WHO, MIL77 is undergoing phase 1 safety trials in China (54).\nConvalescent blood and plasma Largely based on its use during prior outbreaks and the expected availability of suitable donors, in September 2014 the WHO issued a guideline recommending the use of convalescent whole blood or plasma for treatment of patients with early EVD, despite the lack of conclusive evidence of its effectiveness (55) . In their guideline, the WHO noted the lack of a proven treatment for EVD and cited previous use of convalescent material to treat Ebola and other infectious diseases. Interestingly, the collection of convalescent plasma was identi\ufb01ed as a priority for the WHO team responding to the \ufb01rst Ebola outbreak in 1976 (1), and units thus collected were administered to at least two patients during the 1976 outbreak (2).\nEarly administration of convalescent serum or plasma was also documented in a 1976 case involving a researcher at Porton Down who was accidently infected (57). The researcher, who survived, received interferon for 2 weeks plus heat-treated convalescent serum on day 3 and day 6. While there was a decrease in viremia after the \ufb01rst infusion of serum, the decrease cannot be attributed to the serum infusion; viremia decreases in all surviving patients at some time even in the absence of speci\ufb01c treatments. During the 1995 Kikwit outbreak, whole blood from Ebola disease survivors was administered to eight patients (58). There was no clear association between the volume administered or time of administration and survival, and it was noted that a controlled trial would likely be necessary to assess any treatment effect (59).\nThe results from animal studies evaluating the activity of convalescent plasma or other anti-Ebola hyperimmune material have been variable. An equine IgG\n\nDownloaded from https://journals.asm.org/journal/spectrum on 20 August 2024 by 2c0f:f6d0:26:2d:8551:cc20:4633:4ac.\n\n4\n\nASMscience.org/MicrobiolSpectrum\n\nEbola Virus Disease: Therapeutic and Potential Preventative Opportunities\n\npreparation was successful in delaying viremia, clinical signs, and death in a postexposure prophylaxis model using 1,000 plaque forming units of Ebola Zaire challenge in cynomolgus macaques but did not confer a survival bene\ufb01t compared to placebo (60). In contrast, baboons challenged with a lower dose of Ebola Zaire (10 to 30 50% lethal doses) were protected if an equine hyperimmune product similar to the one described above was administered within 60 minutes of infection. No bene\ufb01cial effect was observed in rhesus macaques receiving whole blood with a high titer of anti-Ebola antibodies (measured by ELISA) immediately after an Ebola Zaire challenge (56). Further complicating the overall interpretation are data indicating that neutralizing antibodies protective in one species may not be ef\ufb01cacious in another (61), and protection may correlate with total anti-Ebola IgG titers and not necessarily with neutralizing antibody titers (62). Antibody-dependent cellular cytotoxicity has been proposed as an important mechanism for ef\ufb01cacy of anti-Ebola monoclonals (41, 63) but has not been investigated in the context of the polyclonal nature of convalescent serum or plasma administered for Ebola disease.\nUse of convalescent blood or plasma is not without risks, because it may result in transfusion reactions including transfusion-related acute lung injury (TRALI), hemolytic reactions, anaphylaxis, circulatory overload, and transfusion-transmitted infections. Indeed, acute respiratory distress consistent with TRALI was reported in a patient with EVD who received convalescent plasma and favipiravir on day 10 of her illness (37).\nWhen designing a study to evaluate the ef\ufb01cacy of immune plasma, including a nonimmune control arm in a randomized, controlled trial is important for addressing the question of whether it is the immune component of convalescent plasma or other attributes of infusing plasma such as the hemodynamic support from an infusing \ufb02uid and protein and providing clotting factors that is responsible for the observed effects. Another factor to consider is that if immune plasma is collected from vaccine recipients rather than people who have had Ebola infection, the characteristics of the vaccine-generated hyperimmune plasma may differ from plasma collected from people who have recovered from Ebola infection. Further characterization of the similarities and differences in antibodies generated in response to natural infection and vaccination and preclinical studies may provide insights into similarities and differences and the possible clinical implications.\nThree phase 2/3 uncontrolled clinical trials were initiated in West Africa with convalescent plasma, one each\n\nin Guinea, Liberia, and Sierra Leone (24). The Liberia study was discontinued due to the decline in cases in the country, but as of 19 June 2015, 101 patients had received plasma in the Guinea Ebola-Tx study (54). The investigators for the Guinea convalescent plasma trial, a historically controlled trial, reported that transfusion of convalescent plasma with unknown levels of neutralizing antibodies in 84 patients with con\ufb01rmed EVD was not associated with a signi\ufb01cant improvement in survival (64). The signi\ufb01cant limitations of the Guinea convalescent plasma study (a historically controlled trial using plasma with unknown levels of neutralizing antibody) limit the ability to draw any de\ufb01nitive conclusions about the role of convalescent plasma to treat patients with Ebola. Convalescent plasma was also used under an emergency investigational new drug application for several cases of EVD in the United States prior to the establishment of the randomized, controlled clinical trial to evaluate Ebola therapies (65\u201367).\nSmall molecules AVI-7537, AVI-7539, and AVI-6002 (Sarepta) In 2010 the U.S. Department of Defense\u2019s Joint Project Manager Transformational Medical Technologies program awarded a contract to AVI BioPharma (now Sarepta Therapeutics) to advance their antisense-based PMOplus chemistry for therapeutics against Ebola and Marburg, building upon earlier investments in the company from the Department of Defense\u2019s Defense Threat Reduction Agency (68). Antisense-based therapies function by binding a complementary nucleic acid strand to the mRNA encoding for a target viral protein and were \ufb01rst described nearly 40 years ago (69). Af\ufb01nity, cellular penetration, and stability in the presence of ubiquitous nucleases presented an initial challenge, but the development of chemically modi\ufb01ed oligonucleotides made clinical development possible (70). In 2006 phosphorodiamidate morpholino oligomers (PMOs) designed to target EBOV VP24, VP35, and L were demonstrated to provide pre-exposure prophylaxis against an EBOV challenge in rhesus macaques (71); a mixture of VP24 (AVI-7537)\u2013 and VP35 (AVI-7539)\u2013directed PMOs (this combination is referred to as AVI-6002) provided protection against EBOV infection in an animal model of postexposure prophylaxis (72). AVI-6002 was advanced into a phase 1 study, where it was found to be well tolerated, albeit with a short plasma half-life (2 to 5 hours) (73). Further research indicated that this mixture of PMOs could be narrowed to a single oligomer (VP-7537) targeting EBOV VP24 and still retain potency in the rhesus challenge model (74). To date,\n\nDownloaded from https://journals.asm.org/journal/spectrum on 20 August 2024 by 2c0f:f6d0:26:2d:8551:cc20:4633:4ac.\n\nASMscience.org/MicrobiolSpectrum\n\n5\n\nFisher and Borio\n\nthere are no published data on use of AVI-7537, AVI7539, or AVI-6002 for treatment of EVD in humans, and as of 5 November 2015, further development of these compounds was in question, reportedly due to a lack of funding and intellectual property limitations (75, 76).\nBCX4430 (Biocryst) BCX4430 is a synthetic nucleoside analog of adenosine and has in vitro activity against RNA viruses in many families (including Filoviridae), as might be expected from its mechanism of action as an RNA chain terminator. This compound is active against EVD and Marburg disease in murine models of pre- and postexposure prophylaxis and protects guinea pigs from Marburg infection when dosed as late as 72 hours postinfection. Similarly, six of six cynomolgus macaques survived a challenge with Marburg when treated with 15 mg/kg BCX4430 twice a day starting at 48 hours postchallenge (77). Biocryst was awarded a 2013 National Institute of Allergy and Infectious Diseases contract to develop BCX4430 for treatment of Marburg disease and to investigate the compound\u2019s utility for treating EVD (78). Phase 1 testing started in December 2014 and is ongoing (79); in March 2015 BARDA awarded Biocryst a contract for advanced development including clinical trials and large-scale manufacturing (80).\nFavipiravir (T-705; Toyama Chemical) Favipiravir is a pyrazinecarboxamide derivative that appears to have at least some activity against a number of viruses through the inhibition of RNA-dependent RNA polymerase (81). Approved by the Japanese Ministry of Health, Labor, and Welfare in March 2014 (https://www.toyama-chemical.co.jp/eng/news/news 140324e.html), the compound has been stockpiled in Japan for use against pandemic in\ufb02uenza (http://www .fuji\ufb01lm.com/news/n150722.html). It has demonstrated activity postexposure in animal models of infection against a variety of pathogenic RNA viruses beyond in\ufb02uenza including West Nile virus (82), yellow fever virus (83), Lassa virus (84), and EBOV (85, 86). It has been administered prophylactically (87) to several individuals with EVD (37, 88\u201390). An open label, historically controlled study of favipiravir in patients with EVD (91) (the JIKI trial; NCT02329054) was sponsored by INSERM in Guinea. Based on an interim analysis of 69 patients, the authors suggest possible bene\ufb01t in the subset of patients who present to care with lower EBOV viral load (92). However, limitations in the study design as well as improvements in supportive care over the course of the epidemic (32) preclude drawing any\n\nmeaningful conclusions about its role in the treatment of patients with EVD.\nTKM-Ebola (TKM-100802; Tekmira) and TKM-Ebola-Guinea TKM-Ebola is a lipid-stabilized siRNA targeting EBOV VP35 and polymerase (93), while TKM-Ebola-Guinea is a version of TKM-Ebola modi\ufb01ed to remove mismatches in the EBOV Makona variant (http://investor.arbutusbio .com/releasedetail.cfm?releaseid=907998). The use of nucleic acid\u2013lipid particles, whose development was funded in part by the U.S. Department of Defense, had been demonstrated to be an effective postexposure therapeutic in guinea pigs and nonhuman primates, although different viral targets were examined in each study: an siRNA against L was used in the guinea pig studies, and a combination of siRNAs against VP24, VP35, and L was used in the nonhuman primate studies (94, 95). TKM-Ebola was used on an infected French M\u00e9decins Sans Fronti\u00e8res nurse (96) and administered to two U.S. patients under an emergency investigational new drug application (66). In December 2014 Tekmira partnered with the University of Oxford and the Wellcome Trust to perform a phase 2, single-arm clinical ef\ufb01cacy trial using TKM-Ebola-Guinea in Sierra Leone (http://www .sec.gov/Archives/edgar/data/1447028/0001171843140 06000/newsrelease.htm), but the study reportedly was terminated in June 2015 after reaching a statistical futility boundary (97). Differences in outcomes between animal EVD models and human cases of EVD highlight the dif\ufb01culty in extrapolating to humans data derived from animal studies. As of August 2015 clinical development work for TKM-Ebola has been terminated (98), and Tekmira Pharmaceuticals has changed its corporate name to Arbutus Biopharma and suspended work on \ufb01loviruses to concentrate on chronic hepatitis B (http:// investor.arbutusbio.com/releasedetail.cfm?ReleaseID= 925130).\nGS-5734 (Gilead) A late breaker abstract session at the 2015 ID Week Conference described a prodrug of an adenine nucleotide analog that was effective at inhibiting growth of multiple \ufb01loviruses in cell culture (99). The presumptive target is the \ufb01lovirus polymerase; a surrogate RNA polymerase was inhibited by GS-5734 with a 50% inhibitory concentration value of 1 \u03bcM. A survival bene\ufb01t was demonstrated above that of placebo (50% survival in GS-5734-treated animals versus 0% survival in placebo-treated animals) when GS-5734 was administered to EBOV-infected rhesus macaques with systemic\n\nDownloaded from https://journals.asm.org/journal/spectrum on 20 August 2024 by 2c0f:f6d0:26:2d:8551:cc20:4633:4ac.\n\n6\n\nASMscience.org/MicrobiolSpectrum\n\nEbola Virus Disease: Therapeutic and Potential Preventative Opportunities\n\nviremia. The compound was administered to a patient with Ebola-related meningitis (100), but the contribution of GS-5734 to her recovery remains unknown.\nVaccines\nVSV-ZEBOV\nThe recovery of recombinant vesicular stomatitis virus (VSV) from cells transfected with DNA plasmids was pioneered by Lawson et al. in 1995 and provided an ideal mechanism for rapidly growing large stocks of high-titer recombinant virus carrying foreign genes that could be used as a vaccine (101). This platform was used to generate VSV\u0394G/ZEBOVGP, a live-virus vaccine created by substituting EBOV GP for the GP normally present in VSV (102). A single intramuscular injection of VSV\u0394G/ZEBOVGP vaccine induced both humoral and cellular immunity and was effective at preventing EVD in cynomolgus macaques (103) challenged a month after vaccination. Protection was also observed in cynomolgus macaques challenged with the West African EBOV Makona strain as early as 3 days postvaccination (104). The VSV-based vaccine was effective in postexposure prophylaxis animal models of infection in mice, guinea pigs, and to a lesser degree, rhesus macaques (105). This vaccine, now called VSV-EBOV, has been administered as postexposure prophylaxis to a physician who had a high-risk potential exposure to EBOV as the result of a needlestick (106). VSV-EBOV was also offered as a prophylactic measure to close contacts of the Scottish Ebola patient who experienced a recurrence of symptoms.\nVSV-EBOV, originally developed by the Public Health Agency of Canada, was licensed by NewLink Genetics in 2010 and entered advanced development with support from the Department of Defense (http://investors.linkp .com/releasedetail.cfm?ReleaseID=864161) with a phase 1 study initiated in late 2014 (http://investors.linkp.com /releasedetail.cfm?ReleaseID=869082). Responding in part to concerns about scaling up production and overcoming testing delays, NewLink granted exclusive rights to VSV-EBOV to Merck (107), and BARDA provided additional funding to support manufacturing (108). VSV-EBOV elicits neutralizing antibodies and was well tolerated in healthy volunteers (109), and it is currently being evaluated in three ongoing clinical trials in West Africa: the PREVAIL, STRIVE, and Ebola \u00e7a Suf\ufb01t trials. The dose of VSV-EBOV used in these trials (2 \u00d7 107 plaque forming units) is consistent with that used in the rhesus and cynomolgus macaque challenge studies (1 \u2212 5 \u00d7 107 plaque forming units [103\u2013105]). PREVAIL is a three-arm, double-blind, randomized phase 2 clinical\n\nstudy to compare the safety and ef\ufb01cacy of VSV-EBOV and ChAd3-EBOZ to placebo in the general population in Liberia (110). STRIVE also seeks to evaluate safety and ef\ufb01cacy, although the target population is composed of health care workers in Sierra Leone. The trial design also differs in that it is unblinded and participants are randomized to immediate vaccination or deferred vaccination (approximately 6 months later [111]). Safety and immunology results are pending for the PREVAIL and STRIVE studies, but the rapid decline in EVD cases in West Africa as the trials were ramping up in early 2015 will require ef\ufb01cacy assessments to be based on immunogenicity instead of disease.\nThe Ebola \u00e7a Suf\ufb01t study utilizes an open-label ring vaccination strategy, where close contacts of Ebola patients are clustered into an epidemiologically de\ufb01ned \u201cring,\u201d and each ring is randomized to either immediate vaccination with VSV-EBOV or to receive vaccination 2 weeks later, with a 1:1 ratio between the study arms (112). This design allows estimation of vaccine ef\ufb01cacy by comparing the hazard ratio between the two groups. As of November 2015 there are promising results from this trial (113), although there are concerns that the interim analysis may overestimate ef\ufb01cacy in the ring vaccination strategy. Speci\ufb01c concerns identi\ufb01ed include failing to meet the pre-established statistical test of ef\ufb01cacy between study arms and an analytical bias due to population differences between the study arms (114).\nChAd3-EBOZ\nAnother approach for recombinant vaccines is based on the use of replication-defective adenovirus (Ad) expressing the antigen of interest. Like VSV, Ad vectors can be grown to high titers and induce a strong immune response, especially when used as the boost component in a heterologous prime-boost system; an EBOV nucleoprotein/glycoprotein (NP/GP) DNA prime/Ad-GP boost vaccine protected cynomolgus macaques against a low-dose EBOV challenge (115). However, this approach required multiple priming immunizations, which presents considerable logistical problems for effective deployment of a vaccine. An accelerated vaccination strategy was investigated, where the DNA prime was abolished and Ad vectors expressing EBOV GP or NP were used concomitantly. A single injection of the Ad-GP/Ad-NP mixture was suf\ufb01cient for protection against a high-challenge dose of EBOV in the cynomolgus macaque model, and a comparison of the CD4/ CD8 response pre- and postchallenge suggests that a CD8 response is important in mediating protection for this vaccine (116). Additional research indicated\n\nDownloaded from https://journals.asm.org/journal/spectrum on 20 August 2024 by 2c0f:f6d0:26:2d:8551:cc20:4633:4ac.\n\nASMscience.org/MicrobiolSpectrum\n\n7\n\nFisher and Borio\n\nthat the NP component was unnecessary, and Ad-GP alone could provide robust protection in the cynomolgus macaque model (117) and also depended upon CD8 cells (118). To avoid ubiquitous pre-existing immunity to human adenovirus (which had been demonstrated to impact humoral responses to the rAd5 vaccine [119]), the vaccine backbone was changed to a chimpanzee Ad 3 (Ch3Ad). A single immunization with 1011 particles of this construct was successful at protecting 50% of cynomolgus macaques when challenged 10 months postvaccination. GlaxoSmithKline (GSK) and the National Institutes of Health partnered to move this vaccine candidate into clinical trials, and a phase 1 study found that while the vaccine was well tolerated, the magnitude of the immune response was less in humans than in the nonhuman primate models (120). As mentioned above, ChAd3-EBOZ is currently being evaluated in the PREVAIL trial, although results will be limited to safety and immunogenicity.\nAd26.ZEBOV (Crucell/ Johnson & Johnson) and MVA-BN Filo (Bavarian Nordic) While EBOV GP expressed in an Ad26 vector rapidly induced a T-cell response and protected against EVD in the cynomolgus macaque model (121), pre-existing immunity and duration of immunity remained a concern for the adenovirus-based vaccines. The heterologous primeboost approach, where an adenovirus-based EBOV construct was used as the initial vaccine followed by a boost with EBOV GP expressing modi\ufb01ed vaccinia Ankara (MVA), was promising since it provided durable immunity (122). The Crucell subsidiary of Johnson & Johnson had developed a monovalent Ebola vaccine using an Ad26 virus vector (Ad26.ZEBOV), while Bavarian Nordic had pursued a multivalent \ufb01lovirus vaccine (containing the GP from Ebola, Sudan, and Marburg viruses) expressed in an MVA vector (MVA-BN Filo). An interesting collaboration to leverage the heterologous prime-boost strategy was formalized in October 2014 between these two companies (123), and multiple clinical trials are underway (http://id .bavarian-nordic.com/pipeline/\ufb01lovirus.aspx).\nOther vaccines Several other vaccine candidates are also in the development pipeline. Profectus BioSciences Inc. approached the use of VSV through a different strategy than that utilized by GSK. Instead of replacing VSV G with that of EBOV, Profectus modi\ufb01ed the vector by swapping EBOV GP for the VSV N gene, relocating the VSV N gene to a region proximal to VSV G and truncating VSV G. The resulting recombinant had a decreased\n\ngrowth rate in vitro and produced lower viremias in vaccinated cynomolgus macaques but was still effective at preventing EVD in this primate model with a single dose of vaccine (124). In contrast to the virally vectored vaccines advanced by Merck, GSK, Crucell, Bavarian Nordic, and Profectus, Novavax is developing a proteinbased vaccine to be used with an adjuvant (Matrix-M). A two-dose regimen of the vaccine is effective at preventing EVD in cynomolgus macaques (http://novavax. com/download/\ufb01les/presentations/Novavax_EBOV_GP _Vaccine_2015_07_21_FINAL.pdf), and Novavax initiated a phase 1 study for a recombinant GP protein vaccine in February 2015 (125).\nCONCLUSIONS\nThe development and evaluation of investigational therapies for an emerging infectious disease such as Ebola requires a number of elements to be in place, ranging from the ability to produce or manufacture suf\ufb01cient quantities of good-quality investigational agents so that clinical trials to evaluate the investigational agents can be conducted, infrastructure to provide care for patients and to support the conduct of clinical trials, engagement of the affected communities in the response effort, information on the disease and its major manifestations, and properly designed clinical trials that have the capacity to draw scienti\ufb01cally valid conclusions and protect patient safety. The epidemic of EVD in West Africa revealed serious weaknesses in international preparedness and response efforts to emerging threats.\nThe manufacturing or production of suf\ufb01cient supplies of investigational product to support the conduct of clinical trials was a major challenge in the response to the Ebola epidemic. The reasons for these delays differ for the different types of investigational products that are being developed for Ebola. For example, delays in planning, organizing, and equipping health care providers with the means to collect convalescent serum and blood (identi\ufb01ed as one of the highest therapeutic priorities by the WHO [126]) was one factor that impeded the evaluation of these potential therapies. When developing therapies for an emerging infectious disease such as Ebola, it is important to consider the product characteristics, the ability to scale up production, and the time required to deliver adequate supplies in response to an outbreak. For products that are not currently stockpiled, availability and scalability of the production process must be taken into account when prioritizing MCMs or developing contingency plans for their use. These risks can be mitigated in part by\n\nDownloaded from https://journals.asm.org/journal/spectrum on 20 August 2024 by 2c0f:f6d0:26:2d:8551:cc20:4633:4ac.\n\n8\n\nASMscience.org/MicrobiolSpectrum\n\nEbola Virus Disease: Therapeutic and Potential Preventative Opportunities\n\nensuring that appropriate mechanisms exist for enlisting additional manufacturing facilities and/or \ufb01lling lines when necessary.\nThe characteristics of an investigational product and the setting in which it will be used impact the utility of the product in the response effort. The drugs and vaccines developed by the United States were initially envisioned for use in the context of prophylaxis of military personnel or for a bioterrorist event resulting in cases of EVD, and their use assumed a high degree of coordination between local, state, and federal partners within the United States. Monoclonal antibodies such as ZMapp must be slowly administered over several hours to decrease the risk of infusion-related adverse events. For outbreaks where the ability of health care providers to provide and monitor the administration of compounds such as ZMapp is compromised, other product classes that can be delivered orally or as an intramuscular injection may be preferable. Likewise, stability can complicate deployment efforts in areas with sporadic electricity and refrigeration. For example, the VSVEBOV vaccine requires ultra-low (\u221270\u00b0C) storage for long-term stability; experience with polio and smallpox vaccination campaigns demonstrated the importance of a thermally stable vaccine. In the absence of such a stable vaccine, the ability to maintain a cold chain is essential and must be part of contingency planning. This planning should also consider how the vaccines and drugs can be evaluated for safety and ef\ufb01cacy during the outbreak to rapidly identify the safest and most ef\ufb01cacious MCMs.\nFor therapies against an emerging infectious disease such as Ebola, it is important to consider biological diversity and that the infectious agent may acquire mutations that could alter targets for countermeasures. It would be ideal to have multiple countermeasures with different mechanisms of action in order to have therapies that will remain active in the setting of mutations of the infectious agent or species differences that may impact the activity of some countermeasures. For example, monovalent vaccines developed based on EBOV strains such as Mayinga or Kikwit protect against the Mayinga strain, but had the outbreak been triggered by a separate Ebola virus species (Sudan ebolavirus or Bundibugyo ebolavirus), cross-neutralization would be unlikely and the vaccines would require modi\ufb01cation. Similarly, MCMs that are highly speci\ufb01c to speci\ufb01c strains and species of virus are vulnerable to the emergence of sequence variants. TKM-Ebola was modi\ufb01ed to TKM-Ebola-Guinea to more closely match the sequence of the Makona strain of EBOV. Therefore,\n\nstockpiles of monovalent MCMs may be less valuable for response than those composed of multivalent vaccines and antivirals with broad activity.\nDemonstrating the safety and ef\ufb01cacy of a new drug or vaccine can be a challenging endeavor under normal circumstances. These challenges were ampli\ufb01ed by the rapidly evolving Ebola epidemic (a communicable agent causing severe illness with signi\ufb01cant mortality), the unprecedented stress on the West African health care system and society in general, and the need to establish infrastructure to support clinical trials to identify bene\ufb01cial therapies. Properly designed clinical trials are an essential component of the response effort. The \ufb01ndings from such trials can help patients by identifying whether an investigational therapy bene\ufb01ts patients. Conducting clinical trials that are not properly designed can delay the identi\ufb01cation of effective therapies, lead to uninterpretable or misleading conclusions, and impede the ability to adequately monitor patient safety. For example, the lack of well-designed clinical trials and information about the characteristics of the convalescent plasma tested have impeded the ability to evaluate the role of convalescent plasma in treating patients with EVD. Fortunately, there were some successful and partially successful efforts during the epidemic. Some well-controlled trials of vaccines and a therapeutic were implemented during the epidemic. While these trials were implemented very quickly compared to the usual time to launch a clinical trial, it is apparent that we need to continue to build on this effort so that the evaluation of countermeasures can be implemented even more rapidly in the response to any future outbreak.\nThe importance of the advance preparation of protocols to allow the evaluation of safety and ef\ufb01cacy of MCMs in an outbreak setting cannot be overstated. Randomized clinical trials offer advantages in terms of rapidly providing interpretable, robust data on the safety and ef\ufb01cacy of an MCM (127). Careful consideration must be given to inclusion and exclusion criteria, and if need be, \ufb02exibility must be introduced into the protocol to allow for adaptation; for example, the PREVAIL I protocol was modi\ufb01ed in November 2015 to include an open-label cluster vaccination component in response to new clusters of EVD in Liberia. The use of a common protocol that allows the \ufb02exibility for evaluating multiple unproven MCMs is one potential solution to quickly identify the most effective therapy or prophylactic while weeding out those that actually cause harm, and it can help communicate a standard level of supportive care that is expected for patients (22). If there are debates about how to ethically conduct certain trials,\n\nDownloaded from https://journals.asm.org/journal/spectrum on 20 August 2024 by 2c0f:f6d0:26:2d:8551:cc20:4633:4ac.\n\nASMscience.org/MicrobiolSpectrum\n\n9\n\nFisher and Borio\n\nDownloaded from https://journals.asm.org/journal/spectrum on 20 August 2024 by 2c0f:f6d0:26:2d:8551:cc20:4633:4ac.\n\nthese conversations should take place before an outbreak instead of during the outbreak. Community engagement also has a role to play in trial design. When the purpose for randomized, controlled trials\u2014to evaluate the safety as well as ef\ufb01cacy in untested products\u2014was articulated to local populations by individuals trusted by those in the community, there was acceptance as evidenced by enrollment and high visit compliance in the Ebola \u00e7a Suf\ufb01t and PREVAIL vaccine trials (110, 113). In addition, the above-mentioned cluster vaccination component of the PREVAIL I protocol was implemented by the Liberian partners, building on the training they had received for the initial trial.\nSadly, for the \ufb01rst time in history, a suf\ufb01cient number of EVD cases existed to allow for the collection of data on the natural history of disease in humans. Applied research to address knowledge gaps bene\ufb01tted from a great deal of collaborative work accomplished through partnerships between nongovernmental organizations, industry, academia, and government agencies. While the data thus generated will be analyzed and subjected to scrutiny and debate for years to come, we do not have the same luxury of time in which to apply the lessons of how the world responded to this unprecedented outbreak. Considering severe acute respiratory syndrome\u2013 associated coronavirus, pandemic in\ufb02uenza, and now EVD, we must do more in terms of preparedness and contingency planning for every stage of the MCM product development cycle. Instead of asking \u201cWhat do we have?\u201d during an outbreak, the question should be reframed to \u201cWhich contingency plan is appropriate?\u201d for any given outbreak situation.\nACKNOWLEDGMENTS\nThis book chapter re\ufb02ects the views of the authors and should not be construed to represent the FDA\u2019s views or policies.\nREFERENCES\n1. World Health Organization. 1978. Ebola haemorrhagic fever in Sudan, 1976. Bull World Health Organ 56:247\u2013270.\n2. World Health Organization. 1978. 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Ledgerwood JE, DeZure AD, Stanley DA, Novik L, Enama ME, Berkowitz NM, Hu Z, Joshi G, Ploquin A, Sitar S, Gordon IJ, Plummer SA, Holman LA, Hendel CS, Yamshchikov G, Roman F, Nicosia A, Colloca S, Cortese R, Bailer RT, Schwartz RM, Roederer M, Mascola JR, Koup RA, Sullivan NJ, Graham BS, the VRC 207 Study Team. 2014. Chimpanzee adenovirus vector Ebola vaccine: preliminary report. N Engl J Med 373:775\u2013776.\n120. Rampling T, Ewer K, Bowyer G, Wright D, Imoukhuede EB, Payne R, Hartnell F, Gibani M, Bliss C, Minhinnick A, Wilkie M, Venkatraman N, Poulton I, Lella N, Roberts R, Sierra-Davidson K, Kr\u00e4hling V, Berrie E, Roman F, De Ryck I, Nicosia A, Sullivan NJ, Stanley DA, Ledgerwood JE, Schwartz RM, Siani L, Colloca S, Folgori A, Di Marco S, Cortese R, Becker S, Graham BS, Koup RA, Levine MM, Moorthy V, Pollard AJ, Draper SJ, Ballou WR, Lawrie A, Gilbert SC, Hill AVS. 2015. A monovalent chimpanzee adenovirus Ebola vaccine: preliminary report. N Engl J Med. 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N Engl J Med 371:2350\u20132351.\n\nDownloaded from https://journals.asm.org/journal/spectrum on 20 August 2024 by 2c0f:f6d0:26:2d:8551:cc20:4633:4ac.\n\n14\n\nASMscience.org/MicrobiolSpectrum\n\n\n",
    "authors": [
      "Robert Fisher",
      "Luciana Borio"
    ],
    "doi": "10.1128/microbiolspec.EI10-0014-2016",
    "date": "2016-05-06",
    "item_type": "journalArticle",
    "url": "https://journals.asm.org/doi/10.1128/microbiolspec.EI10-0014-2016"
  },
  {
    "key": "83VDFQW2",
    "title": "The emergence of antibody therapies for Ebola",
    "abstract": "This review describes the history of Ebola monoclonal antibody (mAb) development leading up to the recent severe Ebola outbreak in West Africa. The Ebola virus has presented numerous perplexing challenges in the long effort to develop therapeutic antibody strategies. Since the \ufb01rst report of a neutralizing human anti-Ebola mAb in 1999, the straightforward progression from in vitro neutralization resulting in in vivo protection and therapy has not occurred. A number of mAbs, including the \ufb01rst reported, failed to protect non-human primates (NHPs) in spite of protection in rodents. An appreciation of the role of effector functions to antibody ef\ufb01cacy has contributed signi\ufb01cantly to understanding mechanisms of in vivo protection. However a crucial contribution, as measured by post-exposure therapy of NHPs, involved the comprehensive testing of mAb cocktails. This effort was aided by the use of plant production technology where various combinations of mAbs could be rapidly produced and tested. Introduction of appropriate modi\ufb01cations, such as speci\ufb01c glycan pro\ufb01les, also improved therapeutic ef\ufb01cacy. The resulting cocktail, ZMappTM, consists of three mAbs that were identi\ufb01ed from numerous mAb candidates. ZMappTM is now being evaluated in human clinical trials but has already played a role in bringing awareness to the potential of antibody therapy for Ebola.",
    "full_text": "Human Antibodies 23 (2014/2015) 49\u201356\n\n49\n\nDOI 10.3233/HAB-150284\n\nIOS Press\n\nThe emergence of antibody therapies for Ebola\n\nAndrew Hiatta,\u2217, Michael Paulya, Kevin Whaleya, Xiangguo Qiub, Gary Kobingerb and Larry Zeitlina aMapp Biopharmaceutical, Inc. San Diego, CA, USA bNational Laboratory for Zoonotic Diseases and Special Pathogens, Public Health Agency of Canada, Winnipeg,\nMB, Canada\n\nAbstract. This review describes the history of Ebola monoclonal antibody (mAb) development leading up to the recent severe Ebola outbreak in West Africa. The Ebola virus has presented numerous perplexing challenges in the long effort to develop therapeutic antibody strategies. Since the \ufb01rst report of a neutralizing human anti-Ebola mAb in 1999, the straightforward progression from in vitro neutralization resulting in in vivo protection and therapy has not occurred. A number of mAbs, including the \ufb01rst reported, failed to protect non-human primates (NHPs) in spite of protection in rodents. An appreciation of the role of effector functions to antibody ef\ufb01cacy has contributed signi\ufb01cantly to understanding mechanisms of in vivo protection. However a crucial contribution, as measured by post-exposure therapy of NHPs, involved the comprehensive testing of mAb cocktails. This effort was aided by the use of plant production technology where various combinations of mAbs could be rapidly produced and tested. Introduction of appropriate modi\ufb01cations, such as speci\ufb01c glycan pro\ufb01les, also improved therapeutic ef\ufb01cacy. The resulting cocktail, ZMappTM, consists of three mAbs that were identi\ufb01ed from numerous mAb candidates. ZMappTM is now being evaluated in human clinical trials but has already played a role in bringing awareness to the potential of antibody therapy for Ebola.\nKeywords: Ebola virus, Ebola antibody discovery, effector functions, passive immunotherapy\n\n1. Introduction\nEbola virus (EBOV) is responsible for catastrophic clinical disease and high mortality outbreaks. Some variants of Ebola have been fatal in 90% of infections [2]. Past outbreaks have largely been contained by public health and infection-control measures. The current outbreak in West Africa, which is the largest in history, puts an emphasis on the need for an effective treatment.\nDuring the 2014\u20132015 West Africa outbreak, seriously ill infected individuals received a cocktail of three monoclonal antibodies (mAbs) that were produced in plants (ZMappTM) under compassionate use. ZMappTM contains antibodies against three Ebola glycoprotein (GP) epitopes and is manufactured by transient expression in a relative of cultivated tobacco called Nicotiana benthamiana [3,4]. When tested in non-human primates (NHPs), ZMappTM conferred a\n\u2217Corresponding author: Andrew Hiatt, Mapp Biopharmaceutical, Inc. San Diego, CA, USA. E-mail: andy.hiatt@mappbio.com.\n\n100% survival bene\ufb01t even when the treatment started 4 to 5 days after infection, when the animals were symptomatic. Control animals succumbed to infection in the study between 4 and 8 days post-infection. In the case of the human course of disease which is extended compared to NHPs (challenge is intramuscular for most studies of countermeasures), the average time to death is extended to 6\u201316 days after symptoms appear [6] and in the case of the patients who received ZMappTM, their original infection most likely occurred more than 5 days prior to the onset of immunotherapy (Fig. 1).\nCurrently, ZMappTM is being evaluated for safety and ef\ufb01cacy in randomized clinical trials in West Africa and the US. The ZMappTM antibodies were the result of over \ufb01fteen years of antibody discovery and research, in an effort to identify an effective anti-Ebola therapeutic. The effort represents the collaborations of private research labs (Mapp Biopharmaceutical, Inc. and Defyrus) and various United States (DARPA, USAMRIID, NIH, DTRA, BARDA) and Canadian (PHAC, CSSP) government agencies.\n\nISSN 1093-2607/14/15/$35.00 c 2014/2015 \u2013 IOS Press and the authors. All rights reserved\n\n50\n\nA. Hiatt et al. / The emergence of antibody therapies for Ebola\n\nDays\nFig. 1. Progression of Ebola Virus Disease in Humans and Non-human primates (NHP). (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/HAB-150284)\n2. Early mAbs and surprising results\nOne of the \ufb01rst efforts to isolate a neutralizing mAb (Text Box 1) against Ebola involved panning of phage display libraries that were constructed from the bone marrow of patients who had survived Ebola infection. These initial mAb discovery experiments focused largely on in vitro neutralization for identi\ufb01cation of potential therapeutic mAbs [7]. Speci\ufb01cally, phage libraries were constructed from the bone marrow of 2 convalescent donors [8]. Several months after the onset of disease bone marrow was sampled and RNA was used to prepare cDNA, from which antibody heavy and light chains could be ampli\ufb01ed by PCR. The libraries were panned against an Ebola Zaire-infected Vero cell lysate and positive phage showed strong signals in ELISA when tested with donor sera. After panning against the secreted form of Ebola glycoprotein (sGP) from the supernatant of Ebola-Zaire-infected Vero cells, three antibodies were isolated. This was the \ufb01rst report of the isolation of human monoclonal antibodies to Ebola.\nOne Fab, KZ52, obtained by panning against the virion preparation, had a unique reactivity pattern [7]. In addition to virion binding, it showed signi\ufb01cant cross-reactivity with a supernatant preparation, whereas four other Fabs obtained by panning against the supernatant preparation only showed weak crossreactivity with the virion preparation. When Fabs were tested for their ability to neutralize Ebola virus, KZ52 showed 50% neutralization at 0.4 \u03bcg/ml (8 nM) and none of the other Fabs had any neutralizing ability. KZ52 was the Fab that reacted most effectively with live virus-infected cells. In addition, the KZ52 IgG1 neutralized about fourfold more effectively than Fab\n\nKZ52. It was subsequently found that the KZ52 mAb protects guinea pigs from lethal Ebola Zaire virus challenge [9]. Administration before or up to 1 h after challenge resulted in dose-dependent protection. Interestingly, some antibody-treated animals survived despite developing high-level viremia, suggesting that the mechanism of protection by KZ52 may extend beyond reduction of viremia by virus neutralization.\nSurprisingly, although KZ52 protected against robust EBOV in the guinea pig model [9] attempts to use this mAb to protect rhesus macaques prophylactically (50 mg/kg dose \u22121 and +4 days with respect to challenge) not only failed but also had a minimal effect on the explosive viral replication following infection. The inability of this antibody to impact infection was not due to neutralization escape. It appears that Ebola virus has a mechanism of infection propagation in vivo in macaques that is insensitive even to high concentrations of antibodies that are considered neutralizing in vitro. This result and other failures in NHPs [10], led to a signi\ufb01cant dampening of interest in mAbs as postexposure prophylactics for Ebola virus infection.\nTo better understand targets and mechanisms of neutralization, a panel of mAbs shown previously to react with the envelope glycoprotein were analyzed [11]. While one non-neutralizing mAb recognized a GP epitope in the nonessential mucin-like domain, the rest were speci\ufb01c for GP1, were neutralizing, and could be further distinguished by reactivity with secreted GP. The human mAb KZ52 and monkey JP3K11, were speci\ufb01c for conformation-dependent epitopes comprising residues in GP1 and GP2. Neutralization occurred by two distinct mechanisms; KZ52 inhibited cathepsin cleavage of GP whereas JP3K11 recognized the cleaved, fusion-active form of GP. This result suggested that the effort to \ufb01nd a protective mAb or mAbs for use in humans would require far more analysis than simple neutralization assays and may indeed require the use of multiple antibodies.\nAdditional neutralizing mAbs similar to KZ52 have been described [12] and were thought to recognize a crucial epitope [13,14] bridging GP1-GP2 to neutralize at a post-internalization step perhaps involving viral fusion. However, it was recognized that the ability of mAbs to neutralize virus as well as to protect certain animal models doesn\u2019t necessarily predict ef\ufb01cacy in higher primates. It became clear that multiple epitopes need to be targeted in order to provide suf\ufb01cient ef\ufb01cacy as well as to suppress the occurrence of escape mutations. All of the subsequent reports, in one way or another, begin to approach the problems associated\n\nA. Hiatt et al. / The emergence of antibody therapies for Ebola\n\n51\n\nNeutralization vs. protection In vitro neutralization typically refers to the ability of a mAb to block infection of a cell line in cell culture, with the expectation that the mAb is likely inhibiting binding of the virus to its target receptor or preventing fusion or entry of the virus with or into the target cell. However, in many infections, this is a poor representation of what occurs in vivo. For example, with Ebola:\n1. Typically endothelial cells are used as target cells in neutralization assays. However, the \ufb01rst cells infected in vivo tend to be macrophages, followed by dendritic, Kupfer and endothelial cells with endothelial cells being one of the last infected (1). Further, in vivo, immune effector cells (phagocytes, NK cells, macrophages, etc.) likely play a role in the ef\ufb01cacy of antibodies.\n2. Given the diversity of cell types that Ebola infects, a monoculture of target cells may provide misleading data on the functionality of a mAb. In the HIV \ufb01eld, the presence or absence of T cells and dendritic cells has a dramatic effect on which broadly neutralizing mAb is the most potent in a particular assay.\n3. The fact that roughly 1 pfu can be lethal in non-human primates would suggest the in vitro assay is a poor surrogate (i.e. generally, cell culture is signi\ufb01cantly less sensitive to viral infection than a mammal).\n4. Different populations of antibodies can contribute to host resistance to viruses by mechanisms that do not involve viral neutralization (5). In particular, the effectiveness of protective, non-neutralizing mAbs (PrNabs) could involve a variety of mechanisms including ADCC, interaction with virus infected cell surfaces, secreted proteins, and cryptic or transitional epitopes.\n5. Due to variations in mAb quality, glycosylation, isotypes, and neutralization protocol variations, it is inherently dif\ufb01cult to compare in vitro neutralization results.\nwith a multi-mAb cocktail against Ebola. In fact, the role of effector functions contributing to animal survival was a surprising development in a research environment that focused heavily on neutralization [15].\nIt is generally accepted that the ability of mAbs to inhibit plaque formation by Ebola virus does not necessarily predict their protective ef\ufb01cacy in mice [15]. In one group of anti-GP mAbs reported in 2000 [15], none of the protective mAbs inhibited plaque formation in the absence of complement suggesting an important role for speci\ufb01c antibody dependent effector functions. One reported mAb did not reduce the number of plaques, but did reduce plaque size, suggesting a role for the restriction of infection of adjacent cells. Overall, these results suggested that it might be possible to elicit, by vaccination, or produce for therapeutic use, antibodies protective against all Ebola viruses. Moreover, the idea that an antibody\u2019s reactivity with both sGP and GP would render it ineffectual in protection was found to be unsupported [16,17]. The af\ufb01nity of an antibody for its epitope, possibly in\ufb02u-\n\nenced by posttranslational modi\ufb01cations such as glycosylation, was appreciated as an important determinant of protective ef\ufb01cacy. In general, protective mAbs had higher af\ufb01nity for their epitopes than unprotective mAbs. Clearly though, antibody speci\ufb01city and neutralization in vitro are inadequate as sole predictors of protection. Protection may depend on the proper speci\ufb01city, isotype, and/or af\ufb01nity of a single antibody or a combination of antibodies [18].\n3. The evidence for infection enhancing antibodies\nIn addition to the ability of mAbs to potentially provide protection and therapy against Ebola, the possibility of enhanced infectivity mediated by those mAbs has been a signi\ufb01cant concern. Anti-sera produced by DNA immunization with a plasmid encoding the GP of the Zaire strain of Ebola enhances the infectivity of vesicular stomatitis virus pseudotyped with the GP [19]. Substantially weaker enhancement could be observed with antiserum to the GP of Reston virus, which is much less pathogenic in humans than the Ebola Zaire and Sudan viruses. The enhancing activity was abolished by heat but was increased in the presence of complement system inhibitors, suggesting that heat-labile factors other than the complement system are required for this effect. An anti-Ebola GP monoclonal antibody that appeared to enhance viral infectivity as well as another that neutralized it, suggested the presence of distinct epitopes for these effects. It was proposed that antibody-dependent enhancement of infectivity may account for the extreme virulence of some variants of the virus [19,20].\nThe proposed involvement of enhancing antibodies in Ebola pathogenesis may have precedent in immunization with inactivated Marburg virus antigens, which was associated with earlier deaths in immunized animals [21]. This suggested that passive prophylaxis with GP antibodies should be restricted to neutralizing antibodies [15]. Both neutralizing and enhancing epitopes may exist on GP molecules. In addition, plasma or serum from convalescing patients reportedly enhanced the infection of primate kidney cells by Ebola virus, and this enhancement was mediated by antibodies to the viral glycoprotein and by complement component C1q [19,22].\nIn another report [23] the neutralizing and enhancing activities of Ebola virus mAbs were tested with four murine antibodies speci\ufb01c to GP, a recombinant human mAb speci\ufb01c to GP, a polyclonal equine IgG,\n\n52\n\nA. Hiatt et al. / The emergence of antibody therapies for Ebola\n\nand serum obtained from a convalescent monkey. All but one of these antibodies neutralized Ebola infectivity of primary human monocytes/macrophages or Vero cells. None of the antibodies enhanced Ebola infectivity in these cells. Taken together with in vivo observations that early deaths were not observed in animals immunized with various viral vectors expressing Ebola GP, it appeared unlikely that any Ebola infectivity-enhancing antibodies signi\ufb01cantly affected Ebola pathogenesis. The importance of antibodies and the epitopes that can enhance viral infectivity remains controversial.\n4. Protection with polyclonal IgG\nThe survival of 7 of 8 patients with Ebola infections after transfusions of convalescent-phase blood during a 1995 outbreak of Ebola has frequently been cited as evidence that passive immunotherapy is a viable treatment option [24]. To test whether whole-blood transfusions were more ef\ufb01cacious than passively administered immunoglobulins or monoclonal antibodies, convalescent-phase blood was transfused from Ebolaimmune monkeys into naive animals shortly after challenge with Ebola [10]. Although passively acquired antibody titers comparable to those associated with effective vaccination were obtained, all monkeys that had received transfusions succumbed to infection concurrently with control monkeys. The reasons for this failure are unclear but could relate more to a failure to elicit an appropriate effector response in infected monkeys than an overall failure of passive immunotherapy. More recently, it has been shown that passive transfer of puri\ufb01ed IgG from NHPs that survived Ebola infection (dose of 80 mg/kg, given at days 2, 4 and 8 post-challenge) could protect na\u00efve NHPs against lethal challenge [25]. This was the \ufb01rst demonstration of passive immunity with Ebola virus in NHPs.\n5. Early mAb cocktails\nThe \ufb01rst report that attempted to evaluate the ef\ufb01cacy of a mAb cocktail employed newly discovered mAbs binding to new GP epitopes [26]. Using the mouse and guinea pig animal models, the protective ef\ufb01cacy of two monoclonal antibodies whose epitopes are distinct from those of the antibodies tested by others was evaluated. Treatment of mice with these antibodies 2 days after challenge completely protected\n\nmost of the animals. Surprisingly, treatment 3 or 4 days after challenge was, at least in part, effective. Although antibody treatment in the guinea pig model was not as effective as in the mouse model, single-dose treatment of guinea pigs 1 day before, or 1 to 2 days after challenge did offer some protection. As has been observed numerous times previously, the protection seen in these animal models did not correlate with the in vitro neutralizing activity. In addition, the combination of the two antibodies, using these mAbs, did not enhance the protective bene\ufb01t.\nHowever, two human-mouse chimeric mAbs (ch133 and ch226) with strong neutralizing activity against Ebola Zaire were evaluated for their protective potential in a rhesus macaque model [27]. Reduced viral loads and partial protection were observed in animals given mAbs ch133 and ch226 when combined intravenously at 24 hours before and 24 and 72 hours after challenge. These mAbs were noted circulating in the blood of a surviving animal until virus-induced IgG responses were detected. In contrast, serum mAb concentrations decreased to undetectable levels at terminal stages of disease in animals that succumbed to infection, indicating substantial consumption of these antibodies due to virus replication. The rapid decrease of serum mAbs was clearly associated with increased viremia in non-survivors. The results indicated that Ebola neutralizing antibodies, particularly in combination, might be bene\ufb01cial in reducing viral loads and prolonging disease progression. The bene\ufb01t of a combination of mAbs rather than single mAbs was \ufb01rst made apparent.\n6. Plant-based Ebola mAb production systems\nAs research into passive immunotherapy for Ebola continued to make progress, production systems for generating large quantities of Ebola protective antibodies have been explored in mammalian, fungus, and plant cells. The focus of this review is on transient infection of whole plants.\nIn one system, it was demonstrated that the bean yellow dwarf virus (BeYDV) viral replicon system permits simultaneously ef\ufb01cient replication of two DNA replicons and thus, high-level accumulation of two recombinant proteins in the same plant cell [28]. It was also demonstrated that a single vector that contains multiple replicon cassettes was as ef\ufb01cient as the threecomponent system in driving the expression of two distinct proteins. Using either the non-competing, three-\n\nA. Hiatt et al. / The emergence of antibody therapies for Ebola\n\n53\n\nvector system or the multi-replicon single vector, both the heavy and light chain subunits of a protective IgG mAb 6D8 against Ebola virus GP1 [15] were produced at 0.5 mg of mAb per gram leaf fresh weight within 4 days post-in\ufb01ltration in Nicotiana benthamiana leaves. It was further demonstrated that the full-size tetrameric IgG complex retained its functionality as it bound speci\ufb01cally to inactivated Ebola virus. Thus, a singlevector replicon system was shown to provide highyield production capacity for Ebola mAbs as well as being a useful advance in transient expression technology for antibody production in plants.\nThe attractiveness of the plant approach derives from high yields of recombinant protein obtained within days after transient delivery of viral vectors [29]. Modi\ufb01ed viral genomes of both RNA and DNA viruses have been created. BeYDV has a small, single stranded DNA genome that replicates in the nucleus of an infected plant cell, using the cellular DNA synthesis apparatus and a virus-encoded replication initiator protein. Other systems employ two different noncompeting viral replicons to accomplish expression of multi-chain proteins [3,30]. Delivery of these vectors to leaf cells via Agrobacterium-mediated infection produces very high levels of recombinant DNA that can act as a transcription template, yielding high levels of mRNA for the protein of interest.\n7. Glycoengineering in plants\nIn addition to rapid and high yield mAb production, the plant systems can be manipulated to introduce a variety of nearly homogeneous glycoforms [31,32]. These include GnGn, galactosylated and sialylated glycans. The key to achieving the relatively high level of homogeneity appears to result from the absence of xylose and fucose (\u0394XF), which can be reduced by RNAi technology [33].\nIn the context of mAbs, alteration of glycosylation can profoundly affect ef\ufb01ciency and bioactivity. Production of different glycoforms of an Ebola virus mAb (13F6) using the magnICON expression system resulted in nearly homogeneous human like biantennary N-glycans with terminal N-acetylglucosamine on each branch (GnGn structures) [34]. Therefore, plant production can allow for the ef\ufb01cient generation of different human-like glycoforms at near homogeneity of virtually any antibody.\nThe impact of glycoengineering on Ebola immunotherapy has been demonstrated in vivo [34]. Starting\n\nwith the 13F6 mAb that recognizes the heavily glycosylated mucin-like domain of GP, point mutations were introduced into the variable region to remove predicted human T-cell epitopes, and the variable regions joined to human constant regions to generate a hybrid mAb h-13F6 appropriate for human use. The ef\ufb01cacy of three h-13F6 variants carrying different glycosylation patterns in a lethal mouse Ebola challenge model were evaluated. The glycosylation patterns were found to correlate to the level of protection, with aglycosylated h-13F6 providing the least potent ef\ufb01cacy (ED50 = 33 \u03bcg). A version with typical heterogenous mammalian glycoforms (ED50 = 11 \u03bcg) had similar potency to the original murine mAb. However, h-13F6 carrying complex N-glycosylation lacking core fucose exhibited superior potency (ED50 = 3 \u03bcg). Binding studies using Fc\u03b3 receptors revealed enhanced binding of non-fucosylated h-13F6 to mouse and human Fc\u03b3 RIII. Together the results indicated Fc N-glycans play a role in the protective ef\ufb01cacy of h-13F6, and that mAbs manufactured with uniform glycosylation and a higher potency glycoform offer promise as potent therapeutics.\n8. Recent mAb cocktails\n8.1. Since 2011, the discovery of many new Ebola mAbs has been reported\nRecombinant VSVDeltaG/ZEBOVGP was used to generate mAbs against the EBOV GP [35]. A total of 8 mAbs were produced using traditional hybridoma cell fusion technology, and then characterized by ELISA using EBOV VLPs, Western blotting, immuno\ufb02uorescence assays, and immunoprecipitation. All 8 mAbs worked in IFA and IP, suggesting that they are all conformational mAbs, however six of them recognized linearized epitopes by Western blotting. ELISA results demonstrated that one mAb bound to the secreted GP (sGP 1\u2013295aa); three bound to part of the mucin domain (333\u2013458aa); three mAbs recognized epitopes on the C-terminal domain of GP1 (296\u2013 501aa); and one bound to full length GP (VLPs/GP1,2 DeltaTm). Using a mouse model these mAbs were evaluated for their therapeutic capacity during a lethal infection. All 8 mAbs improved survival rates by 33%\u2013 100% against a high dose lethal challenge with mouseadapted EBOV.\nThe protective ef\ufb01cacy of these 8 mAbs in mice and guinea pigs demonstrated the potential of mAb\n\n54\n\nA. Hiatt et al. / The emergence of antibody therapies for Ebola\n\ncocktails [36]. Immunocompetent mice or guinea pigs were given mAbs i.p. in various doses individually or as pools of 3\u20134 mAbs to test their protection against a lethal challenge with mouse- or guinea pig-adapted Ebola. Each of the 8 mAbs (100 ug) protected mice from a lethal Ebola challenge when administered 1 day before or after challenge. Seven mAbs were effective 2 days post-infection (dpi), with 1 mAb demonstrating partial protection 3 dpi. In the guinea pigs two mAbs showed partial protection at 1 dpi, however the mean time to death was signi\ufb01cantly prolonged compared to the control group. Moreover, treatment with pools of 3\u20134 mAbs completely protected the majority of animals, while administration at 2\u20133 dpi achieved 50\u2013100% protection. This data suggests that the mAbs generated are capable of protecting both animal species against lethal Ebola virus challenge and when used as an oligoclonal set are a potential therapeutic for post-exposure treatment of Ebola infection.\nExtension of this strategy to macaques demonstrated that a combination of three neutralizing mAbs (now referred to as ZMAb) directed against GP resulted in complete survival (four of four cynomolgus macaques) with no apparent side effects when three doses were administered 3 days apart beginning at 24 hours after a lethal challenge with Ebola [37]. The same treatment initiated 48 hours after lethal challenge with Ebola resulted in full recovery of two of four macaques. The survivors demonstrated an Ebola-GP-speci\ufb01c humoral and cell-mediated immune response. In a follow up to this set of experiments using the \ufb01rst mAb cocktail, now referred to as ZMAb, the importance of the endogenous immune response was explored [38]. Since the survivors demonstrated Ebola-GP-speci\ufb01c humoral and cell-mediated immune responses the question of whether this is suf\ufb01cient to protect survivors against a subsequent exposure is key to understanding the participation of the immune response in recovery. Animals that survived the initial challenge were re-challenged after 10 or 13 weeks. The animals re-challenged at 10 weeks all survived whereas 4 of 6 animals survived a re-challenge at 13 weeks. The data indicate that a robust immune response was generated during the successful treatment of Ebola-infected NHPs which resulted in sustained protection against a second lethal exposure.\nSoon after publication of the initial ZMAb macaque study, a report was published of three mAbs (originally produced in reference [15]) produced in plants [39]. This combination of three mAbs, c13C6, h-13F6, and c6D8, was referred to as MB003 where c-refers to\n\nchimeric. It was discovered that MB-003 protected macaques from lethal challenge when administered 1 h post-infection. In a pivotal follow-up experiment, it was discovered that signi\ufb01cant protection (P < 0.05) was afforded when MB-003 treatment began 48 h postinfection (four of six survived vs. zero of two controls). In all experiments, surviving animals that received MB-003 experienced little to no viremia and had few, if any, of the clinical symptoms observed in the controls.\nThis study was further elaborated to test whether the MB-003 cocktail was ef\ufb01cacious as a therapeutic after the onset of symptoms [40]. Rhesus macaques were challenged with Ebola and treatment was initiated upon con\ufb01rmation of infection according to a diagnostic assay, which has a U.S. Food and Drug Administration Emergency Use Authorization, and observation of a documented fever. Of the treated animals, 43% survived challenge (3 of 7), whereas both the controls and all historical controls with the same challenge stock succumbed to infection. These results represented successful therapy (as opposed to post-exposure prophylaxis) of Ebola infection in NHPs.\nIn combination with previous studies evaluating the binding sites of other protective antibodies, these studies suggest that antibodies targeting the GP1-GP2 interface and the glycan cap are often selected for postexposure interventions against Ebola.\n9. Preclinical studies and clinical use of ZMappTM\nContinued evaluation of various mAb combinations from the ZMAb and MB-003 cocktails, representing the collaborative efforts of three companies (Mapp, Kentucky BioProcessing, and Defyrus) and government agencies from the United States and Canada resulted in an optimized set of a three mAb cocktail consisting of c13C6, c4G7, and c2G4 (ZMappTM) that demonstrated superior ef\ufb01cacy in terms of post symptomatic treatment of non-human primates [4]. The ZMappTM cocktail was produced under cGMP by Kentucky Bioprocessing using the MagnICON transient plant system [3,30].\nThis combination of mAbs is able to rescue 100% of rhesus macaques when treatment is initiated up to 5 days post-challenge. High fever, viremia and abnormalities in blood count and blood chemistry were evident in many animals before ZMappTM intervention. Advanced disease, as indicated by elevated liver enzymes, mucosal haemorrhages and generalized pe-\n\nA. Hiatt et al. / The emergence of antibody therapies for Ebola\n\n55\n\ntechia could be reversed, leading to full recovery. ELISA and neutralizing antibody assays indicate that ZMappTM is cross-reactive with the Makona variant of Ebola. ZMappTM exceeds the ef\ufb01cacy of any other therapeutics described so far, and the results warrant further development of this cocktail for clinical use.\nNine individuals with symptoms of Ebola and laboratory con\ufb01rmed diagnoses volunteered to receive ZMappTM requested by their treating physicians under compassionate use. The de\ufb01ned treatment course was three doses of ZMapp at 50 mg/kg at three day intervals. Patients were monitored for infusion reactions during administration and were followed for progression of their Ebola infection. When possible, samples were collected for assessment of viral load. Decreases in viral load were observed after dose administration for each patient from whom pre- and post-dose samples were tested. Data gained from this case series highlights the urgent need for further investigation in a prospective clinical trial for patients diagnosed with Ebola.\n10. Summary\nThe antibody discovery effort to identify a treatment for Ebola has been signi\ufb01cantly assisted by plant-based expression technologies. First, the importance of plant production systems is derived from the rapid turnaround time from experimental results to expression of new or modi\ufb01ed mAbs or different combinations of mAbs. Due to the complexity of the Ebola infection and our lack of a complete understanding of the infection process, an iterative process of testing, modifying and changing mAb combinations was required for identi\ufb01cation of an optimal mAb cocktail. The plant systems are unique in their ability to engage in this iterative process [3]. Second, the ability to optimize the interaction of mAbs with appropriate Fc receptors by glycoengineering can be easily accomplished in plant systems [31,32] and has been speci\ufb01cally demonstrated for Ebola [34]. Finally, the plant system could provide large scale manufacturing [41,42] when plantbased manufacturing infrastructure is expanded.\nReferences\n[1] M.T. Osterhol et al., Transmission of Ebola viruses: What we know and what we do not know, mBio 6(2) (2015), e00137.\n[2] H. Ebihara et al., Molecular determinants of Ebola virus virulence in mice, PLoS Pathogens 2(7) (2006), e73.\n\n[3] A. Hiatt and M. Pauly, Monoclonal antibodies from plants: A new speed record, Proceedings of the National Academy of Sciences of the United States of America 103(40) (2006), 14645\u201314646.\n[4] X. Qiu et al., Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp, Nature 514(7520) (2014), 47\u201353.\n[5] A.L. Schmaljohn, Protective antiviral antibodies that lack neutralizing activity: precedents and evolution of concepts, Current HIV research 11(5) (2013), 345\u2013353.\n[6] M. Goeijenbier, J.J. van Kampen, C.B. Reusken, M.P. Koopmans and E.C. van Gorp, Ebola virus disease: a review on epidemiology, symptoms, treatment and pathogenesis, The Netherlands Journal of Medicine 72(9) (2014), 442\u2013448.\n[7] T. Maruyama et al., Ebola virus can be effectively neutralized by antibody produced in natural human infection, Journal of Virology 73(7) (1999), 6024\u20136030.\n[8] T. Maruyama et al., Recombinant human monoclonal antibodies to Ebola virus, The Journal of Infectious Diseases 179(Suppl 1) (1999), S235\u2013S239.\n[9] P.W. Parren, T.W. Geisbert, T. Maruyama, P.B. Jahrling and D.R. Burton, Pre- and postexposure prophylaxis of Ebola virus infection in an animal model by passive transfer of a neutralizing human antibody, Journal of Virology 76(12) (2002), 6408\u20136412.\n[10] P.B. Jahrling, J.B. Geisbert, J.R. Swearengen, T. Larsen and T.W. Geisbert, Ebola hemorrhagic fever: evaluation of passive immunotherapy in nonhuman primates, The Journal of Infectious Diseases 196(Suppl 2) (2007), S400\u2013S403.\n[11] D.J. Shedlock et al., Antibody-mediated neutralization of Ebola virus can occur by two distinct mechanisms, Virology 401(2) (2010), 228\u2013235.\n[12] J.M. Dias et al., A shared structural solution for neutralizing ebolaviruses, Nature Structural & Molecular Biology 18(12) (2011), 1424\u20131427.\n[13] A. Sanchez and P.E. Rollin, Complete genome sequence of an Ebola virus (Sudan species) responsible for a 2000 outbreak of human disease in Uganda, Virus Research 113(1) (2005), 16\u201325.\n[14] S.I. Okware et al., An outbreak of Ebola in Uganda, Tropical Medicine & International Health: TM & IH 7(12) (2002), 1068\u20131075.\n[15] J.A. Wilson et al., Epitopes involved in antibody-mediated protection from Ebola virus, Science (New York, N.Y.) 287(5458) (2000), 1664\u20131666.\n[16] A. Sanchez, S.G. Trappier, B.W. Mahy, C.J. Peters and S.T. Nichol, The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing, Proceedings of the National Academy of Sciences of the United States of America 93(8) (1996), 3602\u2013 3607.\n[17] V.E. Volchkov, V.A. Volchkova, W. Slenczka, H.D. Klenk and H. Feldmann, Release of viral glycoproteins during Ebola virus infection, Virology 245(1) (1998), 110\u2013119.\n[18] Z. Euler and G. Alter, Exploring the potential of monoclonal antibody therapeutics for HIV-1 eradication, AIDS Research and Human Retroviruses 31(1) (2015), 13\u201324.\n[19] A. Takada, H. Feldmann, T.G. Ksiazek and Y. Kawaoka, Antibody-dependent enhancement of Ebola virus infection, Journal of Virology 77(13) (2003), 7539\u20137544.\n[20] A. Takada, S. Watanabe, K. Okazaki, H. Kida and Y. Kawaoka, Infectivity-enhancing antibodies to Ebola virus glycoprotein, Journal of Virology 75(5) (2001), 2324\u20132330.\n[21] G.M. Ignatyev, Immune response to \ufb01lovirus infections, Cur-\n\n56\n\nA. Hiatt et al. / The emergence of antibody therapies for Ebola\n\nrent Topics in Microbiology and Immunology 235 (1999), 205\u2013217. [22] A. Takada, H. Ebihara, H. Feldmann, T.W. Geisbert and Y. Kawaoka, Epitopes required for antibody-dependent enhancement of Ebola virus infection, The Journal of Infectious Diseases 196(Suppl 2) (2007), S347\u2013S356. [23] T.W. Geisbert, L.E. Hensley, J.B. Geisbert and P.B. Jahrling, Evidence against an important role for infectivity-enhancing antibodies in Ebola virus infections, Virology 293(1) (2002), 15\u201319. [24] K. Mupapa et al., Treatment of Ebola hemorrhagic fever with blood transfusions from convalescent patients, International Scienti\ufb01c and Technical Committee, The Journal of Infectious Diseases 179(Suppl 1) (1999), S18\u2013S23. [25] J.M. Dye et al., Postexposure antibody prophylaxis protects nonhuman primates from \ufb01lovirus disease, Proceedings of the National Academy of Sciences of the United States of America 109(13) (2012), 5034\u20135039. [26] A. Takada, H. Ebihara, S. Jones, H. Feldmann and Y. Kawaoka, Protective ef\ufb01cacy of neutralizing antibodies against Ebola virus infection, Vaccine 25(6) (2007), 993\u2013999. [27] A. Marzi et al., Protective ef\ufb01cacy of neutralizing monoclonal antibodies in a nonhuman primate model of Ebola hemorrhagic fever, PloS One 7(4) (2012), e36192. [28] Z. Huang et al., High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system, Biotechnology and Bioengineering 106(1) (2010), 9\u2013 17. [29] Q. Chen, J. He, W. Phoolcharoen and H.S. Mason, Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants, Human Vaccines 7(3) (2011), 331\u2013338. [30] A. Giritch et al., Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors, Proceedings of the National Academy of Sciences of the United States of America 103(40) (2006), 14701\u201314706. [31] R. Strasser, F. Altmann and H. Steinkellner, Controlled glycosylation of plant-produced recombinant proteins, Current Opinion in Biotechnology 30 (2014), 95\u2013100. [32] A. Castilho et al., In planta protein sialylation through overexpression of the respective mammalian pathway, The Journal\n\nof Biological Chemistry 285(21) (2010), 15923\u201315930. [33] R. Strasser et al., Generation of glyco-engineered Nicotiana\nbenthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure, Plant Biotechnology Journal 6(4) (2008), 392\u2013402. [34] L. Zeitlin et al., Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant, Proceedings of the National Academy of Sciences of the United States of America 108(51) (2011), 20690\u2013 20694. [35] X. Qiu et al., Characterization of Zaire ebolavirus glycoprotein-speci\ufb01c monoclonal antibodies, Clinical Immunology (Orlando, Fla.) 141(2) (2011), 218\u2013227. [36] X. Qiu et al., Ebola GP-speci\ufb01c monoclonal antibodies protect mice and guinea pigs from lethal Ebola virus infection, PLoS Neglected Tropical Diseases 6(3) (2012), e1575. [37] X. Qiu et al., Successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies, Science Translational Medicine 4(138) (2012), 138ra181. [38] X. Qiu et al., Sustained protection against Ebola virus infection following treatment of infected nonhuman primates with ZMAb, Scienti\ufb01c Reports 3 (2013), 3365. [39] G.G. Olinger, Jr. et al., Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques, Proceedings of the National Academy of Sciences of the United States of America 109(44) (2012), 18030\u201318035. [40] J. Pettitt et al., Therapeutic intervention of Ebola virus infection in rhesus macaques with the MB-003 monoclonal antibody cocktail, Science Translational Medicine 5(199) (2013), 199ra113. [41] G.P. Pogue et al., Production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product using plant-based transient expression systems, Plant Biotechnology Journal 8(5) (2010), 638\u2013654. [42] V. Klimyuk, G. Pogue, S. Herz, J. Butler and H. Haydon, Production of recombinant antigens and antibodies in Nicotiana benthamiana using \u2018magnifection\u2019 technology: GMPcompliant facilities for small- and large-scale manufacturing, Current Topics in Microbiology and Immunology 375 (2014), 127\u2013154.\n\n\n",
    "authors": [
      "Andrew Hiatt",
      "Michael Pauly",
      "Kevin Whaley",
      "Xiangguo Qiu",
      "Gary Kobinger",
      "Larry Zeitlin"
    ],
    "doi": "10.3233/HAB-150284",
    "date": "2015-12-23",
    "item_type": "journalArticle",
    "url": "https://www.medra.org/servlet/aliasResolver?alias=iospress&doi=10.3233/HAB-150284"
  },
  {
    "key": "BFH8EQVB",
    "title": "Use of convalescent plasma in Ebola virus infection",
    "abstract": "The recent Ebola virus epidemics which threatened three West African countries (Dec.2014\u2014Apr.2016) has urged global collaborative health organizations and countries to set up measures to stop the infection and to treat patients, near half of them being at risk of death. Convalescent plasma\u2014recovered from rescued West Africans\u2014was considered a feasible therapeutic option. Efficacy was difficult to evaluate because of numerous unknowns (especially evolution of neutralizing antibodies), prior to the cessation of active transmission. This raises a large body of questions spanning epidemiological, virological, immunological but also ethical, sociological and anthropological aspects, alongside with public health concerns, in order to be better prepared to the next outbreak.. This essay summarizes efforts made by a large number of groups worldwide, and attempts to address still unanswered questions on the benefit of specific versus non-specific plasma on altered\u2014leaking\u2014vascular endothelia in Ebola infection.",
    "full_text": "Accepted Manuscript\n\nTitle: Use of convalescent plasma in Ebola virus infection\n\nAuthor: <ce:author id=\"aut0005\" author-id=\"S1473050216302002e16adcff0c44d2a4d4c43ee38805f18d\"> Olivier Garraud\n\nPII: DOI: Reference:\n\nS1473-0502(16)30200-2 http://dx.doi.org/doi:10.1016/j.transci.2016.12.014 TRASCI 2107\n\nTo appear in:\n\nPlease cite this article as: Olivier Garraud, Use of convalescent plasma in Ebola virus infection, http://dx.doi.org/10.1016/j.transci.2016.12.014\nThis is a PDF \ufb01le of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its \ufb01nal form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.\n\nUse of convalescent plasma in Ebola virus infection\nOlivier Garraud1,2\n1EA3064 University of Lyon, Faculty of Medicine of Saint-Etienne, 42023 Saint-Etienne cedex 02, France 2Institut National de la Transfusion Sanguine, 75015, Paris, France\nSummary The recent Ebola virus epidemics which threatened three West African countries (Dec.2014\u2014Apr.2016) has urged global collaborative health organizations and countries to set up measures to stop the infection and to treat patients, near half of them being at risk of death. Convalescent plasma\u2014recovered from rescued West Africans\u2014 was considered a feasible therapeutic option. Efficacy was difficult to evaluate because of numerous unknowns (especially evolution of neutralizing antibodies), prior to the cessation of active transmission. This raises a large body of questions spanning epidemiological, virological, immunological but also ethical, sociological and anthropological aspects, alongside with public health concerns, in order to be better prepared to the next outbreak.. This essay summarizes efforts made by a large number of groups worldwide, and attempts to address still unanswered questions on the benefit of specific versus non-specific plasma on altered\u2014leaking\u2014vascular endothelia in Ebola infection.\nKeywords Ebola virus disease; blood donation; therapeutic plasma; convalescent plasma; plasma therapy; neutralizing antibodies; transfusion; apheresis.\n1\n\nForeword (contextualization) The last outbreak of Ebola Virus Disease (EVD) from December 2013 to April 2016 has called worldwide attention and called attention to both strengths and weaknesses of global preparedness plans against health threats in the XXIst Century. The first cases of this last epidemic were confirmed in March 2014, and the situation has been identified as a priority by the WHO during summer 2014. Three West-African countries were considered epidemic: Guinea, Liberia and Sierra Leone; 36 cases have been exported to other West African countries, Europe and North America; and it is now estimated that there have been more than 28,600 cases of clinical disease, with 11,300 deaths (meaning near 17,300 survivors) [1-4]. However, a non-negligible number of exposed, clinically non-infected individuals have been identified, though with large uncertainty. The EVD epidemic has been declared terminated by the WHO by the end of March 2016, with only erratic cases remaining [4]. During a year and half (summer 2015 to the end of year 2016), several consortia, under the auspices of the WHO and benefiting from special grants by diverse public and private funds, in Europe and North America developed strategies to combat the virus and its spread. In addition to epidemiologic means and containment measures, there were basically three intervention options: the development of antiviral drugs, the rapid development of vaccine strategies, and the design of immune therapies. Regarding this 3rd option, there were several possible lines of action, i.e. principally the use of convalescent plasma (to be infused un-separated or fractionated into specific antibodies), or the engineering of neutralizing monoclonal antibodies [5]. Before these solutions could become available, the epidemic vanished and, for example, a large\n2\n\nnumber of plasma units were recovered from convalescent individuals that have not been effectively transfused to newly infected persons. Thus, clinical trials reporting experiences are limited in size and some investigators acknowledge that conditions were not optimized to effectively obtain definitive data.\nIntroduction Soon after the epidemic of EVD infection was recognized as a threat, public health authorities and the WHO made preparedness plans available; however, some scientific societies such as the World Apheresis Association (WAA) [6] along with other scientists [7,8] raised concerns about excessive delays in meetings the needs. WAA contributed an Open Letter to the Director of WHO to call attention to the potential benefit of plasmapheresis and transfusion of convalescent plasma, but also to the potential benefits of fresh plasma on leaking blood vessels [6]. Interest in the first part of the proposal has largely been shared with several consortia, while the second part gained attention with a more limited number of initiatives. The present State-of-the-Art paper, initially presented on the occasion of the 2016 Congress of WAA jointly with the XVIth Congress of the French Society, on April 27, in Paris, aims at briefly to report the use of convalescent plasma therapy in EVD infection and to introduce some information on the benefits of fresh, non-specific, plasma as well.\nConvalescent plasma and plasma therapy Convalescent plasma refers to a plasma therapy based on plasma or plasma derivatives obtained from donors having recovered from [in general] severe infection and infused into newly infected individuals. This type of therapeutic intervention is not novel, as it has been used regularly over a centenary to deal with most life threatening epidemics\n3\n\n(reviewed in [9]), until other therapeutic means were made available; however, it may still be an operative therapy, as interest has not vanished in more than 100 years (the first well documented occurrence being the Spanish Flu in 1917-1919) [10]). Many reports deal with periods where antiviral drugs were virtually absent, and several recent experiences have tested the effects of convalescent plasma on SARS, MERS, 2005 A/H5N1 flu, 2009 A/H1N1 flu, Chikungunya, etc. [11-13].\nQuestions raised by plasma therapy and convalescent plasma As a large experience has accumulated, questions raised to obtain, process and use convalescent plasma are now listed:\n- How to access blood donors? - Preference of plasma obtained in large volume by repeated apheresis over\nsmaller volumes of recovered plasma from whole blood? - How to guarantee ethical principles? - How not to avoid altering donors\u2019 health, especially if formerly sick? - How to solve technical problems in unfavorable environments (power supply,\nshipping logistics, quarantine and containment, viral biohazards, etc.)? - The value of extending testing for other infectious pathogens that can be sampled\nwith blood or plasma, in endemic countries? And to what extent? - The value of inactivating pathogens in plasma from apheresis or whole blood,\nregarding the risk of [other] infectious pathogen transmission? - How best to identify or select patients to benefit from this therapy: intent to care\nor compassionate? - How to insure that convalescent plasma contains sufficient neutralizing\nantibodies (NAbs)? How to define NAb development: what is the development\n4\n\ncourse, and in particular are there still persistent viruses by the time where Abs can be detected? How long after clinical resolution of symptoms is there a chance to obtain NAbs if any? Are all Abs neutralizing or are there also cross-reactive, potentially facilitating Abs? (as recently seen between Dengue and Zika viruses [14])? - How understanding the issue of concurrent infections and subsequent inflammation, as has been recently emphasized with the suspected benefit conferred by malaria infection [15]? - How to monitor each step to make sure that so-called convalescent donors were indeed infected and that newly infected patients are viraemic? - How to ensure global safety and quality at each step of the process? - Etc.\nPrinciples of plasma therapy for severe viral infections There are two assumptions: One major, and one minor. The major assumption is that convalescent plasma contains protective Abs, ascribed as NAbs, that are transferable from a symptom-free donor\u2014having however recovered from proven or documented infection\u2014to a newly infected patient. The use of plasma from non-human hosts is prohibited for the time being, for immunological but also cross-species infection safety reasons, but the question is still open regarding purified and virus-inactivated Ab preparations. The minor assumption is that plasma can convey other healing factors that may be therapeutic in hemorrhagic fevers even in the absence of NAbs. While the major assumption is not debated at all because it represents the essence of plasma therapy, the second one is theoretical and it postulates that plasma\u2014and neither serum nor purified Igs\u2014is the preferential support therapy; it addresses the issue of leaking vessels\n5\n\n(hemorrhagic fevers) only, as fresh plasma also contains factors that restore the endothelium glycocalyx [16]. It is interesting to consider that few consortia have considered this point, however, despite an interesting lesson that could have been learnt from the Lassa infection episodes which demonstrated benefit from plasma therapy in the 80\u2019s [17], but received\u2014in our opinion\u2014too little attention. A very recent report from the French Army Transfusion Service (CTSA) supports this hypothesis [18].\nPlasma therapy and EVD infections: from the proof of concept to the initiation of therapy The proof of concept for convalescent plasma therapy dates back to 2001 where Gupta et al demonstrated the neutralizing effect of mouse Abs in an experimental model [19]. Before consortia handled the collection and the transfer of Plasma or Abs, initial recommendations were issued by some of us (scientists/specialists), prior or concomitantly to the release of the WHO guidance for plasma collection (2014) [20], and the WHO interim guidance for Ethics review [21]. The \u201cEbola_Tx Consortium\u201d contributed a couple of publications on its organization and on the trial concept, along with other similar initiatives [22-29]. The \u201cEbola_Tx Consortium\u201d released its first report in the New Eng J Med in January 2016 [23], reporting basically a lack of evidence for the prevention of deaths by using convalescent plasma in EVD infection. Van Griensven however acknowledges that information was lacking at this time regarding existence of NAbs, optimal volumes to transfuse and optimal timing to collect, to cite some of the caveats [30]. More or less simultaneously, another American and European consortium reported that 81.5% of infected patients survived, independent of the type of the therapy; and the key message is that resuscitation means are essential in the care of EVD infected persons [31]. Meanwhile, there has been the very recent report\n6\n\nconcerning European care providers, infected in Africa, recovered from very severe clinical situations by careful resuscitation means and \u201cregular\u201d fresh frozen plasma [18].\nWhat next? Since the last outbreak, research has been extensive. There has been accumulated experience, with good news (indications that there are indeed protective NAbs [32]; vaccine perspectives [33,34]) and less good news (long virus persistence; etc. [35,36]). Processes proving effective to inactivate this and other viruses/infectious pathogens not only in plasma but also in whole blood are encouraging [37,38], especially if one considers the curative benefits of non-specific plasma. However, the protection of exposed health care professionals is still an issue. On the one hand, we have also learned from blood donors, as a non-negligible number of them have Abs (despite non necessarily NAbs) in blood, meaning that Ebola virus is not uniformly fatal and that there are exposed clinically non-infected persons, which is very encouraging news [39]. On the other hand, this EVD episode revealed how fragile the people are when they have to face such threat [40], not only physically speaking but also mentally, and this has called much attention to the necessity to be prepared with a more comprehensive plan, when the next outbreak comes (EVD or a similar threat) [41,42]. One would like to urge decision makers to be\u2014or more likely to stay\u2014prepared.\nConclusions This short life threatening virus outbreak has revealed strengths and weaknesses of organizations. Convalescent plasma therapy appears a first line answer to a number of critical sanitary crises: however, efficacy is difficult to evaluate and evidence based trials are challenging to conduct for many reasons. A lesson that has been learned from the\n7\n\nEVD outbreak is that resuscitation is seminal: it is perhaps more important than specific measures; of particular importance is the evidence that fresh plasma is per se therapeutic, irrespectively of its content of NAbs. This does not mean that research on protective Abs must not be further encouraged, as the next outbreak may well depend on NAbs, and not solely on non-immune healing factors; it means however that lessons from pathophysiology must not be ignored, and they must be included in the next preparedness plans.\nDisclosures (conflicts of interests) OG has had received invitations from Cerus-Europe, TerumoBCT-Europe, MacoPharmaFrance and OctaPharma-France over the past five years.\nAcknowledgements The author wishes to express gratitude to the \u201cFrench Doctors\u201d in general and to M\u00e9decins Sans Fronti\u00e8res\u2014MSF in particular, for their tremendous dedication to the sick all around the world, and more particularly during this EBD crisis; he extends his greetings to all allied health personnel having traveled in West Africa to assist local organizations; he also wants to acknowledge convalescent plasma donors. 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    "authors": [
      "Olivier Garraud"
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    "doi": "10.1016/j.transci.2016.12.014",
    "date": "02/2017",
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  },
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    "key": "6UY4K5PV",
    "title": "Ebola and blood transfusion: existing challenges and emerging opportunities",
    "abstract": "METHODS: A search has been performed using the premier scientific databases, WHO documents, and English language search engines. Of 278 potential articles that were identified using a fixed set of criteria, a convenience sample of eighty-two appropriate articles was chosen for this review.\nRESULTS: The current EBO outbreak is predominantly driven by various confounding riskfactors like: (1) frail health care system, (2) unique cultural and religious customs, (3) hugeshortage of skilled professionals, (4) no licensed therapeutic agents, (5) ill-prepared monitoring and early warning systems, and (6) strained budgets; all these have bolstered this epidemic. As lack of neither specific treatments nor reliable interventions to quickly quell this epidemic, WHO has indorsed \u2018blood transfusion as an empirical therapeutic modality\u2019. Currently, several clinical trials are underway, particularly the two Ebola candidate vaccines and several antiviral drugs and it has been observed that the initial results are quite promising. However, there are several daunting ethical and practical challenges ahead to stem off this outbreak.\nCONCLUSIONS: The Ebola-hit poverty stricken West-African countries struggle to contain the outbreak, due to lack of potent therapeutics. Consequently, blood-transfusion could serve as an ideal therapeutic modality to save millions of lives. Therefore, industrialized nations and international agencies must aid them to combat with this catastrophe. Besides, it must warrant further",
    "full_text": "See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/281778763\nEbola and blood transfusion: existing challenges and emerging opportunities\nArticle \u00b7 September 2015\n\nCITATIONS\n15\n2 authors, including:\nA K Karunamoorthi Jazan University 80 PUBLICATIONS 2,702 CITATIONS\nSEE PROFILE\n\nREADS\n338\n\nAll content following this page was uploaded by A K Karunamoorthi on 07 November 2015.\nThe user has requested enhancement of the downloaded file.\n\nEuropean Review for Medical and Pharmacological Sciences\n\n2015; 19: 2983-2996\n\nEbola and blood transfusion: existing challenges and emerging opportunities\n\nS. ABDULLAH1, K. KARUNAMOORTHI2\n1Laboratory Medicine Department, Faculty of Applied medical Sciences, Jazan University, Kingdom of Saudi Arabia 2Unit of Tropical Diseases, Division of Medical Entomology and Vector Control, Department of Environmental Health, Faculty of Public Health and Tropical Medicine, Jazan University, Kingdom of Saudi Arabia\n\nAbstract. \u2013 OBJECTIVE: The deadly Ebola\nvirus has been first known to mankind since 1976. In the past decades, Ebola outbreaks has often been ignored/neglected as erupted in the rural remote/isolated areas of Africa. The recent 2013-2014 epidemic is the most wide-spread with high incidence rates, morbidity and, mortality in the Ebola history. Eventually, the World Health Organization (WHO) has declared it as a \u2018Public Health Concern of the International Community\u2019. This scrutiny was conducted to initiate a serious debate on various aspects of Ebola, particularly blood transfusion as an empirical therapeutic modality.\nMETHODS: A search has been performed using the premier scientific databases, WHO documents, and English language search engines. Of 278 potential articles that were identified using a fixed set of criteria, a convenience sample of eighty-two appropriate articles was chosen for this review.\nRESULTS: The current EBO outbreak is predominantly driven by various confounding riskfactors like: (1) frail health care system, (2) unique cultural and religious customs, (3) hugeshortage of skilled professionals, (4) no licensed therapeutic agents, (5) ill-prepared monitoring and early warning systems, and (6) strained budgets; all these have bolstered this epidemic. As lack of neither specific treatments nor reliable interventions to quickly quell this epidemic, WHO has indorsed \u2018blood transfusion as an empirical therapeutic modality\u2019. Currently, several clinical trials are underway, particularly the two Ebola candidate vaccines and several antiviral drugs and it has been observed that the initial results are quite promising. However, there are several daunting ethical and practical challenges ahead to stem off this outbreak.\nCONCLUSIONS: The Ebola-hit poverty stricken West-African countries struggle to contain the outbreak, due to lack of potent therapeutics. Consequently, blood-transfusion could serve as an ideal therapeutic modality to save millions of lives. Therefore, industrialized nations and international agencies must aid them to combat with this catastrophe. Besides, it must warrant further\n\nmulti-layered interventions and interagency policies, in order to build an Ebola-free safe world in the near future.\nKey Words: Ebola, Blood transfusion, Ebola virus, West African\nCountries, Therapuetic agents.\nIntroduction\nEbola is a deadly infectious disease of humans and other primates and is often fatal. Ebola viruses (EBOV) cause a rash, reddened eyes, hiccups, substantial intravascular volume depletion and marked electrolyte abnormalities attributable to both internal and external bleeding1. It first emerged in a small remote village of the Democratic Republic of Congo (DRC) near the Ebola River, subsequently it\u2019s named as an \u2018Ebola hemorrhagic fever\u20192. Though EBOV was first identified in southern Sudan in 19763, but most likely occurred as early as 1972 in Tandala, DRC (formerly known as Zaire)4.\nThe recent outbreak erupted in Guinea in December 2013 but soon spurted into neighboring Liberia and Sierra Leone with a reported seventy percent of the case fatality rate5. It is unprecedented in terms of number of cases, mortality rate and deleterious socio-economic impact. Thereupon World Health Organization (WHO) declared Ebola as a \u2018Public Health Emergency of International Concern\u2019 on 8th August 20142. To date there is no specific preventive and curative therapeutic agent in existence, henceforth, EBOV remains to pose a serious public health threat to both civilian and military populations as bio-weapons6.\nThough, ever since the mid of 1970s there are several sporadic outbreaks reported, 2014 epidemic has broken down the entire civic society by killing thousands of people, destroying families\n\nCorresponding Author: Kaliyaperumal Karunamoorthi, MD; e-mail: karunamoorthi@gmail.com 2983\n\nS. Abdullah, K. Karunamoorthi\n\nand threatening the world. It is a matter of grave concern that urges us to find a countermeasure quickly to fight against the \u2018common enemy\u2019 of mankind. In these perspectives, the present communication becomes more significant and pertain.\nThis review is an attempt to initiate serious debates on various Ebola related issues like mode of transmission, high risk-groups, symptoms, existing therapeutic modalities, particularly blood transfusions. Furthermore, it is an effort to shed some limelight on the existing practical challenges and emerging opportunities to minimize the Ebola related illness and deaths by adopting blood transfusion as one of the empirical therapeutic modalities. We strongly believe that the outcome of this scrutiny could serve as a baseline data for public health care providers and policymakers to combat with Ebola outbreaks or epidemics effectively in the near future.\nMethods\nData Mining: Evidence Acquisition Identification of studies\nThe search strategy and terms were developed collaboratively with the assistance from the information specialists. An appropriate search was performed in PubMed, Google Scholar, Web of Science, EMBASE, Scopus, World Health Organization\u2019s WHOLIS, Research gate, and Academic Premier databases in English, using the terms: \u201cEbola virus disease /Recent outbreak\u201d OR \u201cEbola and blood transfusion\u201d, \u201cEbola and Public Health\u201d, \u201cEbola transmission\u201d, \u201cEbola and Human Health\u201d \u2018 Ebola and West Africa\u2019 and \u201cEbola vaccine\u201d to include articles published without time limitations i.e., from earliest to most recent (December 2014).\nPotentially relevant articles (in all languages) and websites were accessed in order to review the full-text. Besides, references cited by each relevant paper, review, and book chapters were also scrutinized to identify additional potential papers. Furthermore, the reference lists of all articles were hand-searched, and the full text of those references that appeared relevant to Ebola and blood transfusion were retrieved. The exclusion and inclusion criteria for choosing the appropriate research articles, notes and reviews were shown in Figure 1 for this narrative review, and their bibliographic details (authors, title, full source, document type and addresses) have been downloaded and maintained in a \ufb01le.\n\nResults\nThe study selection process is given in Figure 1 as a flow diagram. Of 228 potentially relevant, unique citations from all literature searches, sixty-one studies met the inclusion criteria. Twentyseven studies were empirical research studies; three were booked chapters, two of them is from conference and three involved systematic reviews and meta-analyses, with the rest twenty-six involving case reports or press releases. The majority of studies was carried out in the WHO defined African region (n=48), and the remaining studies (n=13), covers the rest of the world; the majority of them have been published later than 2012. Meta-analysis was not performed as the included studies were heterogeneous in important aspects, including: populations (different ages and settings), and study designs (cross-sectional, casecontrol, cohort).\nFiloviruses Filoviruses are lager, negative stranded, non-\nsegmented RNA viruses with a characteristic of thread like structure, henceforth the family name is Filoviridae (Latin filum = thread). Over the past decades, several natural outbreaks have been reported in the DRC, Sudan, Uganda, Angola, and Gabon6. Both Marburg and Ebola species are morphologically identical, but vary in length7. They are imposing potential threats to humans as well as non-human primates8. Filovirus illness is characterized by fever, myalgia, headache, and gastrointestinal symptoms, and patients may also develop a maculopapular rash. Death has often been correlated with increased viremia, convulsions, and disseminated intravascular coagulation9.\nEbola Virus: An Old Enemy and Recent Killer\nIt is an ancient one, having split off from other viruses dating back thousands of years with a relatively constant mutation rate10. Filoviridae includes three genera viz., Cuevavirus, Marburgvirus, and Ebola virus. EBOV is a filovirus with a 19-kb, non-segmented, negative-strand RNA genome that encodes seven viral proteins11. Till date, five genetically distinct subspecies have been identified, namely Zaire, Bundibugyo, Sudan, Reston and Ta\u00ef Forest. However, three species [(Bundibugyo Ebola virus (C\u00f4te D\u2019Ivoire); Zaire Ebola virus (Democratic Republic of the Congo, formerly Zaire); and Sudan\n\n2984\n\nEbola and blood transfusion: existing challenges and emerging opportunities\n\nFigure 1. The exclusion and inclusion criteria, for choosing the appropriate research articles, notes and reviews for this narrative review.\n\nEbola virus (Sudan)] have been associated with larger outbreaks in Africa. The recent 2014 West African outbreak was caused by the Zaire Ebola virus (ZEBOV)2, with a ninety percent of mortality rate12 and it cannot further mutate and become more contagious10.\nExisting Ebola Virus Strains In 1976, the first outbreak of Ebola (Ebola-Su-\ndan) was reported in southern Sudan and a few months later, the second Ebola virus emerged from Yambuku, Zaire, Ebola-Zaire (EBOZ). The third strain of Ebola, Ebola Reston (EBOR), was first identified in 1989 when infected monkeys were imported from the Philippines to Reston, Virginia, United States of America. The last\n\nknown strain of Ebola, Ebola Cote d\u2019Ivoire (EBO-CI) was discovered in 1994 when a female ethologist was performing a necropsy on a dead chimpanzee from the Tai Forest, Cote D\u2019Ivoire, got accidentally infected herself during the necropsy13.\nThe Deadly History of Ebola Outbreaks It is not a new disease of mankind and typical-\nly occurs as deadly outbreak in the resource-constrained tropical Sub-Saharan Africa. Since mid of 1970s to until December 2013, 1,716 confirmed cases have ever been reported2,10. To date, the ongoing 2014 West African Ebola outbreak is the largest, which affects many African countries viz., Guinea, Sierra Leone, Liberia, Mali, and\n\n2985\n\n2986\n\nFigure 2. The previous Ebola virus disease outbreaks.\n\nS. Abdullah, K. Karunamoorthi\nNigeria. As of 7th January 2015, 20 747 suspected cases and 8235 deaths had been reported14. Past Ebola outbreaks have been elaborately illustrated in terms of number of cases and deaths in Figure 22.\nHigh Risk Group Whoever in close contact with Ebola patients\nare at the highest risk of infection, notably healthcare providers, close family members, and friends as they have higher probabilities to be infected by blood or body fluids of patients10. Fortunately, Ebola survivors cannot serve as a source of infection. It spreads rapidly during outbreaks within healthcare settings and could be easily evaded by wearing appropriate personal protective equipment10. As of 23 October 2014, nearly 450 health care workers (HCWs) [eighty-inGuinea; 228 in Liberia; eleven in Nigeria; 127 in Sierra Leone; one in Spain; and three in the US] are known to have been infected and a total of 244 HCWs have died during 2014 outbreak alone15,16. Quite a considerable number of people have acquired infection by means of attending the burial ceremonies of Ebola victims17.\nEbola Recent outbreak: a Public Health Emergency of International Concern Ground Zero in Guinea: the outbreak Smoulders\nCurrently, West Africa is experiencing the world\u2019s largest Ebola outbreak18. It has erupted on 6th December 2013, when a two-year-old male child Emile (the first known case of 2014 Ebola epidemic) in Meliandou village, Gu\u00e9ck\u00e9dou Prefecture, in the Nz\u00e9r\u00e9kor\u00e9 Region of southern Guinea got infected19-21. It is one of the most difficult villages to reach by road and it is a two-day drive from the national capital of Conakry, followed by a long walk through the dense rain-forest22. This outbreak\u2019s \u2018hot zone\u2019 (Meliandou) is located in a triangle-shaped forested area, where the borders of Guinea, Liberia and Sierra Leone shared. As a result of decades of civil wars and unrest, these countries has deeply been impoverished and the health infrastructures have severely been damaged19.\nDeath Toll and Morbidity As of 7th January 2015, a total of 20 747 cases\n(confirmed, probable, and suspected cases of Ebola) and 8 235 deaths have been reported in five Ebola-hit countries viz., Guinea, Liberia, Mali, Sierra Leone, and the USA and in three\n\nEbola and blood transfusion: existing challenges and emerging opportunities\n\npreviously affected countries viz., Nigeria, Senegal and Spain14. Reported case incidence is slightly increasing in Guinea, declining in Liberia, and may still be increasing in Sierra Leone. The case fatality rate across the three most-affected countries in all reported cases with a recorded definitive outcome is seventy-six percent; in hospitalized patients the case fatality rate is sixty-one percent23. WHO has estimated that it may take nearly six-to-nine months to contain the ongoing outbreak and during this period of time it may likely to claim another 20,000 lives. Recent WHO (2014)24 report indicates that cases in Liberia are under-reported. It may be due to asymptomatic infections, which have also been observed in earlier outbreaks too25. Ebola imposes an urgent public health threat not only to Africa but also to the rest of the world.\nDriving Forces of Recent Outbreak Past outbreaks have flared up in the remote\nand isolated forested communities and these outbreaks die out, like the flare-up in the central African countries like DRC in the past few months26. However, the recent 2013-2014 outbreak is the largest and most complex in terms of duration, number of people affected, and geographic extent27. Currently, Ebola has been considered as not only as a public health emergency, but also as a \u2018poverty\u2019, \u2018infrastructure\u2019, as well as a \u2018educational\u2019 crisis26. This epidemic (20142015) driven by various sociological, ecological, and environmental determinants, has both directly and indirectly influenced the emergence of Ebola in the poverty pervasive West African countries.\nHigh-risk of confounding factors (Alexander et al, 2014)27:\n\u2022 Free people movement \u2013 rural-to-urban migration as well as extensive movement of people within and across borders.\n\u2022 Decades of civil war/unrest \u2013 have severely weakened the basic and health care sector infrastructures.\n\u2022 Behavioral and cultural practices \u2013 traditional customs/practices like burial ceremony traditions and rites.\n\u2022 Bush meat consumption \u2013 principal mechanism of EBOV spillover from wildlife reservoirs to humans.\n\u2022 Traditional medicine and cures \u2013 it delays the Ebola patient to access life-saving quality healthcare.\n\nNatural Host and Mode of Transmission Fruit Bats (Family: Pteropodidae) are serving\nas a natural host. EBOV introduced into the human through close contact through body fluids and other secretions of infected wild-animals viz., chimpanzees, gorillas, fruit bats, monkeys, antelope and porcupines found ill or dead5. Once infection is established, it can be transmitted through human-to-human via direct contact with the blood, secretions, organs or other bodily fluids (including but not limited to urine, saliva, sweat, feces, vomit, breast milk, and semen) of infected human28, and animals [fruit bats or primates (apes and monkeys)]. Human handling and consumption of contaminated bushmeat (Ebola infected animals) has been cited as a major source of transmission to human5.\nA study found that the case mortality rates highly correlate with the mode of transmission. Overall, hundred and eighty percent mortality was observed among those exposed via contaminated needles, and close-contact with infected people, respectively29. Aerosol transmission has also been reported but only in the laboratory settings30 and is rare or absent in natural outbreaks31. The contaminated surfaces, materials (bedding, clothing), and objects (needles and syringes) could also transmit infection. Therefore, healthcare professional are at the highest risk of infection, when the transmission control protocols (TCP) are not strictly practiced5. People remain infectious as long as their blood and body fluids (including semen and breast milk), contain the Ebola virus5. There is no evidence that mosquitoes or other insects can transmit EBOV10.\nMode of Disease Causing Mechanism and Pathophysiology\nEbola virus kills cells, making some of them to explode. Subsequently, it wrecks the immune system, causes heavy internal hemorrhage and electrolytes abnormalities. It virtually damages every organ32, often leading to death31,33. Distinguishing the symptoms of Ebola from other infectious diseases is quite a challenging task. However, it can be diagnosed by performing standard serological diagnostic tests like antibody-capture enzymelinked immunosorbent assay (ELISA), antigencapture detection, serum neutralization, reverse transcriptase polymerase chain reaction (RT-PCR) assay, electron microscopy and virus isolation by cell culture5. Since, samples are potential biohazard risks, diagnosis should be performed under highest-level of biosafety containment conditions.\n\n2987\n\nS. Abdullah, K. Karunamoorthi\n\nFigure 3. Ebola virus transmission cycle (Source: CDC 2014)10.\n\nEbola virus Infection Treatment and Management\nAs there are currently no licensed vaccines or therapeutics to prevent or treat any filovirus infection, particularly Ebola30, it has emerged as a potential global public health threat34. Prior EVD outbreaks have been controlled by implementation of integrated strategies like identification of cases, contact tracing, quarantine, early diagnosis, supportive care, infection control, and safe burial practices. Fortunately, the negative impact of current 2014 epidemic illustrate the importance of effective novel preventive and curative arsenal35 to fight against the Ebola. Though to date no approved therapeutic agents exist to treat or prevent Ebola, the following therapeutic options have been administered to curb Ebola-associated illness and death (Table I).\nImportance of Blood Transfusion to Treat EVD cases\nDue to the absence of effective vaccine and reliable drugs to neutralize the Ebola virus, the treatment or management of Ebola patients have\n\nbeen mainly focused on symptomatic treatments with available antiviral agents and blood transfusion17. Besides, early supportive care with rehydration and electrolyte correction, monitoring basic physiological functions, protein deficiencies, has often been administered to improve the survival of Ebola patients5.\nIn 1995, eight Ebola patients were transfused with convalescent whole blood (CWB) and plasma (CP) of EVD survivors to treat illness in the DRC. Donated blood contained only IgG EBO antibodies and no antigen. However, antigens were detected in all the recipients just prior to the transfusion and this empirical study found that among the eight only one patient (12.5%) died. The percent of the mortality rate is significantly lower than other EBO epidemic in Congo and rest of the world45. The reason for the lower- mortality rate remains to be scientifically authenticated and warranted by further clinical studies to evaluate and elucidate the passive immune therapy.\nIn addition, currently several blood transfusion studies are underway by enrolling patients of 2014 epidemic and the preliminary results are\n\n2988\n\nEbola and blood transfusion: existing challenges and emerging opportunities 2989\n\nTable II. Existing therapeutic options to treat Ebola virus disease.\n\nName of the S. No. therapeutic agent\n\nNature\n\nMode of action\n\nRemarks\n\nReference (s)\n\n1.\n\nAVI 6002 (AVI-7537) Drug\n\nBlocking viral protein\n\nIt helps monkeys tosurvive from Ebola for some sixty-to-eighty percent. It has shown human tolerability in early studies.\n\nAxtelle et al. 201236\n\n2.\n\nHyperimmune globulin Prepared by purifying and\n\nNeutralizing antibody\n\nProtective in monkeys, but are not currently\n\nKudoyarova-\n\nconcentrating plasma of\n\nagainst Ebola virus\n\navailable and would not be expected before\n\nZubavichene et al.\n\nimmunized animals or previously\n\nmid of 2015\n\n199917; Maron\n\ninfected humans with high titers.\n\n201437\n\n3.\n\nKZ52\n\nA neutralizing human monoclonal antibody\n\nIt protects guinea pigs from lethal Ebola Zaire virus challenge\n\nKZ52 is a promising candidate for immunoprophylaxis of Ebola virus infection\n\nParren et al. 200238\n\n4.\n\nFavipivavir\n\n(T-705 or Avigan)\n\nAntiviral drug approved for influenza in Japan\n\nBlocks the replication of many RNA viruses, particularly it inhibits the RNA-dependent RNA polymerase (RdRP) of influenza\n\nNeed to be used at much higher doses. Though the efficiency of this drug against human Ebola infection remains unclear, some positive results are emerging in animal experiments\n\nFuruta et al. 200939, 201340\n\n5.\n\nInterferons\n\nTargeting and disabling the VP24 protein\n\nThough they did not increase overall survival, but delay the death. Nevertheless, it remains unclear which interferon to use, when and at what dosage regimen to obtain optimal results.\n\nBasler and Amarasinghe 200941\n\n6.\n\nBioCryst\n\nDrug\n\nIt needs more animal treatment data before it could be considered.\n\n7.\n\nZMapp (a mixture of\n\nUnder development as\n\nthree antibodies)\n\na treatment for Ebola\n\nvirus disease\n\nLike intravenous immunoglobulin therapy, ZMapp contains neutralizing antibodies that provide passive immunity to the virus by directly and specifically reacting with it in a \"lock and key\" fashion.\n\nIt was first used experimentally to treat some people with Ebola virus disease during the 2014 West African Ebola outbreak, but as of August 2014 it had not yet been tested in a clinical trial to support widespread usage in humans; it is not known whether it is effective to treat the disease, nor if it is safe\n\nQiu et al. 201342; WHO 2014g, h43,44\n\nS. Abdullah, K. Karunamoorthi\n\nquite promising and encouraging too46-48. The unprecedented attack of Ebola on world community has created a high demand of blood transfusion than ever before2. It remains to be considered as the best option to treat Ebola virus disease in the absence of effective therapeutic agents.\nAccordingly, during the ongoing 2014 EVD outbreak, whole blood and plasma obtained from Ebola recovered patients have been prioritized for reconnaissance, as one of the empirical treatment modalities49 for the patients with early EVD clinical symptoms and manifestations. As convalescent plasma has been successfully administered to treat various infectious diseases, this modality is biologically plausible50 and could be efficacious too. In addition, by reviewing all the existing experimental potential therapies and vaccines, the WHO experts indicate that treating Ebola patients with blood transfusions from survivors of EVD is the top most priority17 to save hundreds of thousands of lives.\nEbola and Blood Transfusions Mode of Mechanism\nSince the survivors of Ebola infection typically produce effective antibodies against the virus, the transfusions of their blood into a newly Ebola infected individual could save life17. As none of the existing/considered Ebola regimes have been adequately tested in humans, the convalescent wholeblood (CWB) and serum could serve as therapeutic agents against Ebola virus. Indeed, it has to be transfused safely with careful screening17, or else mismatched blood type and infected blood could cause a few severe reactions that may be lifethreatening too. Therefore, both WHO and public health experts debated on the pros and cons of treating Ebola patients with transfusions of whole blood and plasma of EBVD survivors51.\nIn the past, CWB modality has been adopted empirically among a small group of patients, with promising results45. However, the use of CWB is technically more complex and demands more facilities and skills. Consequently the eventual use of CP in Guinea, Sierra Leone and the Democratic Republic of Congo will depend on the availability of technical expertise51. Finally, WHO (2014)49 has issued an interim guidance to national health authorities and blood transfusion services to outline the steps required to collect CWB or CP from EVD recovered patients for transfusion to patients with early EVD, as an empirical treatment modality. It includes the following phases:\n\nKey phases for the Blood Transfusion Identification\nThe EVD recovered patients (those have been discharged from the recognized Ebola treatment centers or units) could serve as potential donors (after 28 days of discharge) for CWB/CP. It is important to note that the Ebola neutralizing antibodies are expected to be most effective when CWB/CP is sourced from the epidemic/endemic areas of active Ebola virus (EBOV) transmission. However, in circumstances where the demand is high and the system is challenged by an overwhelming number of active EVD patients, and CWB/CP could also be sourced from the places linked to the current EVD outbreak in West Africa where the outbreak has come under control49.\nScreening/Pre-Donation Tests (WH O 2014)49 \u2022 Estimation of donors hemoglobin concentration \u2022 ABO (A, B, AB and O) grouping and RhD\ntype screening tests for blood borne infectious diseases like human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), syphilis co-infections and other locally transmitted infections like malaria, as applicable \u2022 Titration of total Ebola neutralizing antibodies to identify the potential donor, particularly if the donor is willing to continue serving as CWB/CP source.\nReview of Pre-Donation Tests It is one of the critical phases in empirical\nblood transfusion treatment. Results of the predonation tests must be carefully reviewed. Donors, those are shown negative for all TTI tests and fulfilling the remaining all other criteria should be chosen for CWD/CP donations. If the duration between predonation test and donation exceeds forty-eight h, then the routine TTI tests must be repeated49. A minimum period of twelve and sixteen weeks for males and females, respectively is needed for further blood donation.\nSelection of EVD Patients and Blood Sample Collection\nOnly EVD confirmed patients in the early stages should be selected for CWB/CP transfusion49. Two venous blood samples (5 mL) each must be drawned from the patient prior to transfusion; (a) one in EDTA for a plasma, while (b) another one in a plain tube (without anticoagulant) for a serum sample for the (a) ABO and\n\n2990\n\nEbola and blood transfusion: existing challenges and emerging opportunities\n\nRhD blood grouping, and cross-matching and, (b) baseline viral load assay. Plain sample is used to measure the viral-load and other tests. Furthermore, prior to discharge of recovered patients, two additional five mL plain samples have to be collected to analyze the viral-load in the successive days. Residual serum of the blood samples should be stored in aliquots for retrospective antibody or any other future examinations49.\nSelection of Convalescent Whole Blood or Plasma Units\nABO and RhD matched blood or plasma units need to be chosen for transfusion. RhD-ve units should be transfused to RhD-ve women of childbearing age (if possible). However, If the RhD group of the patient is not known or in case of non-availability of RhD specific group, blood matched only for ABO group may be used for treatment49:\n\u2022 In order to minimize the risk associated with handling of infectious blood samples, cross matching of patients\u2019 serum and donors\u2019 red cells, can be omitted if ABO group compatible CWB/CP is selected.\n\u2022 If it is not possible to test the patient\u2019s ABO group or if ABO matched CWB/CP is not available then: (1) for whole blood transfusion - group O convalescent whole blood, ideally from donors with low titer anti-A and anti-B, can be transfused; while (2) for plasma transfusion - group AB convalescent plasma separated by centrifugation should be used.\n\u2022 Non ABO-matched CP could also be considered if group AB plasma is not available, but should preferably be group A or B.\n\u2022 Transfusion needs to be done within twentyfour hours of CP preparation in order to obtain higher RBC concentration.\nTransfusion of Convalescent Whole Blood or Plasma\nCWB/CP units should be transfused to the EVD patients by following the clinical transfusion protocols. One unit of CWB [equals is 450 mL (just under a pint)] have to be transfused for adult patients. In the absence of evidence, 400500 mL of CP in two doses of 200-250 mL each, separated from two different WB donations, should be considered for adult patients, and whereas, in the case of pediatric CWB/CP transfusion, a dose of 10 mL/kg could be ideal based on the considerations of blood volume49.\n\nPatient Monitoring Transfused EVD patients must be closely\nmonitored in order to assess the patient improvement in terms of clinical conditions and the concomitant decline of virus load detected in plasma18, as well as to evaluate the effectiveness of the treatment. In addition to clinical monitoring, Ebola antibody levels and other tests are also extremely important. Besides, standard case reporting forms must also be maintained to monitor all potential interventions for EVD49, 52.\nHowever, a few public health experts have raised their concern regarding adopting blood transfusion as a therapeutic modality for the Ebola treatment: (1) the emergence of a black market trading of blood of EVD survivors2, (2) risk of escalating transmission of several infectious diseases, particularly killer diseases like human immunodeficiency virus (HIV), malaria and viral hepatitis infection and, (3) other infectious diseases46, 53. Nevertheless, blood transfusion is one of the easily adoptable empirical therapeutic modalities with the potential to be implemented immediately on a large scale to address the current epidemic54. If healthcare providers strictly adopt the WHO standard protocol called \u2018Interim guidance to national health authorities and blood transfusion services,\u2019 it shall be one of the safest as well as suitable therapeutic modalities to save lives.\nThough there is as yet no licensed treatment proven to neutralize the virus but a wide-range of blood, immunological and drug therapies are under development. At the moment, two potential Ebola candidate vaccines are undergoing for clinical and immunological evaluation55. One experimental drug viz., ZMapp was administered to some of the patients (including two US aid workers) and a few of them died. Therefore, there is no substantial evidence to prove that the ZMapp has saved the lives of Ebola patients or had no effect17.\nExisting Major Challenges and Emerging Opportunities\nThe potentialities of Ebola fighting antibodies are not identical and it may trigger a few adverse side-effects and negative sequelae17. The edict, from some two-hundred public health experts and WHO scientists, was a follow-up to an earlier Ebola ethics review on October 2014 and concluded that blood transfusion would be ethical to transfuse patients experimental therapies when and if they are easily obtainable17: (1) possible to\n\n2991\n\nS. Abdullah, K. Karunamoorthi\n\nrecruit large number of potential donors, (2) collection and, (3) screening of any infectious diseases to potentially save Ebola patients.\nNecessity of Adequate Healthcare Professionals and Facilities\nA strong health system immensely minimize a country\u2019s vulnerability to potential health risks in terms of disease outbreaks and catastrophes with a high-level of preparedness to mitigate any public health crises. Subsequently, it ensures quality healthcare for their citizens56. However, if health systems are ill-equipped to deal with outbreaks and natural disasters, the populations can be very much vulnerable to several infections ultimately death57. The Ebola hit West African countries like Guinea, Sierra Leone and Liberia have very fragile health systems, lacking human and infrastructural resources to deal with the outbreaks since they have recently emerged from decades of civil war and insurgencies2. The 2014 Ebola outbreak highlights how an epidemic can proliferate rapidly and pose huge problems in the absence of a strong health system56.\nWhen the recent Ebola outbreak erupted, the capacity of health systems in Guinea, Liberia and Sierra Leone was limited56. Essential health-system functions were not performing well and this hampered the development of timely response to the outbreak. Besides, there were inadequate qualified health professionals58 as well as infrastructure, logistics, health information, surveillance, governance and also, the drug supply chains were feeble56.\nSharing Knowledge and Resources: a Key to Saving Lives\nIn the Ebola-hit countries, all the febrile individuals need to be screened for Ebola and even if they are found to be negative, they still need to be treated for Ebola. Besides, normal routine services like paediatric, antenatal, safe delivery and postnatal services should be assured while dealing with the direct and indirect effects of epidemic. Otherwise breakdown of general health services may slay more people than the epidemic56. In the remote rural areas the health system is virtually nonexistent or if it does, it often runs down a clinic with shortage of essential life-saving medicines and these countries must improve with their national healthcare sectors immediately.\nSince these third world countries lack resources and funds to implement effective inter-\n\nvention strategies, appropriate long-lasting workable strategy has to be framed by bringing all potential stakeholders. It is extremely important to fight against the \u2018common enemy\u2019, the so called \u2018Ebola\u2019. It must include various non-governmental organizations, civil society and international organizations to incentivize the national health systems, both to mitigate the direct consequences of the outbreak and ensure all essential health services being delivered56. It could pave the way to combat with the Ebola related illness, deaths as well as to minimize the avoidable deaths due to various other infectious diseases56. Besides, international donors, and agencies must assist them to bolster their health system to develop stronger disaster preparedness for the future outbreaks.\nMaximum Use of Supportive Therapy (MUST)\nThe purpose of the MUST is to save the lives. It includes intravenous (IV) drips to substitute the excessive fluid loss due to diarrhea and vomiting. Balancing of electrolytes such as calcium or potassium could prevent kidney and heart failure. Furthermore, nasogastric tubes for feeding and as well as diagnosis and treatment of secondary infections like malaria could be useful to minimize the Ebola related illness and deaths. In addition, MUST might reveal side-effects of new drugs that would otherwise be masked by Ebola symptoms, and it could reduce the rate of complications that might be incorrectly blamed on a drug59.\nIntegrative Approaches Indeed interagency policies for outbreak de-\ntection and rapid response is extremely important. Besides, understanding the cultural and traditional risk factors within and between nations could be useful to develop appropriate communication strategies to generate awareness among people. In addition, the regional coordination and collaboration, particularly with governments and health ministries throughout Africa27 could pave the way to minimize Ebola related illness and deaths. Pandey et al60 urged the effective integrative approaches like case isolation, contact-tracing with quarantine, and sanitary funeral practices must be strictly implemented in order to contain the Ebola outbreak. It has been reported that the actual number of Ebola infection underreported as much as seventy-five percent are due to several factors and it has torn the society in the\n\n2992\n\nEbola and blood transfusion: existing challenges and emerging opportunities\n\npoverty stricken countries. This has to be addressed immediately unless otherwise a single infected individual is enough to create Ebola outbreak.\nFormulation of Effective Chemotherapy Though currently we have a few anti-viral\ndrugs to treat several viral illnesses, there is no approved specific therapeutics to treat filovirus infections. It is a matter of deep and grave concern and pressing need to develop both preventive and curative antiviral agents against filovirus infection6. The experimental Ebola drug called \u2018ZMapp\u2019 has been administered to a limited number of Ebola victims and the results are quite promising. Notably the drug (ZMapp) has proved as a potent antiviral agent in monkeys. However, further clinical trials are yet to be conducted to demonstrate its safety and efficacy in human27. The WHO expert panel has considered the outlook for another seven potential Ebola drugs, but none of them has been proved of effectiveness against Ebola in humans.\nEbola Vaccine Development and Clinical Trials\nThe unprecedented 2014 EVD epidemic has prompted an international response to accelerate the availability of a preventive vaccine. Two vaccine candidates viz., chimpanzee adenovirus serotype 3 (ChAD3-ZEBOV) and recombinant vesicular stomatitis virus (rVSV-ZEBOV) are currently being tested in humans. Both vaccines have shown to be safe and efficacious in animals. Further Phase 1 results are expected to emerge during December 2014 to January 201555.\n\u2022 Phase I clinical trials of the cAd3-ZEBOV vaccine in healthy adults are nearing completion in the United Kingdom, United States, Mali and Switzerland, whereas, the rVSV-ZEBOV vaccine, trials are well advanced or near completion in Canada, the United States, Gabon, Germany, and Switzerland. Trials in Kenya are begin September 2014.\n\u2022 Phase II clinical trials of the cdA3-ZEBOV will test the safety and potential to induce an immune response in larger numbers and in broader populations, particularly among children. These trials are expected to begin in Cameroon, Ghana, Mali, Nigeria and Senegal in early 2015.\n\u2022 Phase III clinical trials are planned to start in the first quarter of 2015 in Guinea, Liberia and\n\nSierra Leone to assess the extent to which the vaccines protect against EVD and to gauge the feasibility of full deployment.\nBesides, two other vaccines \u2013 one developed by Johnson and Johnson and the other by Novavax \u2013 are in due to enter clinical trials in the very near future55.\nCost-free Ebola Vaccination WHO Director-General (Dr Margaret Chan)\nhas indicated that developing Ebola vaccine is not a profit-driven industry, as the victims are poorest people to procure vaccine. At least in the humanity ground the international stake holders and concerned authorities must initiate to develop low-cost potential vaccine and all people must be vaccinated at least in the Ebola-prone African countries. However, with the available existing resources we can substantially contain the Ebola outbreaks by generating awareness and creating supportive community.\nConclusions\nIndeed, EVD remains one of the most dreadful infectious diseases and the recent 2014 outbreak is the most devastating in terms of fatality rate in recorded Ebola history. At the moment, there is no effective curative agents to fight against our common enemy. WHO scientists and a group of key public health experts endorse the blood transfusion as an empirical modality by considering various existing therapeutic options to contain this deadliest outbreak. Nevertheless, it is one of the challenging tasks to implement in the resource-limited settings of tropical sub-Saharan Africa. Therefore, the following interventions must be adopted to address the existing major challenges effectively and immediately: (1) pinpointing the virus\u2019s source, (2) early case detection, (3) quarantine the infected people, (4) tracking and treating the Ebola patients, (5) providing supportive care, (6) free supply of personal protective equipment (PPE) in Ebola epicenters, (7) development and application of simple diagnostic tools and potent therapeutic interventions, (8) identifying and understanding the emerging viral strains through gene sequencing, (9) building a strong global health infrastructure/network, (10) generating the awareness through print and Emedia (11) patients who died of Ebola should be cremated or buried promptly in a hermetically\n\n2993\n\nS. Abdullah, K. Karunamoorthi\n\nsealed casket and, (12) community engagements through social mobilization.\nBesides, presently several clinical trials are underway to assess the potentialities of various anti-viral agents and vaccines against EBOV and the preliminary results are quite encouraging. Until acquiring reliable therapeutic agents, we can adopt blood transfusion as therapeutic modality by strictly following the WHO standard protocol in order to avoid any undesirable effects. Furthermore, strong regional, national and international multilayered collaborations by bringing all the stakeholders is extremely important to address the existing challenges to build a Ebola-free healthiest society in the near future.\n\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013 Acknowledgements\nThe authors would like to acknowledge Ms. L. Melita for her sincere assistance in editing the manuscript. Our last but not least heartfelt thanks go to our colleagues from the Department of Environmental Health, Faculty of Public Health and Tropical Medicine, Jazan University, Kingdom of Saudi Arabia, for their kind support and cooperation.\n\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013\u2013-\u2013\u2013\u2013 Conflict of Interest\nThe Authors declare that there are no conflicts of interest.\nReferences\n1) RUZEK B. EDITED BY SUNIT K. Singh, Daniel. Viral hemorrhagic fevers. Boca Raton: CRC Press, Taylor & Francis Group, 2014; p. 444.\n2) WHO. Ebola virus disease. Fact sheet N\u00b0103. Updated September 2014. Available at: http://www.who.int/mediacentre/factsheets/fs103/ en/ [accessed on 30th November 2014].\n3) TEAM ROAWIS. Ebola haemorrhagic fever in Sudan, 1976. Bull World Health Organ 1978; 56: 247.\n4) HEYMANN D, WEISFELD J, WEBB P, JOHNSON K, CAIRNS T, BERQUIST H. Ebola hemorrhagic fever: Tandala, Zaire, 1977-1978. 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Science 2014; 346: 991996.\n\n2996\nView publication stats\n\n\n",
    "authors": [
      "S Abdullah",
      "K Karunamoorthi"
    ],
    "doi": "",
    "date": "",
    "item_type": "journalArticle",
    "url": ""
  },
  {
    "key": "WLHNTJCM",
    "title": "The emergence of antibody therapies for Ebola.",
    "abstract": "This review describes the history of Ebola monoclonal antibody (mAb) development leading up to the recent severe Ebola outbreak in West Africa. The Ebola virus  has presented numerous perplexing challenges in the long effort to develop  therapeutic antibody strategies. Since the first report of a neutralizing human  anti-Ebola mAb in 1999, the straightforward progression from in vitro  neutralization resulting in in vivo protection and therapy has not occurred. A  number of mAbs, including the first reported, failed to protect non-human  primates (NHPs) in spite of protection in rodents. An appreciation of the role of  effector functions to antibody efficacy has contributed significantly to  understanding mechanisms of in vivo protection. However a crucial contribution,  as measured by post-exposure therapy of NHPs, involved the comprehensive testing  of mAb cocktails. This effort was aided by the use of plant production technology  where various combinations of mAbs could be rapidly produced and tested.  Introduction of appropriate modifications, such as specific glycan profiles, also  improved therapeutic efficacy. The resulting cocktail, ZMapp\u2122, consists of three  mAbs that were identified from numerous mAb candidates. ZMapp\u2122 \\ is now being  evaluated in human clinical trials but has already played a role in bringing  awareness to the potential of antibody therapy for Ebola.",
    "full_text": "",
    "authors": [
      "Andrew Hiatt",
      "Michael Pauly",
      "Kevin Whaley",
      "Xiangguo Qiu",
      "Gary Kobinger",
      "Larry Zeitlin"
    ],
    "doi": "10.3233/HAB-150284",
    "date": "2015-12-23",
    "item_type": "journalArticle",
    "url": ""
  },
  {
    "key": "MVBG5FX5",
    "title": "Ebola virus disease, transmission risk to laboratory personnel, and pretransfusion testing.",
    "abstract": "As Ebola virus has infected thousands of individuals in West Africa, there is growing concern about the appropriate response of hospitals in developed nations  caring for patients and handling laboratory specimens for patients suspected of  Ebola virus disease (EVD). Guidelines for caring for EVD patients are  proliferating rapidly from national and state public health authorities,  professional societies, and individual hospitals. It is no surprise that they  differ from one another, and some very conservative recommendations call for  suspension of routine laboratory testing, including pretransfusion testing. EVD  is transmitted by direct contact with blood, secretions, organs, and other body  fluids and not by airborne routes. Based on experimental and observational data,  the US Centers for Disease Control and Prevention (CDC) recommends that  clinicians follow contact and droplet precautions. Laboratory personnel are  required to follow the blood-borne pathogen standard, especially the use of  appropriate barriers consisting of gloves, gown, goggles, mask to cover nose and  mouth, and plexiglass shield, where splashes of potentially infectious materials  may be generated. Their recommendations are permissive of clinically appropriate  laboratory testing, including pretransfusion testing, using barrier isolation  precautions. Most individuals with suspected EVD will have a fever of another  etiology, such as Plasmodium\u2009falciparum malaria. We believe that forgoing all  routine pretransfusion laboratory testing may result in a greater increase in  poor clinical outcomes than any diminution in the risks to laboratory personnel  will justify. It is imperative for all laboratory directors, working with  institutional infection control and safety personnel, to evaluate their hospital  policies for potentially infectious patients and provide a safe environment for  their patients and employees.",
    "full_text": "",
    "authors": [
      "Louis M. Katz",
      "Aaron A. R. Tobian"
    ],
    "doi": "10.1111/trf.12913",
    "date": "2014-12",
    "item_type": "journalArticle",
    "url": ""
  },
  {
    "key": "EA3P38QQ",
    "title": "Neutralizing monoclonal antibodies for treatment of COVID-19.",
    "abstract": "Several neutralizing monoclonal antibodies (mAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed and are now under  evaluation in clinical trials. With the US Food and Drug Administration recently  granting emergency use authorizations for neutralizing mAbs in non-hospitalized  patients with mild-to-moderate COVID-19, there is an urgent need to discuss the  broader potential of these novel therapies and to develop strategies to deploy  them effectively in clinical practice, given limited initial availability. Here,  we review the precedent for passive immunization and lessons learned from using  antibody therapies for viral infections such as respiratory syncytial virus,  Ebola virus and SARS-CoV infections. We then focus on the deployment of  convalescent plasma and neutralizing mAbs for treatment of SARS-CoV-2. We review  specific clinical questions, including the rationale for stratification of  patients, potential biomarkers, known risk factors and temporal considerations  for optimal clinical use. To answer these questions, there is a need to  understand factors such as the kinetics of viral load and its correlation with  clinical outcomes, endogenous antibody responses, pharmacokinetic properties of  neutralizing mAbs and the potential benefit of combining antibodies to defend  against emerging viral variants.",
    "full_text": "Reviews\n\nNeutralizing monoclonal antibodies for treatment of COVID-19\nPeter C. Taylor\u200a \u200a1\u2009\u2709, Andrew C. Adams2, Matthew M. Hufford2, Inmaculada de la Torre2, Kevin Winthrop3 and Robert L. Gottlieb\u200a \u200a4,5\nAbstract | Several neutralizing monoclonal antibodies (mAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-C\u200b oV-2) have been developed and are now under evaluation in clinical trials. With the US Food and Drug Administration recently granting emergency use authorizations for neutralizing mAbs in non-h\u200b ospitalized patients with mild-t\u200b o-\u200bmoderate COVID-19, there is an urgent need to discuss the broader potential of these novel therapies and to develop strategies to deploy them effectively in clinical practice, given limited initial availability. Here, we review the precedent for passive immunization and lessons learned from using antibody therapies for viral infections such as respiratory syncytial virus, Ebola virus and SARS-CoV infections. We then focus on the deployment of convalescent plasma and neutralizing mAbs for treatment of SARS-\u200bCoV-2. We review specific clinical questions, including the rationale for stratification of patients, potential biomarkers, known risk factors and temporal considerations for optimal clinical use. To answer these questions, there is a need to understand factors such as the kinetics of viral load and its correlation with clinical outcomes, endogenous antibody responses, pharmacokinetic properties of neutralizing mAbs and the potential benefit of combining antibodies to defend against emerging viral variants.\n\nMonoclonal antibodies (mAbs). Clonal antibodies recognizing a single epitope on an antigen. Generally used in reference to recombinant sources.\n1Botnar Research Centre, University of Oxford, Oxford, UK. 2Eli Lilly and Company, Indianapolis, IN, USA. 3Oregon Health & Science University, Portland, OR, USA. 4Baylor University Medical Center, Dallas, TX, USA. 5Baylor Scott & White Research Institute, Dallas, TX, USA. \u2709e-\u200bmail: peter.taylor@ kennedy.ox.ac.uk https://doi.org/10.1038/ s41577-021-00542-\u200bx\n\nIn the midst of the current COVID-19 pandemic, a variety of prophylactic and therapeutic treatments are being developed or repurposed to combat COVID-19. Monoclonal antibodies (mAbs) that can bind to and \u2018neutralize\u2019 the virus in infected patients are a novel class of antiviral intervention1,2. Neutralizing mAbs are recombinant proteins that can be derived from the B cells of convalescent patients or humanized mice (Fig. 1). High-\u200bthroughput screening of these B cells permits the identification of antibodies with the necessary specificity and affinity to bind to a virus and block entry of the virus, therefore abrogating pathology associated with productive infection. These mAbs are termed \u2018neutralizing\u2019 and can ultimately be used as a type of passive immunotherapy (detailed later) to minimize virulence. In this Review, we highlight the relative value that neutralizing mAbs can provide for patients and physicians, and go on to examine the role of these agents among the spectrum of potential treatments for COVID-19.\nIn the United States, three anti-\u200bsevere acute respiratory syndrome coronavirus 2 (SARS-C\u200b oV-2) mAb therapies have been granted emergency use authorization (EUA) for treatment of non-\u200bhospitalized patients with mild-\u200bto-\u200bmoderate COVID-19 \u2014 these are bamlanivimab as a monotherapy, and bamlanivimab together with etesevimab or casirivimab with imdevimab as a\n\ncombination therapy3\u20135. Therefore, several questions need to be addressed about the potential clinical use of neutralizing SARS-C\u200b oV-2 mAbs: who should get them; what is the best dose and frequency; when in the course of the infection will they be most effective; what is the duration of the protection they provide; and what is their associated benefit-\u200bto-\u200brisk ratio? In addition, neutralizing mAbs may have a prophylactic role in individuals deemed to be at high risk of severe COVID-19. Indeed, preliminary non-\u200bpeer-\u200breviewed preprint data suggest that mAbs prevent COVID-19 in high-\u200brisk individuals potentially exposed to SARS-\u200bCoV-2 in nursing homes or within households6,7.\nWhile vaccines remain the best strategy to prevent COVID-19, mAbs could potentially benefit certain vulnerable populations before or after exposure to SARS-C\u200b oV-2, such as the unvaccinated or recently vaccinated high-\u200brisk patients. The antiviral activity seen with neutralizing mAb treatment emphasizes the importance of early intervention to help counter the devastating impact the virus has had in such vulnerable populations and in other high-r\u200b isk patients. However, mAbs are complicated to produce and may be limited in initial supply. Furthermore, any protection offered would be temporary, and the duration of effective protection remains to be determined. Answers to these questions will allow\n\n382 | June 2021 | volume 21\n\n0123456789();:\n\nwww.nature.com/nri\n\nProcess Source material\nConvalescent patient\nHumanized mouse that received target antigen\n\nReviews\n\nSearch Screening for RBD-speci\ufb01c single B cells\n\nSequence and identity Analyse\n\nSelect\n\nCloning and expression Validate and characterize\n\nHigh-con\ufb01dence sequences clustered by sequence identity\n\nBinding validation\n\nMultiplexed bead-based assay\nLive cell-based assay\nFluorescence-activated single-cell sorting\n\nClonal families\n\nFunctional validation Stability\nOrganization Af\ufb01nity\n\nFig. 1 | Neutralizing monoclonal antibodies: identification, selection and production. The neutralizing monoclonal antibodies (mAbs) given emergency use authorization for treatment of COVID-19 were derived from either convalescent patients or humanized mice exposed to severe acute respiratory syndrome coronavirus 2 (SARS-C\u200b oV-2) antigens. However, mAbs can be generated by multiple methods, including from vaccinated individuals (not depicted here). The pathways of mAb generation depicted here converge in the process of selection and production. RBD, receptor-b\u200b inding domain.\n\nPassive immunotherapy The introduction of monoclonal or polyclonal antibodies derived from non-\u200bhuman or human blood products to provide protection against infection or envenomation.\nEmergency use authorization (EUA). A mechanism to facilitate the availability and use of medical countermeasures during a public health emergency. US Food and Drug Administration issuance of an EUA permits the use of unapproved medical products or unapproved uses of approved medical products when no adequate alternatives are available.\nConvalescent plasma therapy (CPT). The administration of donated plasma from an individual who has had an illness and recovered from it (for example, previously infected with severe acute respiratory syndrome coronavirus 2 (SARS-\u200bCoV-2) but has now recovered), to an infected individual. The recovered patient\u2019s plasma contains antibodies, which when administered to other patients is thought to boost the ability to fight disease.\n\nthe most efficacious use of these novel and potentially life-\u200bsaving treatments, as we discuss herein.\nPassive immunization\nMore than 125 years ago, the first major success in modern immunological intervention was developed: a therapeutic serum from animals actively immunized against diphtheria toxin8,9. Paul Ehrlich later produced a seminal article tying the curative antiserum to neutralizing antibodies10. Today, passive immunization involves infusion of antigen-\u200bspecific mAbs or polyclonal antibodies derived from non-\u200bhuman or human blood products. While polyclonal antibodies collected from immunized animals are the primary source of antisera, there is a risk of \u2018serum sickness\u2019, especially after repeated exposures, as the recipient may generate an immune response against antibodies of non-h\u200b uman origin. These risks are mitigated with the use of convalescent plasma from human patients. With careful screening (for example, to assess for the presence of infectious agents and to establish antibody titre and neutralizing capacity), convalescent plasma therapy (CPT) can be effective with minimal safety risks.\nBefore the current pandemic, CPT was used to treat infections with influenza virus11,12, respiratory syncytial virus (RSV)13, Ebola virus14 and other coronaviruses12,15\u201317. CPT appears most efficacious when used early after the onset of symptoms, rather than during severe or prolonged infection12,15,18. It also has the potential to provide protection for the immunocompromised or unvaccinated high-\u200brisk individuals recently exposed to infection13,15. Administration of plasma with higher titres of neutralizing antibodies is associated with improved clinical outcomes17; however, the antibody titres of convalescent plasma differ considerably19. CPT can\n\nbe convenient and adaptable for use in resource-\u200bpoor settings14 and can be rapidly deployed to combat novel virus outbreaks.\nThe antipathogen antibodies from convalescent plasma can mitigate infection by two main mechanisms: namely, antibody effector activity and pathogen neutralization. However, in rare cases, pathogen-s\u200b pecific antibodies can augment virulence in a process termed \u2018antibody-\u200bdependent enhancement\u2019 (ADE) (Fig. 2). ADE can occur via two distinct mechanisms. First, pathogen-\u200bspecific antibodies could increase infection by promoting virus uptake and replication in Fc\u03b3 receptor-\u200bexpressing immune cells (for example, as is seen in dengue haemorrhagic virus infection of macrophages). With SARS-\u200bCoV and SARS-\u200bCoV-2 (ref.20), in vitro evidence amassed to date indicate that these non-\u200blymphotropic coronaviruses are unable to productively replicate within haematopoietic cells21. Alternatively, ADE can be mediated via increased immune activation by Fc-\u200bmediated effector functions or immune complex formation22. In the case of respiratory virus infections, the resulting immune cascade can contribute to lung disease. While the hallmarks of severe COVID-19 have features that overlap with this type of ADE, there is currently no definitive evidence to show ADE occurs with SARS-C\u200b oV-2 infection22.\nNonetheless, steps may be considered to mitigate the potential risk of ADE. When feasible (as with neutralizing mAb therapy), the Fc region of the antibody can be modified to render it incapable of engaging effector immune responses. In the case of CPT, the potential risk of ADE can be reduced by administrating high amounts of pathogen-\u200bspecific antibodies and using plasma with high-\u200baffinity neutralizing antibodies20. However, these strategies must be balanced with the\n\nnature Reviews | IMMuNology\n\n0123456789();:\n\nvolume 21 | June 2021 | 383\n\nReviews\n\nAntibody-d\u200b ependent enhancement (ADE). The promotion of viral uptake into cells owing to the presence of suboptimal antibodies. ADE can result in enhanced viral replication and/or aberrant inflammation.\nRandomized controlled trials (RCTs). Studies that randomly assign participants into an experimental group or a control group to measure the effectiveness of a new intervention or treatment.\n\npotential loss of efficacy from effector-\u200bmediated activity. As shown in a non-\u200bprimate model of SARS-\u200bCoV, neutralizing anti-\u200breceptor-\u200bbinding domain (RBD) or anti-\u200bheptad repeat 2 antibodies provided protective immunity, whereas antibodies specific for other S protein epitopes could trigger ADE23. Furthermore, in\n\nrandomized controlled trials (RCTs), passive immunization with anti-\u200bS protein-\u200bneutralizing mAbs did not provide clinical evidence of ADE in non-\u200bhospitalized patients with COVID-19 (refs3,24,25).\nThe shortage of large RCTs of CPT has limited our understanding of the relative benefit-\u200bto-\u200brisk profile of\n\na Antibody-dependent\ncellular phagocytosis\n\nMacrophage\n\nComplement-dependent cytotoxicity\nC1q Antibody\n\nNeutralization\n\nPhagocytosis Fc\u03b3R\n\nInfected cell\n\nAntigen\nMAC Lysis\n\nE\ufb00ector cell\n\nRelease of granzyme and perforin-mediated cell apoptosis\n\nAntibody-dependent cellular cytotoxicity\n\nOpsonization\n\nb\nVirus\n\nAntibodies can enhance uptake of virions\n\nIncreased immune activation\n\nIncreased cytokine production\n\nIncreased immune cell activation and in\ufb01ltration\n\nProductive viral replication and release\n\nMacrophage\n\nIncreased infection\n\nComplement cascade activation\n\nIn\ufb02ammation\n\nFig. 2 | Mechanism of action of monoclonal antibodies for viral infection and antibody-dependent enhancement. a | Monoclonal antibodies can directly interfere with viral pathogenesis in multiple ways. First, binding of a neutralizing antibody to the virion can prevent target cell binding and/or fusion. Furthermore, antibody binding opsonizes the virions or infected cells for phagocytic uptake. If viral proteins are intercalated into target cell membranes during viral egress, monoclonal antibodies can facilitate target cell death via complement fixation and membrane attack complex (MAC) activation or antibody-\u200bdependent cytotoxicity. These mechanisms may result in apoptosis or necrosis of the infected cell. b | In some instances, opsonization of a virion can facilitate viral pathogenesis in a process termed \u2018antibody-d\u200b ependent enhancement\u2019 (ADE). ADE can occur via two distinct mechanisms. First, pathogen-s\u200b pecific antibodies could increase infection via viral uptake and replication in Fc\u03b3 receptor (Fc\u03b3R)-\u200bexpressing immune cells. Secondly, ADE can be mediated via increased immune activation by Fc-m\u200b ediated effector functions or immune complex formation. The process of ADE and its potential impact during severe acute respiratory syndrome coronavirus 2 (SARS-C\u200b oV-2) infection is expertly reviewed by Lee et al.22.\n\n384 | June 2021 | volume 21\n\n0123456789();:\n\nwww.nature.com/nri\n\nReviews\n\nImmune globulin A sterilized solution made from plasma and containing antibodies.\nViral variants Mutations arising in viruses resulting in genetic variation and the emergence of different versions of the virus.\nSpike (S) protein Protein found on the coronavirus cell surface, responsible for binding of the virus to host cells and subsequent entry of the virus. The receptor-\u200bbinding domain of the S protein is the preferred target of neutralizing monoclonal antibody therapies for severe acute respiratory syndrome coronavirus 2 (SARS-\u200bCoV-2) infection.\n\nthis treatment option. Furthermore, logistical difficulties can complicate the application of CPT. According to the EUA from the US Food and Drug Administration for CPT in patients with COVID-19, convalescent patients should be symptom-\u200bfree for a minimum of 2 weeks and have high titres of anti-\u200bSARS-\u200bCoV-2 antibodies; low-\u200btitre donations could be used for therapy following careful consideration by the health-\u200bcare provider26,27. Thus, widespread use of CPT is dependent on a readily available pool of recovering patients with high antibody titres who are willing to donate plasma, on sufficient local facilities to ensure adequate processing, screening and administration of the therapy, and on governmental coordination to regulate effective implementation.\nAdvantages of monoclonal antibodies\nThere is an increasing focus on replacing CPT with neutralizing mAbs, where dosing to ensure appropriate neutralizing capacity of the antibodies can be more precise. Today, the process to mass-\u200bproduce recombinant mAbs has become scalable to meet demand and is cost-\u200bcompetitive with other treatments. Neutralizing mAbs overcome limitations intrinsic to CPT (for example, the risk of blood-\u200bborne diseases, time to development of detectable high-\u200baffinity antibodies and risk of low antibody titres, as well as variable epitope specificity28). Furthermore, a high titre of neutralizing antibodies \u2014 which current evidence indicates is necessary for the efficacy of CPT \u2014 is inherent with neutralizing mAbs. As of March 2021, at least 20 neutralizing mAb therapies were being tested in late-\u200bstage clinical trials or had already been approved for use in nine infectious diseases, including RSV infection and Ebola29,30 (ClinicalTrials.gov).\nPalivizumab, a neutralizing mAb to the fusion protein of RSV, was initially approved in 1998 as a prophylaxis for severe RSV infection in high-r\u200b isk infants31\u201333. Previously, the standard of care for prophylaxis in these patients was monthly infusions of RSV immune globulin13,31. When administered via monthly intramuscular injections, palivizumab reduced the frequency of hospitalization and severity of RSV disease relative to placebo and was well tolerated31,32. However, palivizumab was not demonstrated in RCTs to improve clinically meaningful outcomes in infants with severe RSV infection in advanced disease stages34\u201336. Furthermore, monthly administration is required to maintain detectable levels of neutralizing mAbs, and as many as five doses may be needed to prevent severe or deadly infection37. A newer medication with a longer half-\u200blife (MEDI8897) is currently in phase II/III trials33.\nDuring the Ebola virus disease outbreak in the Democratic Republic of the Congo in 2018, an openlabel RCT (PALM) investigated four intravenously administered treatments in 681 patients actively infected with Ebola virus: the antiviral remdesivir, the triple mAb cocktail ZMapp, the single mAb MAb114, and the triple mAb combination REGN-\u200bEB3 (ref.38). After an interim analysis, the first two treatments were discontinued as MAb114 monotherapy and REGN-\u200bEB3 were superior with respect to the primary outcome, patient mortality38. One potential factor the PALM study team proposed to\n\nexplain the distinction between the therapeutics was that the full treatments for MAb114 and REGN-\u200bEB3 were administered as a single dose, thereby facilitating a rapid response, while ZMapp was given as three infusions. Indeed, patients treated with MAb114 and REGN-\u200bEB3 had faster rates of viral clearance, lending credence to this hypothesis. Overall, survival was higher in those who were treated early during symptom onset and had lower baseline viral loads. The relatively low efficacy of the ZMapp triple cocktail also serves as a reminder that the number of mAbs is not necessarily a predictor of efficacy per se, and that specific epitopes may also matter.\nTwo main uncertainties persist with passive immunization, spanning both neutralizing mAbs and CPT. First, does their use as a prophylactic or treatment potentially affect natural long-\u200bterm immunity? Considering the large doses used and the relative half-\u200blife of antibodies (~3 weeks for IgG molecules), there is a pertinent consideration whether the presence of circulating neutralizing mAbs could impact active immunity, whether through memory from infection or vaccination. In RSV infection, rodent and primate infection models indicate that the passive transfer of antibodies does diminish the development of humoral immunity in the recipient; however, long-\u200bterm memory was sufficient to protect the hosts from reinfection, largely owing to an intact T cell memory compartment39,40. Considering the limitations in translating data from animal models (where RSV replication is attenuated relative to that in its human host), additional data, particularly from clinical trials, will provide critical insight with regard to this potential challenge.\nSecond, could resistant viral variants emerge that limit the effectiveness of the therapies? The polyclonal nature of CPT, in which a spectrum of differentiated antibodies target multiple epitopes of the pathogen, may help to reduce this risk. Nevertheless, emerging preclinical data suggest SARS-\u200bCoV-2 spike (S) protein mutations escape from polyclonal serum41, and convalescent plasma has reduced neutralizing activity against some viral variants42. For mAbs, however, depending on the infectious agent and the epitope targeted, combinations of mAbs may be necessary to maintain efficacy and prevent treatment failure. Experience with mAbs targeting human immunodeficiency virus (HIV), which has a very high mutation rate, suggests that it may be more effective and durable to use multiple neutralizing antibodies (that is, combinational mAb therapy) rather than a single one43\u201346. These particular mAbs to HIV also need to be broadly neutralizing and target epitopes generally conserved among viral variants. On the other hand, infections involving pathogens with lower mutation rates and/or accessible broadly conserved epitopes may not require combinational mAb therapy; for example, MAb114 monotherapy, which targets a broadly conserved epitope on the Ebola virus\u2019s RBD, was as effective as the combination therapy REGN-E\u200b B3 (ref.38) and more effective than the ZMapp triple cocktail. It is important to note though that the global nature of the COVID-19 pandemic presents a larger risk of escape variants emerging than during the Ebola outbreak, owing to the sheer number of infections and high levels of circulating virus among populations.\n\nnature Reviews | IMMuNology\n\n0123456789();:\n\nvolume 21 | June 2021 | 385\n\nReviews\n\nNucleocapsid (N) protein Protein that encloses and protects a viral genome, such as the severe acute respiratory syndrome coronavirus 2 (SARS-C\u200b oV-2) genomic RNA.\n\nFrom the collective clinical data with MAb114, REGN-\u200bEB3 and palivizumab, the general benefits and risks associated with neutralizing mAbs are similar to those observed with traditional passive immunization against infectious agents. The agents themselves are relatively tolerable for patients, efficacious during the early onset of disease symptoms and in certain cases as a prophylactic, but with limited efficacy once infections are severe. The distinctions between these therapies are largely logistical; CPT is more rapidly implemented during an emerging pandemic when few therapeutic options are yet available, while neutralizing mAbs take time to discover and it takes time for regulatory approval for their use to be obtained as well as to scale up manufacturing capacity. The use and promise of passive immunization during the coronavirus outbreaks of the twenty-\u200bfirst century (that is, with SARS-\u200bCoV, Middle East respiratory syndrome-\u200brelated coronavirus and SARS-\u200bCoV-2) have re-\u200bemphasized these past lessons while also highlighting additional insights, as we discuss next.\nPassive immunization for coronaviruses\nDuring the SARS epidemic in 2003, immune system kinetics following SARS-\u200bCoV infection were different in patients who recovered compared with those who finally succumbed to the viral infection and sequelae. In patients with fatal outcomes, the levels of endogenous neutralizing antibodies peaked at 15 days from symptom onset and then decreased drastically until the time of death. By contrast, in patients who went on to recover, peak neutralizing antibody responses were observed at 20 days from symptom onset47,48. Those who recovered tended to develop antibody responses with diverse isotypes (IgM, IgG and IgA) against two proteins on the virus, the nucleocapsid (N) protein and the S protein, while patients with fatal outcomes had restricted antibody responses to the N protein only. In a serological survey of confirmed convalescent serum samples, 88% had anti-\u200bSARS-\u200bCoV antibodies 31\u2013180 days after the onset of symptoms; the geometric mean of the neutralizing antibody titre was 1:61 (ref.19).\nOwing to the brief duration of the SARS epidemic in 2003, few observational trials examining CPT were conducted. The largest involved 80 patients and was conducted at the Prince of Wales Hospital in Hong Kong15. Those given CPT before day 14 following onset of symptoms had a better outcome than those given CPT after day 14 (P\u2009<\u20090.001); mortality was also lower in the former group (6.3% versus 21.9%). Patients who tested positive for SARS-\u200bCoV by PCR had better outcomes if they were seronegative when given CPT than those who were already seropositive (66.7% versus 20%; P\u2009<\u20090.001). A summary of eight observational studies using CPT during the SARS 2003 outbreak (including the aforementioned Prince of Wales Hospital study) suggested CPT was associated with reduced mortality, shorter hospital stays and reduced overall viral loads in the respiratory tract12. The treatments were considered tolerable12; however, information on minor complications may have been under-\u200breported. Importantly, robust studies on the effectiveness and safety of CPT were not completed,\n\nand thus these results must be interpreted with caution, particularly as patients treated at later times with CPT may demonstrate selection bias for an already refractory pathophysiology.\nIn SARS-\u200bCoV-2 infection, plasma collected from 175 patients who had recovered from mild COVID-19 demonstrated neutralizing antibody and S-\u200bbinding antibody titres that correlated with increased age, greater inflammation (that is, higher C-\u200breactive protein levels) and lower lymphocyte counts; the vast majority of the SARS-\u200bCoV-2 neutralizing antibodies were not cross-\u200breactive with SARS-\u200bCoV49. Several reports indicate convalescent patients can maintain high titres of neutralizing antibodies several weeks after infection49\u201351.\nObservational studies have reported that CPT has been associated with improved outcomes in COVID-19 (ref.52). For example, in a small case series in China, five critically ill patients with acute respiratory distress syndrome showed improved clinical status following CPT. Thirty-\u200bseven days after transfusion, three patients had been discharged and two were in a stable condition53. In a cohort analysis of 35,222 patients hospitalized with COVID-19 from the United States Convalescent Plasma Expanded Access Program, reduced mortality was associated with earlier time to transfusion (after diagnosis) and convalescent plasma with higher antibody levels18. Because these studies were observational, there was limited procedural control including standardization of the level of neutralizing antibodies.\nIn an open-\u200blabel RCT of CPT for patients (n\u2009=\u2009103) with severe or life-\u200bthreatening COVID-19 in China, donors were required to have high levels of antibodies specific to the RBD of the S protein54. However, the study was terminated early; the hazard ratio for the time to clinical improvement within 28 days in the CPT group versus the standard treatment control group was 1.4 (favouring CPT) but was not statistically significant. The proportion of patients with severe disease who achieved the primary end point was significantly higher in the CPT group (21 of 23 patients) versus the standard treatment group (15 of 22 patients; P\u2009=\u20090.03), but no distinction was noted in patients with life-\u200bthreatening COVID-19 (ref.53). In a blinded RCT in Argentina, CPT (with a median titre of 1:3,200 anti-\u200bSARS-\u200bCoV-2 antibodies) also failed to demonstrate benefit in patients with COVID-19-\u200bassociated severe pneumonia55. Furthermore, in an open-\u200blabel RCT in India (PLACID) in hospitalized patients with hypoxaemia (generally comparable with the definition of severe COVID-19 from other trials), CPT did not demonstrate benefit in terms of patient mortality or transition to worsening disease56. Similarly, preliminary data from the RECOVERY RCT among 10,406 hospitalized patients showed no proof of mortality benefit in the primary end point of 28-\u200bday mortality in the CPT group versus the standard treatment group57,58.\nDespite inconsistent clinical efficacy, there is evidence that CPT was associated with greater viral clearance than standard-\u200bof-\u200bcare treatment54,56; collectively, this indicates increased viral clearance alone was not sufficient to clearly improve the clinical outcomes in patients with established COVID-19 (that is, in patients\n\n386 | June 2021 | volume 21\n\n0123456789();:\n\nwww.nature.com/nri\n\nReviews\n\nTime-w\u200b eighted average An average that takes both the numerical level and the time of a particular variable into consideration.\nMedically attended visits Medical visits such as telemedicine visits, in-p\u200b erson outpatient visits to or from a medical provider, urgent care or emergency department visits, or hospitalization.\n\nhospitalized with COVID-19). Because the currently available data on CPT are derived predominantly from inpatient (severe or critical) COVID-19 RCTs, the suitability of CPT as prophylaxis or treatment at the onset of COVID-19 symptoms remains to be determined by appropriately controlled clinical trials.\nMonoclonal antibodies for COVID-19 The primary antigenic epitope on SARS-\u200bCoV and SARS-\u200bCoV-2 is the S protein, which facilitates target cell binding and fusion upon engaging the cell-\u200bsurface angiotensin-c\u200b onverting enzyme 2 (ACE2) receptor, which is found on cells in the respiratory system, gastro\u00adintestinal tract and endothelium59\u201363. Thus, antibodies directed\n\nSARS-CoV-2\n\nImdevimab Casirivimab\nRBD\n\nS protein RBD\n\nAntibody prevents viral binding and/or fusion with host cell\n\nBamlanivimab Etesevimab\n\nACE2\n\nFig. 3 | Inhibition of SARS-CoV-2 target cell engagement by neutralizing monoclonal antibodies. Neutralizing monoclonal antibodies (mAbs) being developed to combat COVID-19 are generated against the receptor-b\u200b inding domain (RBD) of the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-\u200bCoV-2). The anti-R\u200b BD mAbs prevent binding of the S protein to its cognate receptor, angiotensin-c\u200b onverting enzyme 2 (ACE2), on target host cells. Three neutralizing mAb regimens have been given emergency use authorization for treatment of COVID-19. (1) Casirivimab and imdevimab bind distinct epitopes on the RBD with dissociation constants KD of 46 and 47 pM, respectively. Imdevimab binds the S protein RBD from the front or lower-l\u200beft side, while casirivimab targets the spike-\u200blike loop from the top direction (overlapping with the ACE2-\u200bbinding site3,68). (2) Bamlanivimab binds an epitope on the RBD in both its open confirmation and its closed confirmation with dissociation constant KD\u2009=\u200971pM, covering 7 of the approximately 25 side chains observed to form contact with ACE2 (ref.4). (3) Bamlanivimab and etesevimab bind to distinct, but overlapping, epitopes within the RBD of the S protein of SARS-\u200bCoV-2. Etesevimab binds the up/active conformation of the RBD with dissociation constant KD\u2009=\u20096.45 nM (ref.5); it contains the LALA mutation in the Fc region, resulting in null effector function.\n\nto the S protein can neutralize the ability of the virus to bind and fuse with the target host cell. Humanized murine technology or convalescent plasma from recovered patients has been used to derive neutralizing mAbs targeted to the RBD of the S protein64\u201366 (Fig. 3). To date, most advanced research efforts for therapeutic use of neutralizing mAbs are focusing on a handful of pro\u00ad ducts in clinical development, some of which are already authorized on the basis of phase I/II and phase II data for emergency use (Table 1).\nREGN-\u200bCOV2 therapy. REGN-\u200bCOV2 is a combination of two potent neutralizing mAbs \u2014 namely, casirivimab and imdevimab, which are IgG1 mAbs with unmodified Fc regions. These two mAbs were chosen from a pool of more than 200 neutralizing mAbs present in the initial isolation of thousands of antibodies and were derived from parallel efforts using humanized mice and the sera of patients recovering from COVID-19 (refs67,68). The antibodies bind two distinct and non-\u200boverlapping sites on the RBD3,67. The rationale for this antibody combination is that it is unlikely that a mutation in the S protein of SAR-\u200bCoV-2 will simultaneously render both antibodies ineffective. In extensive in vitro testing, this combination retained its ability to neutralize all known S protein mutations67. Further, casirivimab and imdevimab combination therapy initiated antibody-\u200b mediated cytotoxicity and cellular phagocytosis in virally infected cells in vitro3. This product was tested in rhesus macaques and golden hamsters infected with SARS-C\u200b oV-2, which serve as models for mild and severe disease, respectively69. In both models, prophylactic and therapeutic treatment with casirivimab and imdevimab not only resulted in a reduction in viral load but also diminished the incidence and severity of lung disease relative to a placebo.\nAn ongoing phase I/II/III placebo-\u200bcontrolled trial (NCT04425629) is investigating the safety and efficacy of a single infusion of casirivimab and imdevimab \u2014 2,400\u2009mg (n\u2009=\u2009266, interim), 8,000\u2009mg (n\u2009=\u2009267, interim) or matching placebo (n\u2009=\u2009266) \u2014 for symptomatic adults who have not previously been hospitalized within 3 days of a positive active SARS-C\u200b OV-2 diagnosis (and within 7 days of the first symptoms)3. In the modified full analysis set for the phase I/II analysis, the median age was 42 years (7% aged 65 years or older), 85% of patients were white, 9% were Black and 34% were considered at high risk (for example, they were elderly, had obesity or had underlying chronic medical conditions). Pooled treatment achieved the primary end point of time-\u200b weighted average change from the baseline in viral load (log10 copies per millilitre), collected from a nasopharyngeal swab, in patients with a positive baseline for viral RNA (n\u2009=\u2009665). The difference in time-\u200bweighted average from day 1 through day 7 for the pooled doses of casirivimab and imdevimab compared with placebo was \u22120.36 log10 copies per millilitre (P\u2009<\u20090.0001). The combination was reported to reduce viral load particularly in patients with higher viral loads who were seronegative at the baseline3,70. On a key clinical end point, a lower proportion of patients treated with casirivimab and imdevimab had COVID-19-\u200brelated medically attended visits\n\nnature Reviews | IMMuNology\n\n0123456789();:\n\nvolume 21 | June 2021 | 387\n\nReviews\n\nTable 1 | Neutralizing monoclonal antibodies for SARS-C\u200b oV-2 currently in development up to 11 December 2020\n\nSponsors\n\nDrug code/International Status proprietary name\n\nTrial ID\n\nActual starta Estimated primary completiona\n\nJunshi Biosciences and Eli JS016, etesevimab Lilly and Company\n\nEUA when used in combination with bamlanivimabb\n\nNCT04441918 NCT04441931 NCT04427501\n\n5 Jun. 2020 19 Jun. 2020 17 Jun. 2020\n\n11 Dec. 2020 2 Oct. 2020c 20 Sep. 2020c\n\nTychan Pte Ltd\n\nTY027\n\nPhase I; phase III pending\n\nNCT04429529 NCT04649515\n\n9 Jun. 2020 4 Dec. 2020d\n\n19 Nov. 2020c 31 Aug. 2021\n\nBrii Biosciences\n\nBRII-196\n\nPhase I\n\nNCT04479631\n\n12 Jul. 2020 Mar. 2021\n\nBrii Biosciences\n\nBRII-198\n\nPhase I\n\nNCT04479644\n\n13 Jul. 2020 Mar. 2021\n\nAbbVie\n\nABBV-47D11\n\nPhase I pending\n\nNCT04644120\n\n10 Dec. 2020 5 Sep. 2021\n\nSorrento Therapeutics Inc. COVI-\u200bGUARD (STI-1499) Phase I\n\nNCT04454398\n\nSep. 2020d\n\nJan. 2021\n\nMabwell (Shanghai) Bioscience Co. Ltd\n\nMW33\n\nPhase I\n\nNCT04533048\n\n7 Aug. 2020 16 Nov. 2020c\n\nHiFiBiO Therapeutics\n\nHFB30132A\n\nPhase I\n\nNCT04590430\n\n20 Oct. 2020 Apr. 2021\n\nOlogy Bioservices\n\nADM03820\n\nPhase I pending\n\nNCT04592549\n\n4 Dec. 2020 30 Sep. 2021\n\nHengenix Biotech Inc\n\nHLX70\n\nPhase I pending\n\nNCT04561076\n\n9 Dec. 2020d 6 Sep. 2021\n\nUniversity of Cologne and DZIF-10c Boehringer Ingelheim\n\nPhase I/II pending\n\nNCT04631705 NCT04631666\n\n14 Dec. 2020 8 Dec. 2020\n\n31 Jul. 2021 31 Jul. 2021\n\nSorrento Therapeutics Inc. COVI-\u200bAMG (STI-2020)\n\nPhase I/II pending NCT04584697\n\nDec. 2020c\n\nApr. 2021\n\nBeigene\n\nBGB DXP593\n\nPhase I; phase II pending\n\nNCT04532294 (phase I)\nNCT04551898 (phase II pending)\n\n8 Sep. 2020 2 Dec. 2020\n\n19 Feb. 2021 25 Jan. 2021c\n\nSinocelltech Ltd\n\nSCTA01\n\nPhase I; phase II/III NCT04483375\n\npending\n\nNCT04644185\n\n24 Jul. 2020 10 Feb. 2021d\n\n17 Nov. 2020c 10 May 2021\n\nAstraZeneca\n\nAZD7442 (AZD8895 and AZD1061)\n\nPhase I; phase III pending\n\nNCT04507256 NCT04625725\n\n18 Aug. 2020 21 Nov. 2020\n\n25 Oct. 2021 21 Apr. 2021\n\nNCT04625972\n\n2 Dec. 2020 21 Jan. 2022\n\nCelltrion\n\nCT-\u200bP59\n\nPhase I; phase II/III NCT04525079\n\n18 Jul. 2020 31 Aug. 2020\n\nNCT04593641\n\n4 Sep. 2020 22 Oct. 2020\n\nNCT04602000\n\n25 Sep. 2020 Dec. 2020\n\nVir Biotechnology Inc and VIR-7831/GSK4182136 GlaxoSmithKline\n\nPhase II/III\n\nNCT04545060\n\n27 Aug. 2020 Mar. 2021\n\nAbCellera and Eli Lilly and Company\n\nBamlanivimab; combination of bamlanivimab and etesevimab\n\nEUAb\n\nNCT04411628 (phase I) NCT04427501 (phase II) NCT04497987 (phase III)\n\n28 May 2020 17 Jun. 2020 2 Aug. 2020\n\n26 Aug. 2020c 20 Sep. 2020c 8 Mar. 2021\n\nNCT04501978 (phase III) 4 Aug. 2020 Jul. 2022\n\nNCT04518410 (phase II/III) 19 Aug. 2020 May 2023\n\nRegeneron\n\nREGN-\u200bCOV2 (casirivimab EUAb and imdevimab)\n\nNCT04425629 (phase I/II) 16 Jun. 2020 NCT04426695 (phase I/II) 11 Jun. 2020\n\n10 Apr. 2021 16 Apr. 2021\n\nNCT04452318 (phase III) 13 Jul. 2020 15 Jun. 2021\n\nA complete list can be found at COVID-19 Biologics Tracker. EUA, emergency use authorization; SARS-C\u200b oV-2, severe acute respiratory syndrome coronavirus 2. aDates as of 7 April 2021. bHave recieved EUA in the United States. cActual primary completion date. dEstimated start date.\n\nAbsolute risk reduction The difference between the risk of an event in the control group and the risk of an event in the treated group.\n\n(2.8% for pooled doses versus 6.5% for placebo). In post hoc analyses, a lower proportion of patients treated with casirivimab and imdevimab had COVID-19-r\u200b elated hospitalizations or emergency department visits compared with patients who received placebo (2% versus 4%). The absolute risk reduction for casirivimab and imdevimab compared with placebo was greater for patients at high risk of progression to severe COVID-19 and/or hospitalization (3% versus 9%). Collectively, these results supported the EUA of Regeneron\u2019s casirivimab and\n\nimdevimab cocktail in the United States in November 2020 (ref.71).\nBamlanivimab monotherapy. Bamlanivimab is a potent neutralizing mAb (IgG1 with an unmodified Fc region) to the S protein that was derived from the convalescent plasma of a patient who had COVID-19 (refs24,66). Bamlanivimab binds the S protein\u2019s RBD, engaging its cognate epitope in both up and down conformations, which makes this antibody potentially useful as a\n\n388 | June 2021 | volume 21\n\n0123456789();:\n\nwww.nature.com/nri\n\nReviews\n\nPersistent high viral load The continued presence of a high viral load in patients, which is associated with increased risk of hospitalization.\n\nmonotherapy. There have been historical precedents for the effectiveness of neutralizing mAbs as a monotherapy (for example, MAb114 for Ebola)38. To assess theoretical risk of ADE, bamlanivimab was studied in primary human macrophages and immune cell lines exposed to SARS-\u200bCoV-2 at concentrations down to 100-\u200bfold below the effective concentration for half-\u200bmaximum response, and in these studies, it did not demonstrate productive viral infection4. Prophylactic efficacy was tested in rhesus macaques given bamlanivimab 24\u2009hours before a virus challenge66. The symptoms in this model were mild overall, but the treatment significantly decreased viral load and replication in the respiratory tract following inoculation, supporting its antiviral efficacy.\nIn the phase II portion of the ongoing phase II/III BLAZE-1 trial (NCT04427501), ambulatory adults with mild-\u200bto-\u200bmoderate symptoms of COVID-19 within 3 days of a first-\u200bpositive nasopharyngeal swab positive for SARS-\u200bCoV-2 received a single infusion of one of three doses of bamlanivimab (700, 2,800 or 7,000\u2009mg) or placebo in an outpatient setting24. A pre-\u200bplanned interim analysis was conducted of 452 patients who had reached day 11 following infusion (median age 45\u201346 years (12% aged 65 years or older), 88% white, 6% Black and 68% at high risk (for example, they were elderly, had obesity or had underlying chronic medical conditions))24. The study revealed the viral clearance time course via the intrinsic immune response and the enhanced clearance with neutralizing mAb infusion, concomitant with improved clinical response. Viral loads were assayed from serial nasopharyngeal swabs, with postinfusion measurements enabled by a novel partnership with home-\u200bhealth research. Following infusion, the log viral load had begun decreasing relative to the baseline as early as the first postinfusion assessment on day 3 (\u22120.85 for placebo versus \u22121.35 for pooled bamlanivimab doses), continued to decrease on day 7 (\u22122.56 for placebo versus \u22122.90 for pooled bamlanivimab doses) and further decreased by day 11 (\u22123.47 for placebo versus \u22123.70 for pooled bamlanivimab doses). In a post hoc analysis, patients with early persistent high viral load (described as log viral load of 5.27 or greater at trial day 7) had a higher risk of hospitalization, and the risk was further increased for elderly patients and patients with obesity. Clinical evidence demonstrating the efficacy of bamlanivimab came from two predefined secondary end points. First, at day 29, the percentage of patients who were hospitalized with COVID-19 was 6.3% for the placebo group and reduced to 1.6% for the group with pooled bamlanivimab doses. In post hoc analyses, hospitalization among elderly patients (65 years or older) or patients with obesity (body mass index 35\u2009kg\u2009m\u22122 or greater) was 15% for the placebo group and 4% for the group who received pooled bamlanivimab doses. Absolute risk reduction for hospitalizations was more evident for patients with risk factors. Second, amelioration of baseline symptoms was greater for the pooled bamlanivimab doses than for placebo from day 2 through to day 11. Collectively, these results supported the EUA of bamlanivimab monotherapy in the United States and Canada in November 2020 (ref.4).\n\nBamlanivimab and etesevimab. Other treatment arms of the BLAZE-1 trial studied bamlanivimab together with etesevimab (an S protein-\u200bbinding IgG1 with a modi\u00adfied Fc region, resulting in null effector function)25,72. Bamlanivimab and etesevimab together significantly decreased viral load (mean changes from the baseline and percentage of patients with persistent high viral load) compared with placebo at day 3 to day 11 (ref.25). Bamlanivimab- and etesevimab-\u200btreated patients had fewer COVID-19-\u200brelated hospitalizations relative to the placebo group (5.8% for placebo reduced to 0.9% for bamlanivimab together with etesevimab). Recently released placebo-\u200bcontrolled phase III data from 1,035 patients randomized 1:1 to receive bamlanivimab together with etesevimab versus placebo demonstrated that in high-\u200brisk ambulatory patients (including patients aged 12\u201317 years with specific risk factors and patients aged 18 years or older with specific adult risk factors) treatment with bamlanivimab and etesevimab together was associated with a 70% reduction in COVID-19-\u200brelated hospitalizations and deaths relative to placebo treatment (7.0% for placebo reduced to 2.1% for bamlanivimab together with etesevimab)25,73. On the basis of these data, an additional EUA of bamlanivimab together with etesevimab has been issued5.\nMonoclonal antibody therapies in severe COVID-19. There are concurrent studies investigating neutralizing mAbs for patients hospitalized with severe COVID-19. The REGN-\u200bCOV2 trial in hospitalized patients enrols patients with or without supplemental oxygen and is ongoing74,75. In prospectively designed analysis of REGN-\u200bCOV2, there may be clinical benefit in patients treated with casirivimab and imdevimab and who were seronegative at the time of treatment76. In the ACTIV-3 RCT (n\u2009=\u2009326, 1:1 randomization), bamlanivimab added to standard of care (typically including remdesivir) did not demonstrate additional clinical benefit in hospital\u00ad ized patients77. In line with similar studies investi\u00ad gating CPT or neutralizing mAbs for patients with severe viral disease (including COVID-19)15,18,34\u201336,54,56, the evidence indicates that rapid viral clearance, in itself, is insufficient. Rather, additional factors, such as an excessive immune response, are the primary drivers for continued disease in this particular patient population. Thus, early disease seen in outpatients is likely virally driven, whereas the pathophysiology for inpatient advanced disease is predominantly a postviral or periviral phenomenon, with clinical status uncoupled from viral load.\nAdverse events associated with monoclonal antibody therapies. In terms of risk associated with mAb treatment of COVID-19, treatment-a\u200b ssociated adverse events were comparable to those with placebo. The most frequent side effects observed in RCTs include nausea, diarrhoea, dizziness, headache and vomiting24,25,78. One per cent of patients receiving casirivimab and imdevimab reported a grade 2 or higher infusion-r\u200b elated reaction within 4 days of administration (comparable to 1% reported for placebo treatment)78. In the phase II portion of BLAZE-1, nine patients reported an infusion-\u200brelated reaction\n\nnature Reviews | IMMuNology\n\n0123456789();:\n\nvolume 21 | June 2021 | 389\n\nReviews\n\n(1.9% (6/309) with bamlanivimab monotherapy, 1.8% (2/112) with bamlanivimab and etesevimab together, and 0.6% (1/156) with placebo). Most reactions occurred during infusion; these were mild in severity and were not dose related25. Regarding evidence of ADE, in vitro data indicate neutralizing mAbs do not enhance productive infection of immune cells with SARS-\u200bCoV-2 (refs3,4). From the clinical data available to date, there is no clear evidence these therapies result in enhanced immune responses consistent with ADE24,25,78. Furthermore, the safety profiles of modified and modified plus unmodified mAbs to treat SARS-\u200bCoV-2 infection are similar, suggesting that ADE may not play a role in clinical outcomes25.\nEmergence of drug-\u200bresistant SARS-\u200bCoV-2 strains. For patients with COVID-19 who receive neutralizing mAbs, there is potential for the development of drug-\u200bresistant variants, which become more obvious when selective pressure is applied in the setting of drug treatment78,79. For bamlanivimab, non-\u200bclinical studies using serial passage of SARS-\u200bCoV-2 and directed evolution of the SARS-\u200bCoV-2 S protein identified viral variants (E484D/K/Q, F490S, Q493R and S494P, amino acid substitutions in the S protein RBD) that had increased resistance to this drug4.\nIn clinical trials of bamlanivimab, genotypic and phenotypic testing are monitoring SARS-C\u200b oV-2 strains for potential S protein variations that are associated with bamlanivimab resistance. In clinical trials of bamlanivimab, viral sequencing is being performed for all patients, regardless of treatment status/progression. In other studies where only treatment failures are sampled, the selective pressure exerted by the antiviral activity cannot be assessed. In the BLAZE-1 RCT, which was limited to US investigative sites, known bamlanivimab-\u200bresistant variants at the baseline were observed at a frequency of 0.27% to date4. In the same trial, treatment-\u200bemergent variants were detected at S protein amino acid positions E484, F490 and S494 (including E484A/D/G/K/Q/V, F490L/S/V and S494L/P); considering all variants at these positions, 9.2% and 6.1% of participants in the 700-\u200bmg bamlanivimab arm (the EUA dose) harboured such a variant after the baseline at allele fractions of 15% or greater and 50% or greater, respectively, compared with 8.2% and 4.1%, respectively, of participants in the placebo arm. Most of these variants were first detected on day 7 following infusion, and were detected at only a single time point. The clinical impact of these variants is currently unknown4.\nAs with bamlanivimab, casirivimab and imdevimab therapy has the potential to lead to the development of resistant viral variants. In non-\u200bclinical studies, serial passage of vesicular stomatitis virus (VSV) encoding the SARS-C\u200b oV-2 S protein in the presence of the drugs identified escape variants with reduced susceptibility to casirivimab (K417E/N/R, Y453F, L455F, E484K, F486V and Q493K) or imdevimab (K444N/Q/T and V445A)3. Each viral variant showing reduced susceptibility to one mAb remained susceptible to the other mAb; all identified variants retained susceptibility to the combination. In a separate experiment, neutralization assays\n\nwere performed with VSV pseudotyped with 39 variants of the S protein identified in circulating SARS-\u200bCoV-2. The G476S, S494P and Q409E variants had reduced susceptibility (5-\u200bfold, 5-\u200bfold, and 4-f\u200bold, respectively) to casirivimab, and the N439K variant had reduced susceptibility (463-\u200bfold) to imdevimab. The casirivimab and imdevimab combination was active against all individual variants tested3. It has been reported that the combination of mutants at residues 417 and 439 may abrogate the effectiveness of the casirivimab and imdevimab combination80.\nIn the casirivimab and imdevimab RCT NCT04425629, interim data indicated only one variant (G446V) detected in 4.5% of participants at an allele fraction of 15% or greater, each detected at a single time point3. The clinical impact is unknown. In a VSV pseudoparticle neutralization assay, the G446V variant had reduced susceptibility to imdevimab (135-f\u200bold) but retained susceptibility to both casirivimab alone and the casirivimab and imdevimab combination.\nHowever, not all variants must be considered clinically relevant mutations associated with resistance to treatment. During the Ebola outbreak in 2018, a genomic assessment of 48 viral genomes determined that this outbreak was due to a distinct viral variant. The sequence information allowed researchers to evaluate the relevance of the distinct mutations to the available vaccine and therapeutics and to conclude that the neutralizing antibodies MAb114 and ZMapp would likely be effective against the currently circulating variant81. A similar practice for SARS-\u200bCoV-2 surveillance may be prudent to determine whether emergent S protein variants pose a threat to the efficacy of neutralizing mAb therapies.\nIndeed, three SARS-\u200bCoV-2 variants of particular interest have been identified and are circulating globally. In the United Kingdom, a variant called \u2018B.1.1.7\u2019 with a large number of mutations was identified in the autumn of 2020. In South Africa, a variant called \u2018B.1.351 was identified. Originally detected in early October 2020, B.1.351 shares some mutations with B.1.1.7. In Brazil, a variant called \u2018P.1\u2019 was identified that contains a set of additional mutations that may affect its ability to be recognized by first-\u200bgeneration neutralizing mAbs and by the immune responses generated by first-g\u200b eneration vaccines. Although these variants have been detected in the United States, according to real-\u200btime data accessed via the GISAID COVID-19 variant tracker82 these COVID-19 variants do not currently represent a significant proportion of COVID-19 infections in the United States82,83, while recent California (B.1.427/B.1.429) and New York (B.1.526) variants do. To date, the effect of these variants on the neutralizing capacity of vaccines and mAbs is unknown. A recent preprint suggests that the variants identified in the United Kingdom and South Africa are more resistant to CPT and vaccine sera42.\nBamlanivimab and imdevimab maintain full neutralization activity against the primary SARS-\u200bCoV-2 receptor-\u200bbinding site variants (69-70del and N501Y) implicated in the strain originating in the United Kingdom, suggesting that these mAbs should maintain full activity against the new strain originating in the United Kingdom42,84. From what is known about the\n\n390 | June 2021 | volume 21\n\n0123456789();:\n\nwww.nature.com/nri\n\nReviews\n\nstrains that were first identified in South Africa, Brazil as well as the ones in California and New York, it appears that some of the first generation of antibody therapies may not be as effective and it will be important for physicans to refer to the most up to date factsheet3\u20135,42.\nClinical use in COVID-19\nBamlanivimab, bamlanivimab together with etesevimab, and casirivimab with imdevimab decrease viral load when given early on in the course of SARS-\u200bCoV-2 infection and favourably impact clinical outcomes for patients with mild-\u200bto-\u200bmoderate COVID-19 (refs24,70). Although full clinical trial data are pending, top-\u200bline and interim results from multiple trials suggest that the therapies may also function as prophylaxis in at-r\u200b isk patients recently exposed to SARS-\u200bCoV-2 (refs6,7). One signal emerging from early data is that patients with persistently higher viral loads progress more frequently towards medically attended visits, emergency department visits or hospitalization, and this effect is most pronounced for patients with pre-e\u200b xisting risk factors for disease progression3,24. It remains a tenet that antivirals, whether small molecules or neutralizing mAbs, work best when deployed early. By extrapolation from early viral load data, ideally patients would receive treatment as soon as possible (that is, within hours to days following a positive test or symptom onset). In the trial setting, by day 7 to day 11 most patients either are progressing towards clearance of the virus24 or have experienced clinical decline and hospitalization, further emphasizing the need for early intervention. As the clinical trial timelines typically represent an offset of several days from initial diagnosis, corresponding to day 10\u201314 of clinical illness, the actionable message remains unchanged \u2014 treat patients as early as possible to maximize the chance of altering the disease trajectory and promote recovery.\nThe COVID-19 pandemic poses logistical and medical challenges for the distribution of neutralizing mAbs. Up to 10% of initially asymptomatic, minimally symptomatic and mild infections progressed to severe disease including respiratory distress85. While approximately 78% of patients admitted to hospital have at least one documented co-\u200bmorbidity86, there continue to be patients lacking any identified co-\u200bmorbidity who subsequently become critically ill. Thus, the absence of co-\u200bmorbidities does not completely eliminate the risk of severe disease and sequalae, and there is an urgent need for additional insight into a more personalized predictive algorithm to unlock as-\u200byet-\u200bunidentified risk factors. Contrary to the discussion in the lay media, COVID-19 can potentially claim the lives of young adults in their prime, even in the absence of any known underlying risk factors. Given that persistently high SARS-\u200bCoV-2 viral loads may be associated with severe clinical outcomes87\u201389, it is possible that early reassessment of viral loads might help guide who among the \u2018lower-r\u200b isk\u2019 population might be helped by neutralizing mAbs. RCT evidence indicates that the clinical value of neutralizing mAb therapy is more pronounced in individuals who are seronegative at diagnosis70. Collectively, measuring viral load and serology would allow strategic deployment for patients without otherwise identifiable\n\nrisk factors while targeting early supply to the high-\u200brisk population. However, this strategy would be contingent on rapid turnaround of laboratory testing. Meanwhile, it also seems reasonable to use neutralizing mAbs early on during the disease for patients with well-\u200bidentified risk factors for severe disease evolution90.\nAnother way to classify candidate patients for neutralizing mAbs would be to select patients who are expected to have poor antiviral responses (for example, elderly or immunocompromised patients) or to identify patients with poor T cell and/or B cell function via experimental techniques (such as by serology or flow cytometry). Regarding the latter, there is a lack of published evidence on humoral immune response dynamics and correlation with clinical outcomes. Furthermore, technical difficulties in stratifying patients on the basis of antibody production, lymphocyte function and/or viral load might pose a significant impediment to the timely identification of the most appropriate patients for neutralizing mAb therapy.\nFinally, antiviral and antimicrobial therapies are traditionally plagued by their promoting escape variants, and sometimes combination therapy can mitigate this risk. As a first-\u200bgeneration approach for neutralizing mAbs, monotherapies have been developed and have been demonstrated to be efficacious, but it is expected that a greater number of combination therapies will follow. For example, in phase II/III trials involving patients with COVID-19, bamlanivimab and the bamlanivimab and etesevimab combination had similarly improved magnitudes and timings of symptom relief relative to the placebo25. However, to date, the bamlanivimab and etesevimab combination does not appear to lead to the emergence of drug-\u200bresistant variants of SARS-\u200bCoV-2. This is similar to what has been observed for the other authorized neutralizing mAb combination (casirivimab and imdevimab), as already described herein. At present, there remains a role for continued use of monotherapy while transitioning towards combination therapies so as to mitigate the selection pressure for viral escape as manufacturing capacity becomes available, to ensure longevity of the therapies and to reduce the potential rate of treatment failure.\nSummary and conclusions\nThe sudden arrival and devastating spread of the COVID-19 pandemic has stimulated an accelerated programme of international research to identify effective ways to limit the spread of infection and to reduce the morbidity and mortality associated with COVID-19 (refs91,92). The first data are now emerging on vaccines designed to prevent disease93,94. In the context of active SARS-\u200bCoV-2 infection, clinical trials suggest that mortality in infected patients with hypoxia could be reduced with agents such as dexamethasone, baricitinib (in combination with remdesivir) and tocilizumab (data still under review)95\u201399. Furthermore, many trials have been conducted or are under way with various immune-\u200bmodulating medications designed to limit the tissue damage associated with the later stages of COVID-19. However, to date there have been few unequivocal successes. Neutralizing mAbs, particularly in\n\nnature Reviews | IMMuNology\n\n0123456789();:\n\nvolume 21 | June 2021 | 391\n\nReviews\n\ncombination with other medications, are an attractive approach with potential utility in both prophylactic and treatment settings. Encouraging early clinical trial data support further investigation of neutralizing mAbs to determine the optimal dosing regimen. Unanswered questions regarding this novel therapeutic approach set a pressing research agenda; we need to establish which at-r\u200b isk individuals would benefit most from prophylactic\n\nneutralizing mAbs, the duration of protection offered by these mAbs and any potential impact of mAb therapy on subsequent vaccination. It will also be important to determine the optimum timing for administration of neutralizing mAbs on the basis of viral load, serology and other potential clinical factors.\nPublished online 19 April 2021\n\n1. Renn, A., Fu, Y., Hu, X., Hall, M. D. & Simeonov, A. Fruitful neutralizing antibody pipeline brings hope to defeat SARS-\u200bCov-2. Trends Pharmacol. Sci. 41, 815\u2013829 (2020).\n2. Shanmugaraj, B., Siriwattananon, K., Wangkanont, K. & Phoolcharoen, W. Perspectives on monoclonal antibody therapy as potential therapeutic intervention for coronavirus disease-19 (COVID-19). Asian Pac. J. Allergy Immunol. 38, 10\u201318 (2020).\n3. Regeneron Pharmaceuticals Inc. Fact sheet for health care providers: emergency use authorization (EUA) of casirivimab and imdevimab. Regeneron https:// www.regeneron.com/sites/default/files/treatment-\u200b covid19-eua-f\u200bact-sheet-f\u200bor-hcp.pdf (2020).\n4. US Food and Drug Administration. Fact sheet for health care providers emergency use authorization (EUA) of bamlanivimab. FDA https://www.fda.gov/ media/143603/download (2020).\n5. US Food and Drug Administration. Fact sheet for health care providers emergency use authorization (EUA) of bamlanivimab and etesevimab. FDA https:// www.fda.gov/media/145802/download (2021).\n6. Regeneron Pharmaceuticals Inc. Regeneron reports positive interim data with REGEN-\u200bCOV\u2122 antibody cocktail used as passive vaccine to prevent COVID-19. Regeneron https://newsroom.regeneron.com/news-\u200b releases/news-\u200brelease-details/regeneron-\u200breportspositive-i\u200bnterim-data-r\u200begen-covtm-a\u200b ntibody (2021).\n7. Eli Lilly and Company. Lilly\u2019s neutralizing antibody bamlanivimab (LY-\u200bCoV555) prevented COVID-19 at nursing homes in the BLAZE-2 trial, reducing risk by up to 80 percent for residents. Eli Lilly and Company https://investor.lilly.com/news-\u200breleases/news-\u200breleasedetails/lillys-\u200bneutralizing-antibody-\u200bbamlanivimably-c\u200b ov555-prevented (2021).\n8. Llewelyn, M. B., Hawkins, R. E. & Russell, S. J. Discovery of antibodies. BMJ 305, 1269\u20131272 (1992).\n9. Behring, E. A. & Kitasato, S. Ueber das Zustandekommen der Diphtherie-\u200bimmunitat und der Tetanus-i\u200bmmunitat bei Thieren. Dtsch. Med. Wochenschr. 28, 1321\u20131332 (1890).\n10. Kaufmann, S. H. Immunology\u2019s foundation: the 100-year anniversary of the Nobel Prize to Paul Ehrlich and Elie Metchnikoff. Nat. Immunol. 9, 705\u2013712 (2008).\n11. Hung, I. F. et al. Convalescent plasma treatment reduced mortality in patients with severe pandemic influenza A (H1N1) 2009 virus infection. Clin. 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Baricitinib plus remdesivir for hospitalized adults with Covid-19. N. Engl. J. Med. https://doi.org/10.1056/NEJMoa2031994 (2020).\n97. US Food and Drug Administration. Fact sheet for healthcare providers emergency use authorization (EUA) of baricitinib. FDA https://www.fda.gov/ media/143823/download (2020).\n98. The REMAP-C\u200b AP Investigators et al. Interleukin-6 receptor antagonists in critically ill patients with Covid19\u2013preliminary report. Preprint at medRxiv https://doi.org/10.1101/2021.01.07.21249390 (2021).\n99. RECOVERY Collaborative Group et al. Tocilizumab in patients admitted to hospital with COVID-19 (RECOVERY): preliminary results of a randomised, controlled, open-\u200blabel, platform trial. Preprint at medRxiv https://doi.org/10.1101/2021.02.11. 21249258 (2021).\nAcknowledgements This work was supported by Eli Lilly and Company. C. J. Antalis and H. Green (Eli Lilly and Company) provided editorial assistance.\nAuthor contributions The authors contributed equally to all aspects of the article.\nCompeting interests P.C.T. has received research grants, consultation fees and/or speaking fees from AbbVie, Biogen, Bristol-\u200bMyers Squibb, Celgene, Celltrion, Fresenius, Galapagos, Gilead, GlaxoSmithKline, Janssen, Eli Lilly and Company, Sanofi, Nordic Pharma, Pfizer, Roche and UCB. A.C.A., I.d.l.T. and M.M.H. are employees and shareholders of Eli Lilly and Company. K.W. has received research grants from BristolMyers Squibb and Pfizer and consulting fees from AbbVie, AstraZeneca, Bristol-\u200bMyers Squibb, Eli Lilly and Company, Galapagos, GlaxoSmithKline, Gilead, Novartis, Pfizer, Regeneron, Roche, Sanofi and UCB. R.L.G. reports nonfinancial support from Gilead Sciences Inc. and personal fees from Gilead Sciences Inc. outside the submitted work.\nPeer review information Nature Reviews Immunology thanks the anonymous reviewers for their contribution to the peer review of this work.\nPublisher\u2019s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\nRelated links ClinicalTrials.gov: https://clinicaltrials.gov COviD-19 Biologics Tracker: https://www.antibodysociety. org/covid-19-biologics-tracker/ GisAiD COviD-19 variant tracker: https://www.gisaid.org/\n\u00a9 The Author(s), under exclusive licence to Springer Nature Limited. 2021\n\nnature Reviews | IMMuNology\n\n0123456789();:\n\nvolume 21 | June 2021 | 393\n\n\n",
    "authors": [
      "Peter C. Taylor",
      "Andrew C. Adams",
      "Matthew M. Hufford",
      "Inmaculada de la Torre",
      "Kevin Winthrop",
      "Robert L. Gottlieb"
    ],
    "doi": "10.1038/s41577-021-00542-x",
    "date": "2021-06",
    "item_type": "journalArticle",
    "url": ""
  },
  {
    "key": "672L5N3W",
    "title": "Convalescent Plasma Therapy for COVID-19: State of the Art.",
    "abstract": "Convalescent plasma (CP) therapy has been used since the early 1900s to treat emerging infectious diseases; its efficacy was later associated with the evidence  that polyclonal neutralizing antibodies can reduce the duration of viremia.  Recent large outbreaks of viral diseases for which effective antivirals or  vaccines are still lacking has renewed the interest in CP as a life-saving  treatment. The ongoing COVID-19 pandemic has led to the scaling up of CP therapy  to unprecedented levels. Compared with historical usage, pathogen reduction  technologies have now added an extra layer of safety to the use of CP, and new  manufacturing approaches are being explored. This review summarizes historical  settings of application, with a focus on betacoronaviruses, and surveys current  approaches for donor selection and CP collection, pooling technologies, pathogen  inactivation systems, and banking of CP. We additionally list the ongoing  registered clinical trials for CP throughout the world and discuss the trial  results published thus far.",
    "full_text": "REVIEW\ncrossm\n\nConvalescent Plasma Therapy for COVID-19: State of the Art\nDaniele Focosi,a Arthur O. Anderson,b Julian W. Tang,c Marco Tuccorid,e\naNorth-Western Tuscany Blood Bank, Pisa University Hospital, Pisa, Italy bDepartment of Respiratory Mucosal Immunity, US Army Medical Research Institute of Infectious Diseases, Frederick, Maryland, USA cRespiratory Sciences, University of Leicester, Leicester, United Kingdom dDivision of Pharmacology and Pharmacovigilance, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy eUnit of Adverse Drug Reaction Monitoring, Pisa University Hospital, Pisa, Italy\n\nSUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 CP DONOR RECRUITMENT STRATEGIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 CONVALESCENT PLASMA AND PATHOGEN INACTIVATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3\nTechnologies To Virally Reduce Plasma (Pathogen Inactivation) . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5\nLarge-pool products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 MPFS into immunoglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 CP BANKING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 LESSONS FROM SARS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 LESSONS FROM MERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 CONVALESCENT PLASMA FOR COVID-19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 MONITORING RESPONSE TO CP TREATMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 CONCERNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 SIDE BENEFITS FROM CP IN COVID-19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 AUTHOR BIOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17\n\nSUMMARY Convalescent plasma (CP) therapy has been used since the early 1900s to treat emerging infectious diseases; its ef\ufb01cacy was later associated with the evidence that polyclonal neutralizing antibodies can reduce the duration of viremia. Recent large outbreaks of viral diseases for which effective antivirals or vaccines are still lacking has renewed the interest in CP as a life-saving treatment. The ongoing COVID-19 pandemic has led to the scaling up of CP therapy to unprecedented levels. Compared with historical usage, pathogen reduction technologies have now added an extra layer of safety to the use of CP, and new manufacturing approaches are being explored. This review summarizes historical settings of application, with a focus on betacoronaviruses, and surveys current approaches for donor selection and CP collection, pooling technologies, pathogen inactivation systems, and banking of CP. We additionally list the ongoing registered clinical trials for CP throughout the world and discuss the trial results published thus far.\nKEYWORDS Ebola virus disease, Middle East respiratory syndrome, antibodydependent enhancement, convalescent blood product, convalescent plasma, convalescent whole blood, coronavirus disease 2019, enzyme-linked immunosorbent assay, intravenous immunoglobulins, plaque reduction neutralization test, SARS\n\nINTRODUCTION\nThe recent COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1) has demonstrated the fragility of our health systems in tackling emergency situations related to the spread of new infectious agents that require the rapid development of effective care strategies. Unfortunately, there are several potentially pandemic viruses, such as \ufb02aviviruses (e.g., West Nile virus [WNV],\n\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nClinical Microbiology Reviews\n\nCitation Focosi D, Anderson AO, Tang JW, Tuccori M. 2020. Convalescent plasma therapy for COVID-19: state of the art. Clin Microbiol Rev 33:e00072-20. https://doi.org/10.1128/CMR .00072-20. Copyright \u00a9 2020 American Society for Microbiology. All Rights Reserved. Address correspondence to Daniele Focosi, daniele.focosi@gmail.com. Published 12 August 2020\ncmr.asm.org 1\n\nFocosi et al.\ndengue virus, and Zika virus) (2), chikungunya virus (3), in\ufb02uenza viruses A [e.g., A(H1N1) and A(H5N1)] (4), Ebola virus (EBOV) (5), and respiratory betacoronaviruses (SARS-CoV and Middle East respiratory syndrome-CoV [MERS-CoV]), which could put us in situations very similar to the situation with the current pandemic and which require the development of speci\ufb01c intervention protocols.\nWhile vaccination strategy is undoubtedly a viable goal, development of a vaccine requires a time frame not compatible with an emergency situation. It is also a prophylactic approach that has no use in the therapeutic setting. On the other hand, the use of antivirals is valuable for the therapeutic setting (6, 7). For the limited number of antiviral agents currently available, unless provided free of charge to developing countries, \ufb01nancial cost is an issue. Additionally, manufacturing is hard to scale up in short time frames.\nIn situations in which the new pathogen is able to induce an immune response with the production of neutralizing antibodies, passive transfusion of convalescent blood products (CBPs), in particular, convalescent plasma (CP), has proven to be a winning and logistically feasible therapeutic strategy (8). CBPs can be manufactured by collecting whole blood or apheresis plasma from a convalescent donor. This approach has been used since 1900 (9), and previous experiences have been reported elsewhere (10).\nThe main accepted mechanism of action for CBP therapy is clearance of viremia, which typically happens 10 to 14 days after infection (11). So CBP has been typically administered after the appearance of early symptoms to maximize ef\ufb01cacy. Convalescent whole blood (CWB), in addition to antibodies, provides control of hemorrhagic events, as in Ebola virus disease, if transfusion occurs within 24 h to maintain viable platelets and clotting factors. Nevertheless, CP best \ufb01ts settings where only antibodies are required.\nIn this review, we have described current technologies for CP collection, manufacturing, pathogen inactivation, and banking of CP. Then we have summarized historical settings of CBP application, with a speci\ufb01c focus on applications for COVID-19 and other future pandemics. Several articles included in this review are available as preprints which have not yet passed peer review, as indicated in the reference section.\nCP DONOR RECRUITMENT STRATEGIES Convalescent donor testing for neutralizing antibodies is mandatory in upstream\ndonor selection. Donor selection is generally based on neutralizing antibody titer, as assessed with a plaque reduction neutralization test (PRNT) (12), which requires a viable isolate, replication-competent cell lines, and skilled personnel. Since PRNT takes time to be set up and requires expensive facilities, in resource-poor settings or in time-sensitive scenarios, collection based on a retrospective PRNT or, alternatively, on an enzymelinked immunosorbent assay (ELISA) targeting the recombinant receptor binding domains (RBDs) of the viral antireceptor has often been implemented; under these circumstances, studies have suggested that ELISA ratios/indexes have good correlations with PRNT titers; e.g., the Euroimmun ELISA IgG score detected 60% of samples with PRNT titers of \u03fe1:100, with 100% speci\ufb01city using a signal/cutoff reactivity index of 9.1 (13). The current understanding of neutralization suggests that the virus-blocking effect is related to the amount of antibodies against different epitopes coating the virion, whose stoichiometry is in turn affected by antibody concentration and af\ufb01nity.\nThe donor should preferably live in the same area as the intended recipient(s) to allow consideration of mutations of the target viral antigens. SARS-CoV-2 S protein has already mutated after a few months of viral circulation (14), with one mutation outside the receptor-binding motif (23403A\u00a1G single nucleotide polymorphism, corresponding to a D614G amino acid change) currently de\ufb01ning a dominant clade (15) characterized by reduced S1 shedding and increased infectivity (16). Nevertheless, it should be considered that preferring indigenous donors could represent a drawback in areas with epidemics of other infectious diseases (e.g., malaria).\nThree approaches are theoretically available to recruit CP donors, with each having pros and cons. The least cost-effective approach is screening the general regular blood\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nClinical Microbiology Reviews cmr.asm.org 2\n\nConvalescent Plasma Therapy for COVID-19\ndonor population for the presence of anti-SARS-CoV-2 antibodies. In areas of endemicity, such a strategy provides many \ufb01t donors with the additional bene\ufb01t of seroprevalence study in the general population (80% of cases being asymptomatic) but requires a large budget.\nAlternatively, recruitment of hospital-discharged patients is highly cost-effective (patients can be easily tested before discharge and tracked), but patients who have required hospitalization are highly likely to be elderly with comorbidities and, hence, un\ufb01t to donate.\nThe intermediate approach, whenever allowed by privacy regulations, is making calls to positive cases under home-based quarantine to solicit donations; given the large numbers of such cases, some of them are likely to be regular donors, and home-based convalescence suggests that they are \ufb01t enough to donate. Nevertheless, lessons from MERS (17) and preliminary evidence with COVID-19 (18\u201320) suggest that patients with mild symptoms may develop low-titer antibodies, making antibody titration even more important in the population-wide and home-based approaches. Plasma samples collected an average of 30 days after the onset of symptoms had undetectable half-maximal neutralizing titers in 18% of donors (21).\nUnder emergency settings, it has often happened that donors are not screened for high-titer neutralizing antibodies or that low-titer donations are collected; nevertheless, as soon as the urgent requests are satis\ufb01ed and a buffer stock has been created, repeat donations should preferably focus on donors with high titers (22).\nAs recently suggested, plasmapheresis could additionally bene\ufb01t the convalescent COVID-19 donor by reducing the prothrombotic state via the citrate-based anticoagulants administered during donation and by removal of high-molecular-weight viscous components (23).\nIn addition to interventional trials, in the United States several trials have been initiated to create registries (e.g., ClinicalTrials.gov registration no. NCT04359602) or collect plasma with titers of \u03fe1:64 from immune donors for banking purposes, without immediate reinfusion (e.g., trial NCT04360278, NCT04344977, or NCT04344015). These approaches should be encouraged to better face the next waves of the COVID-19 pandemic.\nCONVALESCENT PLASMA AND PATHOGEN INACTIVATION CP should be collected by apheresis in order to ensure larger volumes than available\nwith whole-blood donations and more frequent donations and to avoid causing unnecessary anemia in the convalescent donor. Double \ufb01ltration plasmapheresis (DFPP) using fractionation \ufb01lter 2A20 is under investigation as an approach to increase IgG yield by 3 to 4 times (Table 1, trial NCT04346589 in Italy); since DFPP-derived plasma is not an ordinary blood component but, rather, a discard product, additional regulations could apply in different countries. A very exploratory approach is under investigation in a Chinese trial collecting immunoglobulins from convalescent donors by immunoadsorption (trial NCT04264858), which could be an alternative to plasma fractionation.\nTechnologies To Virally Reduce Plasma (Pathogen Inactivation) Although neither the U.S. Food and Drug Administration (FDA) (24) nor the Euro-\npean Center for Disease Control (ECDC) is recommending pathogen reduction technologies (PRT) for CP (25), several national authorities consider that, under emergency settings, donor screening and conventional viral nucleic acid testing (NAT) (i.e., HIV, hepatitis C virus [HCV], and hepatitis B virus [HBV] NAT) would not be enough to ensure CP safety (12). Under this scenario, additional virological testing and PRT approximately double the \ufb01nal cost of the therapeutic dose. Several technologies for PRT have been approved and are currently marketed.\nSolvent/detergent (S/D)-\ufb01ltered plasma provides quick inactivation of \u03fe4 logs of most enveloped viruses; although the technology was developed and is widely used for large plasma pools, small-scale reduction has been reported. The technology relies on several steps: addition of 1% tri(n-butyl) phosphate\u20131% Triton X-45, elimination of solvent and detergent via oil extraction and \ufb01ltration, and \ufb01nally sterile \ufb01ltration (26).\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nClinical Microbiology Reviews cmr.asm.org 3\n\nFocosi et al.\n\nClinical Microbiology Reviews\n\nTABLE 1 Ongoing interventional clinical trials of convalescent plasma in COVID-19 patientsa\n\nPhase(s) and indication I/II\nExposed or con\ufb01rmed children\nAll patients with COVID-19\nNon-critically ill patients\nSevere or critically ill patients\n\nTrial no.\nNCT04377672\nNCT04292340 NCT04376788\nNCT04345679 NCT04397523 NCT04356482 NCT04357106 NCT04384497\nNCT04389944 NCT04343755 NCT04360486 NCT04354831 NCT04408040 NCT04355897 NCT04332380 NCT04375098 NCT04327349 IRCT20200325046860N1 NCT04365439 NCT04374565 NCT04348877 NCT04408209 NCT04346589 NCT04333355 NCT04352751\nNCT04347681 NCT04353206 NCT04343261 NCT04388527 NCT04389710 NCT04338360 NCT04374370 NCT04358211 NCT04363034 NCT04372368 NCT04340050\n\nCountry\nUSA\nChina Egypt\nHungary North Macedonia Mexico Mexico Sweden\nSwitzerland USA\nColombia Chile Iran Iran Switzerland USA Egypt Greece Italy Mexico Pakistan\nSaudi Arabia USA\n\nStudy population (no. of participants per arm)b\n30\n15 15\n20 20 90 10 50\n15 55 EAP 131 700 100 10 30 30 200 10 29 20 60 10 20 2,000\n40 90 15 50 100 NA EAP EAP EAP up to 100 EAP up to 150 10\n\nSchedule (vs control arm)c\n5 ml/kg, equivalent to 1\u20132 U (200\u2013250 ml/U)\nNA Exchange transfusion by venesection\nof 500 ml of blood replacement by 1 U of PRBC \u03e9 1 mg/kg methylene blue i.v. over 30 min \u03e9 200 ml of CP 1 U of CP (200 ml) NA Different amounts of CP 1 U of CP (200 ml) Up to 7 infusions (200 ml each), dose-\ufb01nding study 2 U of CP (200 ml each) NA NA 1\u20132 U of CP (\u03fd7 ml/kg adjusted IBW) 200\u2013425 ml of CP 500 ml 2 U of CP (250 ml each)/24 h 200 ml of CP on days 1 and 2 NA NA NA 2 U of CP (200 ml each) in 1\u20132 days 1 400-ml unit of CP 3 doses of CP DFPP-collected CP 1\u20132 U of CP (250 ml/24 h) Children \u03fd35 kg, 15 ml/kg over 4\u2013 6 h; adults, \u03fd450\u2013500 ml over 4\u20136 h 10\u201315 ml of CP/kg body wt 1\u20132 U of CP on days 0 and 6 2 U of CP 2 U of CP 1\u20132 U of CP (200/600 ml) 1 U of CP (200/250 ml) 1\u20132 U (200\u2013400 ml/U), not to exceed 550 ml total\n1 U of CP (300 ml)\n\nDonor titerd\n\u03fe1:320\nNA NA\n\u03fe1:320 \u03fe5 AU/ml NA NA NA\nNA \u03fe1:64 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA\nNA NA NA NA NA NA NA \u03fe160 NA NA NA\n\nIII Exposed within 96 h of enrollment and 120 h of receipt of plasma\n\nNCT04323800 NCT04390503\n\nAll patients with COVID-19\n\nNCT04377568 ChiCTR2000030039 NCT04345289\n\nUSA\nCanada China Denmark\n\nNCT04372979\n\nFrance\n\nNCT04374487\n\nIndia\n\nNCT04346446\n\nNCT04380935 IRCT20200310046736N1 NCT04342182 NCT04366245\n\nIndonesia Iran Netherlands Spain\n\n150 (Exp, 75; Ctr, 75)\n200 (Exp, 100; Ctr, 100)\n100 90 (Exp, 30; Ctr, 60) 1,500 (6 arms)\n80\n100\n40\n60 45 426 72\n\n1 U of CP (200\u2013250 ml) vs nonconvalescent plasma\n1 U of CP (200\u2013250 ml) vs 5% albumin i.v.\n10 ml/kg, up to 500 ml, vs BSC 2 U of CP (200/500 ml/24 h) vs BSC 1 600-ml unit of CP vs sarilumab vs\nbaricitinib vs hydroxychloroquine vs injective placebo vs oral placebo 2 U of 200\u2013230 ml of CP vs nonconvalescent plasma Up to 3 200-ml doses of CP 24 h apart vs BSC 1\u20133 U (200 ml) of CP vs nonconvalescent plasma NA vs BSC CP vs PDIES 1 U of CP (250 ml) vs BSC NA vs BSC\n\n\u03fe1:64 NA\nNA NA NA\nNA \u03fe1:40 NA\nNA NA NA NA\n\n(Continued on next page)\n\nOctober 2020 Volume 33 Issue 4 e00072-20\n\ncmr.asm.org 4\n\nConvalescent Plasma Therapy for COVID-19\n\nClinical Microbiology Reviews\n\nTABLE 1 (Continued)\n\nPhase(s) and indication\n\nTrial no.\nNCT04344535 NCT04333251 NCT04355767 NCT04373460\n\nNCT04362176\n\nNCT04376034\n\nNon-critically ill patients NCT04356534\n\nNCT04348656 ChiCTR2000030702 ChiCTR2000030929 ChiCTR2000030010 NCT04332835 NCT04345991\n\nNCT04374526\n\nNCT04393727 NCT04358783 NCT04345523 NCT04364737 NCT04361253\n\nNCT04397757 NCT04359810\n\nSevere or critically ill patients\n\nChiCTR2000029850 ChiCTR2000030179 ChiCTR2000030627 NCT04346446\n\nNCT04385043 NCT04381858\n\nNCT04388410 NCT04405310\n\nCountry USA\nBahrain Canada China\nColombia France Italy Italy Mexico Spain USA\nChina\nIndia Italy Mexico\n\nStudy population (no. of participants per arm)b 500 115 206 1344 (Exp, 772; Ctr, 772)\n500 (Exp, 250; Ctr, 250)\n240\n40 (Exp, 20; Ctr, 20)\n1,200 50 (Exp, 25; Ctr, 25) 80 (Exp, 30; Ctr, 30) 100 (Exp, 50; Ctr, 50) 80 120\n182\n126 (Exp, 63; Ctr, 63) 30 (Exp, 20; Ctr, 10) 278 (Exp, 139; Ctr, 139) 300 220\n80 (Exp, 40; Ctr, NA) 105 (Exp, 70; Ctr, 35)\n20 (Exp, 10; Ctr, 10) 100 (Exp, 50; Ctr, 50) 30 (Exp, 15; Ctr, 15) 40\n400 (Exp, 200; Ctr, 200) 500 (Exp, 340: Ctr, 160)\n250 (Exp, 125; Ctr, 125) 80 (Exp, 40; Ctr, 40)\n\nSchedule (vs control arm)c\n450\u2013550 ml of CP vs BSC 1\u20132 U of CP (250 ml/24) vs BSC 1\u20132 U of CP (200\u2013600 ml) vs placebo 1 U of CP (200\u2013250 ml) vs\nnonconvalescent plasma 1 U of CP (250 ml at a rate of\n500 ml/h) vs placebo 1 (moderate) or 2 (severe) U of CP\nvs BSC 2 U of CP, 200 ml each, over 2 h in 2\nconsecutive days vs BSC 500 ml of CP within 12 h vs BSC NA vs BSC NA vs BSC NA vs BSC 2 U of CP (250 ml/24 h) vs BSC Up to 4 U of CP (200\u2013220 ml each)\nvs BSC 200 ml/day for 3 consecutive days vs\nBSC 1 U (200 ml) of CP vs BSC 1 U (200 ml) of CP vs BSC CP vs BSC 1\u20132 U (250 ml each) vs i.v. placebo 2 U of CP (250 ml each) within 24 h\nvs nonconvalescent plasma 2 U of CP vs BSC 1 U (200\u2013250 ml) of CP vs\nnonconvalescent plasma NA vs BSC NA vs BSC NA vs BSC 1\u20133 U (200 ml each) of CP vs\nnonconvalescent plasma NA vs BSC 2 U (200 ml each) of CP vs\npolyclonal IVIg at 0.3 gr/kg/day (5 doses) 2 U of CP vs masked i.v. saline 1 U of CP vs 20% albumin\n\nDonor titerd \u03fe1:320 \u03fe1:64 \u03fe1:80 \u05461:320\nNA\nNA\nNA\nNA NA NA NA NA NA\nNA\nNA NA NA NA NA\nNA NA\nNA NA NA NA\nNA NA\nNA NA\n\naTrials included are listed in the World Health Organization International Clinical Trial Registry Platform (ICTRP) databases (https://www.who.int/docs/default-source/ coronaviruse/covid-19-trials.xls; accessed 7 July 2020), NIH ClinicalTrials database (www.clinicaltrials.gov; accessed 7 July 2020), and Cytel Global Coronavirus COVID-19 Clinical Trial Tracker (www.covid-trials.org; accessed 7 July 2020). bWhen the information was available, the numbers of participants in the experimental group (Exp) and control group (Ctr) are given in parentheses. EAP, expanded access program; NA, not available. cNA, not available; PRBC, packed red blood cells; IBW, ideal body weight; BSC: best supportive care; PDIES, plasma-derived immunoglobulin-enriched solution; i.v., intravenous. dAU, arbitrary units.\n\nFiltration across 75- to 35-nm-pore-size hollow \ufb01bers could remove large viruses (such as betacoronaviruses) while preserving IgG (27), but this has not been implemented yet.\nIn recent years photoinactivation in the presence of a photosensitizer has become the standard for single-unit inactivation; approved technologies include combinations of methylene blue and visible light (28) (Thera\ufb02ex), amotosalen (S-59) and UV A (29) (Intercept), and ribo\ufb02avin and UV B (30) (Mirasol). These methods do not affect immunoglobulin activity.\nFatty acids are also an option. In 2002 it was reported that caprylic acid (31) and octanoic acid (32) were as effective as S/D at inactivating enveloped viruses.\nHeat treatment of plasma has been used in the past (33, 34) but comes with a risk of aggregation of immunoglobulins (35, 36).\nPooling Figure 1 represents how CP and intravenous immunoglobulin (IVIg) can be obtained\nunder modern fractionation procedures. As per CP collection, two approaches can be pursued.\nOctober 2020 Volume 33 Issue 4 e00072-20\n\ncmr.asm.org 5\n\nFocosi et al.\n\nClinical Microbiology Reviews\n\nFIG 1 Summary of possible convalescent blood products (CBP). (Adapted from reference 153 with permission of Elsevier.)\nLarge-pool products. Pharmaceutical-grade facilities typically pool 100 to 2,500 donors to manufacture S/D-inactivated plasma. IVIgs are similarly prepared from pools of 2,000 to 4,000 liters of plasma (or 100 to 1,000 liters in the case of hyperimmune IVIg) (37, 38). Such volumes can hardly be obtained from CP donors, and timely creation of dedicated CP production chains pose dif\ufb01cult good manufacturing practice (GMP) issues within plasma vendor plants (38).\nMPFS into immunoglobulins. In order to be economically sustainable, contract (private-run) fractionation typically requires well over 10,000 liters of plasma per year, and domestic (state-owned) fractionation typically requires over 100,000 to 200,000 liters per year in addition to starting up a fractionation facility. An \u201con-the-bench\u201d minipool fractionation scale (MPFS) process (5 to 10 liters of plasma, i.e., approximately 20 recovered plasma units) using disposable devices and based on caprylic acid precipitation has been under development in Egypt since 2003 and has proved effective at purifying coagulation factors (39) and immunoglobulins (6-fold enrichment) (40). The same disposable bag system has also been combined with S/D reduction (26).\nCP BANKING CP can be either frozen or transfused as a fresh product. Aliquots of 200 to 300 ml\ncan be easily achieved from a single unit using modern PRT kits. Banking CP at temperatures below \u03ea25\u02daC (according to European Directorate for the Quality of Medicines [EDQM] or FDA guidelines for ordinary plasma for clinical use [41]) is encouraged in order to produce CP as an off-the-shelf, ready-to-use product. Most regulatory systems require that CP be tracked informatically as a blood component\nOctober 2020 Volume 33 Issue 4 e00072-20\n\ncmr.asm.org 6\n\nConvalescent Plasma Therapy for COVID-19\ndifferent from ordinary plasma for clinical use. The \ufb01nal validation label should report that the donor has tested negative by PCR for the convalescent disorder and additional microbiological tests and should describe the inactivation method. A single cycle of freezing and thawing does not signi\ufb01cantly affect the quantity or function of immunoglobulins (42). Given that COVID-19 AB blood group recipients can receive CP units only from scarce matched blood group AB donors, to increase the pool of compatible units several authors have recommended titration of anti-A and anti-B isoagglutinins and transfusion of low-titer (\u03fd1:32) non-ABO-compatible CP units (i.e., O, A, and B) to AB recipients (22, 43).\nLESSONS FROM SARS SARS-speci\ufb01c neutralizing antibodies usually persist for 2 years (44), and a decline in\nprevalence and titers occurs in the third year (45). Convalescent anti-SARS immunoglobulins were manufactured on a small scale (8, 46). Three infected health care workers with SARS progression despite the best supportive care (BSC) survived after transfusion with 500 ml of CP; viral load dropped to zero at 1 day after transfusion (47). Soo et al. reported in a retrospective nonrandomized trial that treatment with CP (titer of \u03fe1:160) in 19 patients was associated with a shorter hospital stay and lower mortality than continuing treatment with high-dose methylprednisolone (48). Amotosalen photochemical inactivation of apheresis platelet concentrates demonstrated a \u03fe6.2 log10 mean reduction of SARS-CoV (49). Thera\ufb02ex reduces infectivity of SARS-CoV in plasma (50). Heating at 60\u00b0C for 15 to 30 min reduces SARS-CoV from plasma without cells (51), while maintaining 60\u00b0C for 10 h is required for plasma products (52). In addition, SARS-CoV was found to be sensitive to S/D (51, 53).\nLESSONS FROM MERS Antibody responses to MERS persist for less than 1 year, and the magnitude corre-\nlates with the duration of viral RNA shedding in sputum (but not with viral load). Patients with mild disease have very low antibody titers, making CP collection challenging in MERS convalescents (54). A study reported that only 2.7% (12 out of 443) exposed cases tested positive by ELISA, and only 75% of them had reactive microneutralization assay titers (17). CP with a PRNT titer of \u05461:80 provides clinical bene\ufb01t in MERS (55). A case of transfusion-related acute lung injury (TRALI) following CP transfusion in a patient with MERS was reported (56, 57). MERS-CoV load in plasma was reduced by Thera\ufb02ex (58), Intercept (59), Mirasol (60), and heating at 56\u00b0C for 25 min (61); in all cases, passaging of inactivated plasma in replication-competent cells showed no viral replication.\nCONVALESCENT PLASMA FOR COVID-19 As soon as the COVID-19 pandemic appeared (62, 63), several authors suggested CP\nas a potential therapeutic agent (64, 65). Of interest, the most critically ill patients show prolonged viremia (strongly correlated with serum interleukin-6 [IL-6] levels) (10), which makes feasible therapeutic intervention with antiviral agents and immunoglobulins even at late stages. Viral shedding in survivors can last as long as 37 days (62), mandating SARS-CoV-2 RNA screening in CP donors. Serum IgM and IgA antibodies appear in COVID-19 patients as early as 5 days after symptom onset (66), while IgG can be detected at day 14 (67). IgGs are generally detected after 20 days (68, 69). Severely ill female patients generate IgG earlier and at higher titers (70, 71); the greatest part of the neutralizing antibody response has been shown to be associated with the IgG1 and IgG3 subclasses (72, 73). Duration of anti-SARS-CoV-2 antibodies in plasma is currently unknown; while the overall antibody responses for other betacoronaviruses typically declines after 6 to 12 months (74), SARS-speci\ufb01c neutralizing antibodies usually persist for 2 years (44). So, in the vast majority of countries, a suitable donor could donate 600 ml of plasma (equivalent to 3 therapeutic doses under most current trials) every 14 days for a minimum of 6 months. Up to 7 plasma donations have been proven not to decrease antibody titers in convalescent donors (18). In contrast to SARS and MERS\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nClinical Microbiology Reviews cmr.asm.org 7\n\nFocosi et al.\npatients, most COVID-19 patients exhibit few or no symptoms and do not require hospitalization; this could suggest that the majority of convalescent donors are best sought in the general population although speci\ufb01c studies on antibody titers in mildly symptomatic patients suggest low titers (18\u201320).\nSARS-CoV-2 is reduced by \u03fe3.4 logs by Mirasol (75) (and likely by other PRTs); nevertheless, SARS-CoV-2 viral RNA (vRNA) is detectable at low viral loads in a minority of serum samples collected in acute infection but is not associated with infectious SARS-CoV-2 (76). Intercept treatment has been proven not to reduce SARS-CoV-2 neutralizing antibody titers (77).\nThe main contraindications to CP therapy are allergy to plasma protein or sodium citrate, selective IgA de\ufb01ciency (\u03fd70 mg/dl in patients 4 years old or older), leading to anaphylaxis from IgA-containing CP (78), or treatment with immunoglobulins in the last 30 days (because of a risk of developing serum sickness). As in many other trial settings, concurrent viral or bacterial infections, thrombosis, poor compliance, short life expectancy (e.g., multiple-organ failure), and pregnancy or breastfeeding are also contraindications (79).\nIn an early case series from China, \ufb01ve patients under mechanical ventilation (4 of 5 with no preexisting medical conditions) received transfusions of CP with an ELISA IgG titer of \u03fe1:1,000 and a PRNT titer of \u03fe40 at days 10 to 22 after admission. Four patients recovered from acute respiratory disease syndrome (ARDS), and three were weaned from mechanical ventilation within 2 weeks of treatment, with the remaining patients being stable (80).\nAnother Chinese pilot study (ChiCTR2000030046) of 10 critically ill patients showed that one dose of 200 ml of CP with a neutralizing antibody titer of \u03fe1:640 resulted in an undetectable viral load in 7 patients, with radiological and clinical improvement (81).\nA third series of 6 cases with COVID-19 pneumonia in Wuhan showed that a single 200-ml dose of CP (with titers of anti-S antibodies determined by chemiluminescent immunoassay [CLIA] only) administered at a late stage led to viral clearance in 2 patients and radiological resolution in 5 patients (82). Pei et al. reported successful treatment of 2 out of 3 patients with 200- to 500-ml doses of CP (83). Recovery from mechanical ventilation was also reported by Zhang et al. in a single patient after antibodies in CP were titrated with an anti-N protein ELISA (84). No improvement in mortality despite viral clearance was reported in a retrospective observational study recruiting 6 late-stage, critically ill patients treated with gold-immunochromatographytitrated CP, compared to results in 13 untreated controls (85). One case of recovery in a centenarian patient who received 2 CP units (S-RBD-speci\ufb01c IgG titer of \u03fe1:640) was also reported (86).\nMany more case reports and small case series are accumulating in the literature; successful treatment was reported in 3 cases with ARDS and mechanical ventilation using two 250-ml CP doses (titrated with ELISA only) in South Korea (43, 87), in 2 cases from Iraq (88), in 8 out 10 severe cases from Mexico (89), in 20 out of 26 severe cases from Turkey (90), in a kidney transplant recipient from China (91), in a case with severe aplastic anemia in Poland (92), in a case with X-linked agammaglobulinemia in Spain (93), and in 1 patient with marginal-zone lymphoma treated with bendamustine and rituximab in the United Kingdom (94). Centers in the United States reported successful treatment with CP in 18 out of 20 patients in a series (95), in 27 out of 31 patients with severe to life-threatening disease in another series (96), in one case with myelodysplastic syndrome (97), in a critically ill obstetric patient (in combination with remdesivir) (98), and in an allogeneic stem cell transplant recipient (99).\nIn a single-arm phase II trial (NCT04321421 [100]) run in Lombardy, 49 patients with moderate to severe disease were treated with up to 3 units of PRT-treated CP (250 to 300 ml/48 h) having neutralizing antibody titers of \u05461:160 in 96% of cases. Importantly, the viral inoculum was 50 50% tissue culture infective doses (TCID50) instead of the usual 100 TCID50. Seven-day mortality was 6% versus 16% in a historical cohort. One case of TRALI was reported (101).\nIn a large case series from Wuhan, 138 patients were transfused with 200 to 1,200 ml\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nClinical Microbiology Reviews cmr.asm.org 8\n\nConvalescent Plasma Therapy for COVID-19\nof CP at a median of 45 days after symptom onset and experienced a 50% lower intensive care unit (ICU) admission rate and mortality than the group treated with best supportive care. Responders had higher lymphocyte counts, lower neutrophil counts, and lower lactate dehydrogenase (LDH), type B natriuretic peptide (BNP), urea nitrogen, procalcitonin, glucose, and C-reactive protein (CRP) levels. Complete data on neutralizing antibody titers in COVID-19 convalescent plasma (CCP) units were not available, but responders tended to have received CP units with higher antibody levels (102).\nIn the \ufb01rst retrospective, randomized controlled trial published to date, 39 patients in New York with severe COVID-19 were transfused with 2 units of ABO-type matched CP with anti-Spike antibody titers of \u05461:320 (measured by a two-step Spike proteindirected ELISA). CP recipients were more likely than control patients to not increase their supplemental oxygen requirements by posttransfusion day 14 (odds ratio [OR], 0.86), but survival improved only for nonintubated patients (hazard ratio [HR], 0.19) (103).\nAnother prospective, multicenter randomized controlled trial from China (ChiCTR2000029757) enrolled 103 patients with severe to life-threatening COVID-19. The study was underpowered because of earlier than expected (200 cases) termination. CP (9 to 13 ml/kg from donors with S-RBD IgG titer of \u05461:640) was associated with a negative SARS-CoV-2 PCR test at 72 h in 87.2% of the CP group versus 37.5% of the BSC group, but clinical improvement at 28 days was statistically different only in patients with severe, but not in life-threatening, disease (104).\nTable 1 lists the other ongoing CP trials in COVID-19 patients collected from different web portals. The United States has developed a speci\ufb01c platform for facilitating clinical trials (https://ccpp19.org/), while the International Society of Blood Transfusion created a resource library (https://isbtweb.org/coronaoutbreak/covid-19 -convalescent-plasma-document-library/). At the same time, in the United States an expanded-access program (EAP) has been approved by the FDA and coordinated by Mayo Clinic and has led to treatment of more than 30,000 patients as of 8 July 2020 (https://www.uscovidplasma.org). A preliminary report on the \ufb01rst 20,000 patients (66% from intensive care units) con\ufb01rms safety (\u03fd1% severe adverse events and 14.9% mortality at 14 days) and suggests a bene\ufb01t compared to results with historical cohorts, especially if CP is administered before mechanical ventilation (105, 106); donor titers were not disclosed, and evidently some donations were not titrated before reinfusion. Largely similar data have been reported from a 25-patient case series from Houston, Texas, where CP has been used as an emerging investigational new drug (eIND) (107).\nTypically, 1 or 2 doses of 200 ml are administered (if 2 doses are used, they are administered at least 12 h apart), with infusion rates of 100 to 200 ml/h. The cumulative dose should be targeted according to body weight and antibody titer (22).\nSeveral authors have suggested plasma exchange with CP (i.e., high-volume therapeutic plasmapheresis followed by CP transfusion) rather than CP transfusion alone in order to clear proin\ufb02ammatory cytokines from the bloodstream (108, 109), and several successful case reports deploying nonconvalescent plasma have been reported (110\u2013 112). One randomized controlled trial (NCT04374539) is ongoing in patients with severe COVID-19, but unfortunately no trial to date is testing plasmapheresis followed by CP.\nUnfortunately, most trials in westernized countries (in contrast to ones ongoing in China) have no control arm, which will impair ef\ufb01cacy interpretation. When present, the control arm consists of the best supportive care alone (typically oxygen and hydroxychloroquine at 400 mg twice a day [b.i.d.] for 10 days) or combined with intravenous placebo or standard (nonconvalescent) plasma (eventually of pharmaceutical grade). Since other plasma components (e.g., aspeci\ufb01c immunoglobulins or isoagglutinins; see below) could contribute to clinical bene\ufb01t, the latter approach is ideal for dissecting the speci\ufb01c contribution of neutralizing antibodies although concerns could be raised by the prothrombotic nature of COVID-19 pathology (see Side Bene\ufb01ts from CP in COVID19, below). Even using a placebo control in late-stage patients (refractory to former lines) could pose some ethical concerns because it denies treatment opportunities to an unresponsive disease. Future trials should investigate combined antiviral and CP therapies.\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nClinical Microbiology Reviews cmr.asm.org 9\n\nFocosi et al.\nNotably, several plasma manufacturers are attempting to develop SARS-CoV-2speci\ufb01c hyperimmune sera (e.g., Takeda\u2019s TAK-888 merged with Biotest, BPL, LFB, Octapharma, and CSL Behring into the Convalescent Plasma Coalition [113]; Kedrion and Kamada have joint ventures [81]).\nMONITORING RESPONSE TO CP TREATMENT CP is considered an experimental therapy, and, as such, phase 3 randomized\ncontrolled trials should be encouraged. Despite this recommendation, in emergency settings phase 2 trials are usually started, hampering ef\ufb01cacy analysis. Response in published trials is generally measured clinically (PaO2/FiO2 ratio) or radiologically according to target organs. Nevertheless, surrogate endpoints can include anti-SARSCoV-2 antibody titer or absolute lymphocyte count increases in recipients, as well as decreases in recipients\u2019 SARS-CoV-2 viral load or IL-6 levels. Whenever quantitative PCR is not available, cycle threshold (CT) value increases in qualitative PCR after transfusion could be a proxy for reduced viral load.\nCONCERNS The \ufb01rst concern is transfusion-transmitted infection (TTI). Modern performance\nimprovement (PI) technologies, combined with NAT, reduce the risk for contracting additional TTIs. Most regulatory systems require additional tests (e.g., for hepatitis A virus [HAV] RNA, hepatitis E virus [HEV] RNA, or parvovirus B19 DNA) to be performed on CP for additional transfusion safety. CBP obtained from donors in the United Kingdom may be problematic for a couple of reasons. Currently, CBP obtained from individuals who lived for at least 6 months in the United Kingdom during the 1980-1996 outbreak of \u201cmad cow disease\u201d (bovine spongiform encephalopathy [BSE]) may not be acceptable in some countries (114) or by some individuals. In addition, there is a now a recognized risk of hepatitis E the within the U.K. blood donor population (115), most likely due to the consumption of poorly cooked pork products (116, 117), for which screening has only relatively recently been initiated (71). Although this does not preclude such SARS-CoV-2 convalescent plasma/serum from being used therapeutically within the United Kingdom, these other risks should be considered during larger clinical trials or with compassionate use in individual patients. Respiratory betacoronaviruses produce only a mild and transient viremia. With SARS-CoV, limited replication in lymphocytes (118) leads to signi\ufb01cant risk only for recipients of blood products with high concentrations of donor lymphocytes (peripheral blood stem cells, bone marrow, granulocyte concentrates, etc.). Preliminary reports have shown that SARS-CoV-2 viremia persists only in critically ill patients (10).\nThe second concern is TRALI, which can be life-threatening in patients who are already suffering from ALI. Male donors are usually preferred in order to avoid the risk of transfusing anti-HLA/HNA/HPA antibodies from parous women. In the case of COVID-19, where female patients have been shown to have higher IgG levels, this could be detrimental, and anti-HLA/HNA/HPA antibody screening could be implemented.\nAntibody-dependent enhancement (ADE) is also a theoretical concern related to passive or active antibodies (targeting S protein domains other than the RBD) facilitating IgG-coated virion entry into macrophages via Fc\u2425 receptors and/or complement receptors (119, 120), leading to activation of the RNA sensing Toll-like receptors (TLR) 3, 7, and 8 and \ufb01nally to elevated production of tumor necrosis factor (TNF) and IL-6 (a so-called cytokine storm). ELISAs discriminating the difference between total and RBD-binding antibodies could be useful to inspect the occurrence of ADE. Genetic polymorphisms (e.g., Fc\u2425RIIa [121]) can also contribute to ADE. To date, potential evidence supporting a role for ADE in COVID-19 include the following: (i) the correlation between disease severity and total anti-SARS-CoV-2 antibody levels (70, 122\u2013124), including neutralizing antibodies (125, 126); (ii) the low prevalence of symptoms in COVID-19 patients younger than 20 (who have likely not been primed by infection with the other common cross-reacting coronavirus 229E or OC43 or anyway have low-af\ufb01nity anti-coronavirus IgG [127, 128]); (iii) the occurrence, in SARS, of ADE at low antibody titers in vitro (129) and\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nClinical Microbiology Reviews cmr.asm.org 10\n\nConvalescent Plasma Therapy for COVID-19\ncorrelation in patients of high IgG titers and early seroconversion with disease severity (130). Overall, these \ufb01ndings raise concerns for usage of low-titer CP units (131). Other evidence is the high level of afucosylated IgG against S protein, facilitating FcR binding, that is produced in the most severely ill patients (132, 133).\nA last, COVID-19-speci\ufb01c, concern is worsening of the underlying coagulopathy (134) from clotting factors in transfused plasma (not only CP but also nonconvalescent plasma in control arms); since this has not been reported to date, it remains a theoretical concern.\nSIDE BENEFITS FROM CP IN COVID-19 Obviously, patients with humoral immune de\ufb01ciencies can bene\ufb01t from polyclonal\nantibodies contained in CP, and patients with hemorrhagic diathesis can bene\ufb01t from clotting factors.\nPlasma is also likely to contain antibodies against other common betacoronaviruses associated with the common cold, which have been shown to cross-react with SARSCoV-2 antigens in intravenous immunoglobulin (IVIg) preparations (135), likely stemming from recent infection with another human betacoronavirus (128). Accordingly, IVIg led to clinical and radiological recovery in 3 Chinese patients with severe COVID-19 (136), and the same team is now leading a randomized controlled trial (NCT04261426).\nAfter demonstration that blood group O health care workers were less likely to become infected with SARS-CoV (137), a research group proved that anti-A blood group natural isoagglutinins (which can also be found in CP plasma from blood group O and B donors) inhibit SARS-CoV entry into competent cells (138). Such binding could opsonize virions and induce complement-mediated neutralization (139). Since SARSCoV-2 uses the same receptor as SARS-CoV, anti-A isoagglutinins are expected to have similar effects against SARS-CoV-2 (140); accordingly, clusters of glycosylation sites exist proximal to the receptor-binding motif of the S protein from both SARS-CoV (141) and SARS-CoV-2 (142). Several publications showed that the odds ratio for acquiring COVID-19 is higher in blood group A than in blood group O (143\u2013147), and one showed the ABO gene polymorphism to be the most signi\ufb01cant at predicting severity of COVID-19 (147). COVID-19 has more severe clinical presentations and outcomes in the elderly and in males; intriguingly, elderly males are known to experience reductions in isoagglutinin titers (148, 149). Although alternative explanations exist (150, 151), studies are hence ongoing to evaluate correlations between isoagglutinin titers and outcomes in blood group O and B patients (152). If the correlations are con\ufb01rmed, while preserving ABO match compatibility, blood group O and B donors for CP in COVID-19 could be preferred, and their anti-A isoagglutinin titers should be tested.\nCONCLUSIONS CP manufacturing should be considered among the \ufb01rst responses during a pan-\ndemic while antivirals and vaccines are tested. 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Salazar E, Perez KK, Ashraf M, Chen J, Castillo B, Christensen PA, Eubank T, Bernard DW, Eagar TN, Long SW, Subedi S, Olsen RJ, Leveque C,\nOctober 2020 Volume 33 Issue 4 e00072-20\n\nSchwartz MR, Dey M, Chavez-East C, Rogers J, Shehabeldin A, Joseph D, Williams G, Thomas K, Masud F, Talley C, Dlouhy KG, Lopez BV, Hampton C, Lavinder J, Gollihar JD, Maranhao AC, Ippolito GC, Saavedra MO, Cantu CC, Yerramilli P, Pruitt L, Musser JM. 27 May 2020. Treatment of coronavirus disease 19 (COVID-19) patients with convalescent plasma. Am J Pathol https://doi.org/10.1016/j.ajpath.2020.05.014. 108. Kesici S, Yavuz S, Bayrakci B. 2020. Get rid of the bad \ufb01rst: therapeutic plasma exchange with convalescent plasma for severe COVID-19. Proc Natl Acad Sci U S A 117:12526 \u201312527. https://doi.org/10.1073/pnas .2006691117. 109. Keith P, Day M, Choe C, Perkins L, Moyer L, Hays E, French M, Hewitt K, Gravel G, Guffey A, Scott LK. 2020. The successful use of therapeutic plasma exchange for severe COVID-19 acute respiratory distress syndrome with multiple organ failure. SAGE Open Med Case Rep 8:2050313x20933473. https://doi.org/10.1177/2050313X20933473. 110. Zhang L, Zhai H, Ma S, Chen J, Gao Y. 26 May 2020. Ef\ufb01cacy of therapeutic plasma exchange in severe COVID-19 patients. Br J Haematol https://doi.org/10.1111/bjh.16890. 111. Shi H, Zhou C, He P, Huang S, Duan Y, Wang X, Lin K, Zhou C, Zhang X, Zha Y. 13 April 2020. Successful treatment of plasma exchange followed by intravenous immunoglobulin in a critically ill patient with 2019 novel coronavirus infection. Int J Antimicrob Agents https://doi .org/10.1016/j.ijantimicag.2020.105974. 112. Ma J, Xia P, Zhou Y, Liu Z, Zhou X, Wang J, Li T, Yan X, Chen L, Zhang S, Qin Y, Li X. 2020. Potential effect of blood puri\ufb01cation therapy in reducing cytokine storm as a late complication of critically ill COVID-19. Clin Immunol 214:108408. https://doi.org/10.1016/j.clim.2020.108408. 113. Takeda Pharmaceutical Company, Ltd. 2020. Rajeev Venkayya, President, Global Vaccine Business Unit on the latest on the coronavirus and Takeda. https://www.takeda.com/newsroom/featured-topics/rajeev -venkayya-president-global-vaccine-business-unit-on-the-latest-on-the -coronavirus-and-takeda/. 114. Anonymous. 2019. Is it time to rethink UK restrictions on blood donation? EClinicalMedicine 15:1\u20132. https://doi.org/10.1016/j.eclinm.2019 .10.014. 115. Hewitt PE, Ijaz S, Brailsford SR, Brett R, Dicks S, Haywood B, Kennedy IT, Kitchen A, Patel P, Poh J, Russell K, Tettmar KI, Tossell J, Ushiro-Lumb I, Tedder RS. 2014. Hepatitis E virus in blood components: a prevalence and transmission study in southeast England. Lancet 384:1766 \u20131773. https://doi.org/10.1016/S0140-6736(14)61034-5. 116. Said B, Usdin M, Warburton F, Ijaz S, Tedder RS, Morgan D. 2017. Pork products associated with human infection caused by an emerging phylotype of hepatitis E virus in England and Wales. Epidemiol Infect 145:2417\u20132423. https://doi.org/10.1017/S0950268817001388. 117. Tedder RS, Ijaz S, Kitchen A, Ushiro-Lumb I, Tettmar KI, Hewitt P, Andrews N. 2017. Hepatitis E risks: pigs or blood\u2014that is the question. Transfusion 57:267\u2013272. https://doi.org/10.1111/trf.13976. 118. Yilla M, Harcourt BH, Hickman CJ, McGrew M, Tamin A, Goldsmith CS, Bellini WJ, Anderson LJ. 2005. SARS-coronavirus replication in human peripheral monocytes/macrophages. Virus Res 107:93\u2013101. https://doi .org/10.1016/j.virusres.2004.09.004. 119. Takada A, Ebihara H, Feldmann H, Geisbert TW, Kawaoka Y. 2007. Epitopes required for antibody-dependent enhancement of Ebola virus infection. J Infect Dis 196:S347\u2013S356. https://doi.org/10.1086/520581. 120. Takada A, Feldmann H, Ksiazek TG, Kawaoka Y. 2003. Antibodydependent enhancement of Ebola virus infection. J Virol 77:7539 \u20137544. https://doi.org/10.1128/jvi.77.13.7539-7544.2003. 121. Yuan FF, Tanner J, Chan PKS, Bif\ufb01n S, Dyer WB, Geczy AF, Tang JW, Hui DSC, Sung JJY, Sullivan JS. 2005. In\ufb02uence of Fc\u2425RIIA and MBL polymorphisms on severe acute respiratory syndrome. Tissue Antigens 66:291\u2013296. https://doi.org/10.1111/j.1399-0039.2005.00476.x. 122. Zhang B, Zhou X, Zhu C, Feng F, Qiu Y, Feng J, Jia Q, Song Q, Zhu B, Wang J. 2020. Immune phenotyping based on neutrophil-tolymphocyte ratio and IgG predicts disease severity and outcome for patients with COVID-19. medRxiv https://www.medrxiv.org/content/10 .1101/2020.03.12.20035048v1. 123. Tan W, Lu Y, Zhang J, Wang J, Dan Y, Tan Z, He X, Qian C, Sun Q, Hu Q, Liu H, Ye S, Xiang X, Zhou Y, Zhang W, Guo Y, Wang X-H, He W, Wan X, Sun F, Wei Q, Chen C, Pan G, Xia J, Mao Q, Chen Y, Deng G. 2020. Viral kinetics and antibody responses in patients with COVID-19. medRxiv https://www.medrxiv.org/content/10.1101/2020.03.24.20042382v1. 124. Jiang H-W, Li Y, Zhang H-N, Wang W, Men D, Yang X, Qi H, Zhou J, Tao S-C. 2020. Global pro\ufb01ling of SARS-CoV-2 speci\ufb01c IgG/IgM responses of\ncmr.asm.org 15\n\nFocosi et al.\n\nClinical Microbiology Reviews\n\nconvalescents using a proteome microarray. medRxiv https://www .medrxiv.org/content/10.1101/2020.03.20.20039495v1. 125. Wang X, Guo X, Xin Q, Pan Y, Li J, Chu Y, Feng Y, Wang Q. 2020. Neutralizing antibodies responses to SARS-CoV-2 in COVID-19 inpatients and convalescent patients. medRxiv https://www.medrxiv.org/ content/10.1101/2020.04.15.20065623v3. 126. Wu F, Wang A, Liu M, Wang Q, Chen J, Xia S, Ling Y, Zhang Y, Xun J, Lu L, Jiang S, Lu H, Wen Y, Huang J. 2020. Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications. medRxiv https://www.medrxiv.org/content/10.1101/2020 .03.30.20047365v2. 127. Zhou W, Wang W, Wang H, Lu R, Tan W. 2013. First infection by all four non-severe acute respiratory syndrome human coronaviruses takes place during childhood. BMC Infect Dis 13:433. https://doi.org/10.1186/ 1471-2334-13-433. 128. Ng K, Faulkner N, Cornish G, Rosa A, Earl C, Wrobel A, Benton D, Roustan C, Bolland W, Thompson R, Agua-Doce A, Hobson P, Heaney J, Rickman H, Paraskevopoulou S, Houlihan CF, Thomson K, Sanchez E, Shin GY, Spyer MJ, Walker PA, Kjaer S, Riddell A, Beale R, Swanton C, Gandhi S, Stockinger B, Gamblin S, McCoy LE, Cherepanov P, Nastouli E, Kassiotis G. 2020. Pre-existing and de novo humoral immunity to SARS-CoV-2 in humans. bioRxiv https://www.biorxiv.org/content/10 .1101/2020.05.14.095414v1. 129. Wang S-F, Tseng S-P, Yen C-H, Yang J-Y, Tsao C-H, Shen C-W, Chen K-H, Liu F-T, Liu W-T, Chen Y-M, Huang JC. 2014. Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins. Biochem Biophys Res Commun 451:208 \u2013214. https://doi.org/10.1016/ j.bbrc.2014.07.090. 130. Lee N, Chan PKS, Ip M, Wong E, Ho J, Ho C, Cockram CS, Hui DS. 2006. Anti-SARS-CoV IgG response in relation to disease severity of severe acute respiratory syndrome. J Clin Virol 35:179 \u2013184. https://doi.org/10 .1016/j.jcv.2005.07.005. 131. Iwasaki A, Yang Y. 2020. The potential danger of suboptimal antibody responses in COVID-19. Nat Rev Immunol 20:339 \u2013341. https://doi.org/ 10.1038/s41577-020-0321-6. 132. Chakraborty S, Edwards K, Buzzanco AS, Memoli MJ, Sherwood R, Mallajosyula V, Xie MM, Gonzalez J, Buffone C, Kathale N, Providenza S, Jagannathan P, Andrews JR, Blish CA, Krammer F, Dugan H, Wilson PC, Pham TD, Boyd SD, Zhang S, Taubenberger JK, Morales T, Schapiro JM, Parsonnet J, Wang TT. 2020. Symptomatic SARS-CoV-2 infections display speci\ufb01c IgG Fc structures. medRxiv https://www.medrxiv.org/ content/10.1101/2020.05.15.20103341v1. 133. Larsen MD, de Graaf EL, Sonneveld ME, Plomp HR, Linty F, Visser R, Brinkhaus M, Sustic T, deTaeye SW, Bentlage AEH, Nouta J, Natunen S, Koeleman CAM, Sainio S, Kootstra NA, Brouwer PJM, Sanders RW, van Gils MJ, de Bruin S, Vlaar APJ, Zaaijer HL, Wuhrer M, van der Schoot CE, Vidarsson G. 2020. Afucosylated immunoglobulin G responses are a hallmark of enveloped virus infections and show an exacerbated phenotype in COVID-19. bioRxiv https://www.biorxiv.org/content/10.1101/ 2020.05.18.099507v1. 134. Wichmann D, Sperhake JP, Lutgehetmann M, Steurer S, Edler C, Heinemann A, Heinrich F, Mushumba H, Kniep I, Schroder AS, Burdelski C, de Heer G, Nierhaus A, Frings D, Pfefferle S, Becker H, BrederekeWiedling H, de Weerth A, Paschen HR, Sheikhzadeh-Eggers S, Stang A, Schmiedel S, Bokemeyer C, Addo MM, Aepfelbacher M, Puschel K, Kluge S. 6 May 2020. Autopsy \ufb01ndings and venous thromboembolism in patients with COVID-19: a prospective cohort study. Ann Intern Med https://doi.org/10.7326/m20-2003. 135. D\u00edez J-M, Romero C, Gajardo R. 2020. Currently available intravenous immunoglobulin (Gamunex\u00a9-C and Flebogamma\u00a9 DIF) contains antibodies reacting against SARS-CoV-2 antigens. bioRxiv https://www .biorxiv.org/content/10.1101/2020.04.07.029017v1. 136. Cao W, Liu X, Bai T, Fan H, Hong K, Song H, Han Y, Lin L, Ruan L, Li T. 2020. High-dose intravenous immunoglobulin as a therapeutic option for deteriorating patients with coronavirus disease 2019. Open Forum Infect Dis 7:ofaa102. https://doi.org/10.1093/o\ufb01d/ofaa102. 137. Cheng Y, Cheng G, Chui CH, Lau FY, Chan PKS, Ng MHL, Sung JJY, Wong R. 2005. ABO blood group and susceptibility to severe acute respiratory\n\nsyndrome. JAMA 293:1450 \u20131451. https://doi.org/10.1001/jama.293.12 .1450-c. 138. Guillon P, Cl\u00e9ment M, S\u00e9bille V, Rivain J-G, Chou C-F, Ruvo\u00ebn-Clouet N, Le Pendu J. 2008. Inhibition of the interaction between the SARS-CoV Spike protein and its cellular receptor by anti-histo-blood group antibodies. Glycobiology 18:1085\u20131093. https://doi.org/10.1093/glycob/ cwn093. 139. Neil SJ, McKnight A, Gustafsson K, Weiss RA. 2005. HIV-1 incorporates ABO histo-blood group antigens that sensitize virions to complementmediated inactivation. Blood 105:4693\u2013 4699. https://doi.org/10.1182/ blood-2004-11-4267. 140. Breiman A, Ruv\u00ebn-Clouet N, Le Pendu J. 2020. Harnessing the natural anti-glycan immune response to limit the transmission of enveloped viruses such as SARS-CoV-2. PLoS Pathog 16:e1008556. https://doi.org/ 10.1371/journal.ppat.1008556. 141. Han DP, Lohani M, Cho MW. 2007. Speci\ufb01c asparagine-linked glycosylation sites are critical for DC-SIGN- and L-SIGN-mediated severe acute respiratory syndrome coronavirus entry. J Virol 81:12029 \u201312039. https://doi.org/10.1128/JVI.00315-07. 142. Kumar S, Maurya VK, Prasad AK, Bhatt MLB, Saxena SK. 2020. Structural, glycosylation and antigenic variation between 2019 novel coronavirus (2019-nCoV) and SARS coronavirus (SARS-CoV). Virusdisease 31:13\u201321. https://doi.org/10.1007/s13337-020-00571-5. 143. Zhao J, Yang Y, Huang H, Li D, Gu D, Lu X, Zhang Z, Liu L, Liu T, Liu Y, He Y, Sun B, Wei M, Yang G, Wang X, Zhang L, Zhou X, Xing M, Wang PG. 2020. Relationship between the ABO blood group and the COVID-19 susceptibility. medRxiv https://www.medrxiv.org/content/10 .1101/2020.03.11.20031096v2. 144. Li J, Wang X, Chen J, Cai Y, Deng A, Yang M. 2020. Association between ABO blood groups and risk of SARS-CoV-2 pneumonia. Br J Haematol 190:24 \u201327. https://doi.org/10.1111/bjh.16797. 145. Zeng X, Fan H, Lu D, Huang F, Meng X, Li Z, Tang M, Zhang J, Liu N, Liu Z, Zhao J, Yin W, An Q, Zhang X, Hu X. 2020. Association between ABO blood groups and clinical outcome of coronavirus disease 2019: evidence from two cohorts. medRxiv https://www.medrxiv.org/content/ 10.1101/2020.04.15.20063107v1%20. 146. Zietz M, Tatonetti NP. 2020. Testing the association between blood type and COVID-19 infection, intubation, and death. medRxiv https:// www.medrxiv.org/content/10.1101/2020.04.08.20058073v1. 147. Ellinghaus D, Degenhardt F, Bujanda L, Buti M, Albillos A, Invernizzi P, Fern\u00e1ndez J, Prati D, Baselli G, Asselta R, Grimsrud MM, Milani C, Aziz F, K\u00e4ssens J, May S, Wendorff M, Wienbrandt L, Uellendahl-Werth F, Zheng T, Yi X, de Pablo R, Chercoles AG, Palom A, Garcia-Fernandez AE, RodriguezFrias F, Zanella A, Bandera A, Protti A, Aghemo A, Lleo A, Biondi A, Caballero-Garralda A, Gori A, Tanck A, Carreras Nolla A, Latiano A, Fracanzani AL, Peschuck A, Juli\u00e0 A, Pesenti A, Voza A, Jim\u00e9nez D, Mateos B, Nafria Jimenez B, Quereda C, Paccapelo C, Gassner C, Angelini C, Cea C, Solier A, et al. 17 June 2020. Genomewide association study of severe COVID-19 with respiratory failure. N Engl J Med https://doi.org/10.1056/ NEJMoa2020283. 148. Tendulkar AA, Jain PA, Velaye S. 2017. Antibody titers in group O platelet donors. Asian J Transfus Sci 11:22\u201327. https://doi.org/10.4103/ 0973-6247.200765. 149. de Fran\u00e7a NDG, Poli MCC, Ramos P, Borsoi C, Colella R. 2011. Titers of ABO antibodies in group O blood donors. Rev Bras Hematol Hemoter 33:259 \u2013262. https://doi.org/10.5581/1516-8484.20110073. 150. Dai X. 28 April 2020. ABO blood group predisposes to COVID-19 severity and cardiovascular diseases. Eur J Prev Cardiol https://doi.org/ 10.1177/2047487320922370. 151. Delanghe JR, De Buyzere ML, Speeckaert MM. 27 May 2020. C3 and ACE1 polymorphisms are more important confounders in the spread and outcome of COVID-19 in comparison with ABO polymorphism. Eur J Prev Cardiol https://doi.org/10.1177/2047487320931305. 152. Focosi D. 9 June 2020. Anti-A isohemagglutinin titers and SARS-CoV2 neutralization: implications for children and convalescent plasma selection. Br J Haematol https://doi.org/10.1111/bjh.16932. 153. Burnouf T, Seghatchian J. 2014. Ebola virus convalescent blood products: where we are now and where we may need to go. Transfus Apher Sci 51:120 \u2013125. https://doi.org/10.1016/j.transci.2014.10.003.\n\nOctober 2020 Volume 33 Issue 4 e00072-20\n\ncmr.asm.org 16\n\nConvalescent Plasma Therapy for COVID-19\n\nClinical Microbiology Reviews\n\nDaniele Focosi is a hematologist employed as resident transfusion physician at the largest blood bank in Italy since 2009. He has been a transplant immunologist and immunogeneticist, quality assurance manager, and production manager. He has received awards from the European Federation of Immunogenetics, the European Society of Organ Transplantation, and the Italian Society of Hematology. He has a Ph.D. degree in Clinical and Fundamental Virology, and a master\u2019s degree in Clinical Trials. He has authored 124 articles indexed in PubMed, for a global h-index of 24, on topics ranging from emerging viral infections to new markers of immune competence.\nArthur O. Anderson is a physician/scientist, pathologist, and applied ethicist. He was the Director, Of\ufb01ce of Human Use, and Ethics and Research Integrity Of\ufb01cer at the U.S. Army Medical Research Institute of Infectious Diseases from 1974 to 2016. He held faculty appointments at Johns Hopkins University from 1972 to 1974 and at the University of Pennsylvania from 1980 to 1983. An active biomedical researcher for over 40 years, Dr. Anderson has nearly 100 publications on immunology, infectious diseases, and medical research ethics, including one entitled \u201cEthical Issues in the Development of Drugs and Vaccines for Biodefense.\u201d Now retired from his civilian position, Dr. Anderson serves as a member of the Board of Trustees at Hood College and Director at Hospice of Frederick County.\n\nJulian W. Tang is a hospital consultant and medical virologist, with special interests in the diagnosis, treatment, epidemiology, and infection control of in\ufb02uenza and respiratory viruses, congenital viral infections, HIV, and blood-borne viruses. He also has a Ph.D. in zoology. He has formerly been associate professor at the University of Alberta and assistant professor at the Chinese University of Hong Kong.\nMarco Tuccori is a clinical pharmacologist with special interest in pharmacovigilance and pharmacoepidemiology. He also has a Ph.D. in pharmacology and medical physiology. He is currently pharmacovigilance manager at the Unit of Adverse Drug Reactions Monitoring of the University Hospital of Pisa and coordinator of the Tuscan Regional Centre of Pharmacovigilance. He collaborates with the Agenzia Italiana del Farmaco (AIFA) as a member of the Working Group for Signal Detection Analysis on Drugs and Vaccines. He was a member of the Advisory Board (formerly Executive Committee) of the International Society of Pharmacovigilance (ISoP) from 2012 to 2019. He is the author of about 90 articles in peer-reviewed scienti\ufb01c journals and four chapters of books.\n\nOctober 2020 Volume 33 Issue 4 e00072-20\n\ncmr.asm.org 17\n\n\n",
    "authors": [
      "Daniele Focosi",
      "Arthur O. Anderson",
      "Julian W. Tang",
      "Marco Tuccori"
    ],
    "doi": "10.1128/CMR.00072-20",
    "date": "2020-09-16",
    "item_type": "journalArticle",
    "url": ""
  },
  {
    "key": "4XW58FKQ",
    "title": "Use of convalescent plasma in Ebola virus infection.",
    "abstract": "The recent Ebola virus epidemics which threatened three West African countries (Dec.2014-Apr.2016) has urged global collaborative health organizations and  countries to set up measures to stop the infection and to treat patients, near  half of them being at risk of death. Convalescent plasma-recovered from rescued  West Africans-was considered a feasible therapeutic option. Efficacy was  difficult to evaluate because of numerous unknowns (especially evolution of  neutralizing antibodies), prior to the cessation of active transmission. This  raises a large body of questions spanning epidemiological, virological,  immunological but also ethical, sociological and anthropological aspects,  alongside with public health concerns, in order to be better prepared to the next  outbreak. This essay summarizes efforts made by a large number of groups  worldwide, and attempts to address still unanswered questions on the benefit of  specific versus non-specific plasma on altered-leaking-vascular endothelia in  Ebola infection.",
    "full_text": "",
    "authors": [
      "Olivier Garraud"
    ],
    "doi": "10.1016/j.transci.2016.12.014",
    "date": "2017-02",
    "item_type": "journalArticle",
    "url": ""
  },
  {
    "key": "M3TX4BUR",
    "title": "Ebola Virus Disease: Therapeutic and Potential Preventative Opportunities.",
    "abstract": "The 2014 Ebola virus disease (EVD) epidemic in West Africa was unprecedented in its geographical distribution, scale, and toll on public health infrastructure.  Standard public health measures were rapidly overwhelmed, and many projections on  outbreak progression through the region were dire. At the beginning of the  outbreak there were no treatments or vaccines that had been shown to be safe and  effective for treating or preventing EVD, limiting health care providers to offer  supportive care under extremely challenging circumstances and at great risk to  themselves. Over time, however, drugs and vaccines in the development pipeline  were prioritized based on all available research data and were moved forward for  evaluation in clinical trials to demonstrate safety and efficacy. The  armamentarium against EVD eventually included biologics such as monoclonal  antibodies, convalescent plasma, and vaccines as well as small molecule  therapeutics such as small interfering RNAs and nucleoside analogs. This article  provides a high-level overview of the interventions and prophylactics considered  for use in the outbreak and discusses the challenges faced when attempting to  deploy investigational countermeasures in the midst of an evolving epidemic.",
    "full_text": "",
    "authors": [
      "Robert Fisher",
      "Luciana Borio"
    ],
    "doi": "10.1128/microbiolspec.EI10-0014-2016",
    "date": "2016-06",
    "item_type": "journalArticle",
    "url": ""
  },
  {
    "key": "PKXLX89D",
    "title": "Clinical Evaluation of Ebola Virus Disease Therapeutics.",
    "abstract": "Ebola virus disease (EVD) was first described over 40 years ago, but no treatment has been approved for humans. The 2013-2016 EVD outbreak in West Africa has  expedited the clinical evaluation of several candidate therapeutics that act  through different mechanisms, but with mixed results. Nevertheless, these studies  are important because the accumulation of clinical data and valuable experience  in conducting efficacy trials under emergency circumstances will lead to better  implementation of similar studies in the future. Here, we summarize the results  of EVD clinical trials, focus on the discussion of factors that may have  potentially impeded the effectiveness of existing candidate therapeutics, and  highlight considerations that may help meet the challenges ahead in the quest to  develop clinically approved drugs.",
    "full_text": "Author Manuscript\n\nAuthor Manuscript\n\nHHS Public Access\nAuthor manuscript\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\nPublished in final edited form as: Trends Mol Med. 2017 September ; 23(9): 820\u2013830. doi:10.1016/j.molmed.2017.07.002.\nClinical Evaluation of Ebola Virus Disease Therapeutics\nGuodong Liu1,2, Gary Wong1,2,3,4, Shuo Su5, Yuhai Bi3,4, George F Gao3,4, Gary Kobinger2,6, and Xiangguo Qiu1,2,* 1Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada 2Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Manitoba, Canada 3Shenzhen Key Laboratory of Pathogen and Immunity, State Key Discipline of Infectious Disease, Shenzhen Third People\u2019s Hospital, Shenzhen, China 4CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China 5Jiangsu Engineering Laboratory of Animal Immunology, Institute of Immunology and College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China 6D\u00e9partement de microbiologie-infectiologie et d\u2019immunologie, Universit\u00e9 Laval, Qu\u00e9bec, Canada\nAbstract\nEbola virus disease (EVD) was first described over 40 years ago, but no treatment has been approved for humans. The 2013\u20132016 EVD outbreak in West Africa has expedited the clinical evaluation of several candidate therapeutics that act through different mechanisms, but with mixed results. Nevertheless, these studies are important because the accumulation of clinical data and valuable experience in conducting efficacy trials under emergency circumstances will lead to better implementation of similar studies in the future. Here, we summarize the results of EVD clinical trials, focus on the discussion of factors that may have potentially impeded the effectiveness of existing candidate therapeutics, and highlight considerations that may help meet the challenges ahead in the quest to develop clinically-approved drug(s).\nKeywords Ebola virus; therapeutics; small molecule inhibitor; convalescent plasma; monoclonal antibody\n*Correspondence: Xiangguo.Qiu@phac-aspc.gc.ca (X Qiu). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The authors have no conflicts of interest to declare.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 2\n\nEbola Virus Disease Therapeutics Are Urgently Needed\nThe outbreak of Ebola virus (EBOV) in West Africa from December 2013 to March 2016 was the largest ever reported to date, with 28,616 cases and 11,310 deaths (http:// apps.who.int/iris/bitstream/10665/208883/1/ebolasitrep_10Jun2016_eng.pdf?ua=1).\nEBOV belongs to the genus Ebolavirus, which causes EBOV disease (EVD) \u2013 clinically manifested by a spectrum of symptoms including fever, fatigue, muscle pain, vomiting, diarrhea, anorexia, rash, bleeding and multi-organ failure [1, 2]. Disease fatality rate can be up to 90% (http://www.who.int/mediacentre/factsheets/fs103/en/). The re-emergence of EBOV in the future cannot be ruled out because it can cause sporadic infections from unknown natural reservoirs (see Glossary) and potential transmission from EVD survivors, such as those that shed virus through bodily fluids including semen and breast milk [3, 4]. Due to high fatality rates, poorly-defined natural reservoirs and transmission mechanisms, in addition to the potential for weaponization, EBOV constitutes a major public health concern.\nEBOV pathogenesis is currently only partially understood. EBOV is known to evade the Type I interferon (IFN) response through viral proteins VP30, VP35 and VP24 [5, 6], which contribute to initial viral replication and pathogenicity. Studies in non-human primates (NHPs) showed that early cellular targets of EBOV comprise macrophages and dendritic cells [7], which are currently recognized as two key players in pathogenesis [8, 9]. Dendritic cell maturation can be suppressed by EBOV, as evidenced by the failure of these cells to secrete proinflammatory cytokines and by the absence of upregulated co-stimulatory molecules, leading to impairment in antigen presentation to T-cells [10, 11]. Indeed, dysfunctional macrophages and dendritic cells likely cause deregulated innate immunity through the excessive production of proinflammatory cytokines and chemokines, as well as by suppression of adaptive immune responses against EBOV due to compromised presentation of antigen to lymphocytes and inadequate expression of co-stimulatory factors [12]. While there have been studies in mice [13] and humans [14] showing substantial involvement of adaptive immunity in advanced EVD, particularly the activation CD8+ Tcells, the systemic dissemination and robust viral replication stemming from an inability to control the infection at early disease stages eventually leads to multi-organ failure [1]. Therefore, strategies to develop targeted therapies against EVD are mostly focused on blocking viral entry and inhibiting viral replication [15].\nSubstantial efforts have been devoted to the development of EVD therapeutics in animal models over the past two decades, but remain untested in humans. The outbreak in West Africa greatly expedited the clinical evaluation of several promising therapeutics. Current candidate therapeutics mainly fall into 2 major categories: i) small molecule inhibitors, including licensed drugs to be repurposed for EVD treatment and newly developed nucleic acid-based products; and ii) immune-based therapeutics, including IFNs, plasma transfusion and monoclonal antibodies (mAbs). Full, or interim results of clinical trials for a number of experimental therapeutics have recently been reported. In this review, we summarize findings from those clinical trials that have been completed (Table 1, Key Table) and discuss the limitations that need to be overcome for the successful development of EVD-targeting therapies.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 3\n\nSmall Molecule Inhibitors: Direct Intracellular Inhibition of EBOV\nA popular approach in the search for effective therapeutics is the identification and characterization of small molecules that might inhibit EBOV, presumably through different mechanisms, including suppression of viral transcription and replication. Many small molecules, such as brincidofovir, BCX4430, favipiravir, GS-5734 and AVI-6002, have been shown to be protective in cultured cells, or in animal models such as mice and NHPs [15, 16], but remain to be assessed against EVD in humans. Recently, several small molecule drugs licensed for the treatment of other viral diseases, such as influenza and yellow fever [17, 18], or that have been newly developed against EVD, have been evaluated for their efficacy and effectiveness against EBOV infection in non-randomized clinical trials. These drugs include small compounds such as nucleotide analogs and small interfering RNAs (siRNAs) that target specific EBOV viral proteins.\n\nNucleotide Analogs: The Potential of Favipiravir in EVD Patients with Low Viral Loads\nFavipiravir (6-fluoro-3-hydroxy-2-pyrazinecarboxamide), or T-705, is a pyrazine derivative discovered from a screen of chemical compounds against influenza virus A/PR/8/34 (H1N1); it is modified intracellularly to form a purine nucleotide analog with inhibitory activity against viral RNA-dependent RNA polymerase (RdRP), but exhibits low or no inhibition of canine DNA and RNA polymerase, or human DNA polymerase [19, 20]. Favipiravir has shown inhibitory effects against a wide range of RNA viruses [17, 18, 20\u2013 25]. Recent studies in mouse models demonstrated post-exposure protection against EBOV through oral administration of favipiravir [26, 27]. In addition, favipiravir was shown to be well-tolerated in healthy or ill adults with uncomplicated influenza in phase 1\u20133 clinical trials [28].\nIn mid-November 2014, favipiravir was given to 39 patients with severe EVD admitted to the Sierra Leone-China Friendship Hospital [29]. Patients (17\u201339 years old) received oral favipiravir at doses of 800 mg bid on day 1, and 600 mg bid on day 2, based on recommendations for use in influenza infections [29]. Patients also received supportive treatments in the following days until recovery, hospital transfer or death. Survival rate and viremia in the favipiravir cohort were compared to control patients who were admitted to the same center earlier and treated with only supportive treatments. Results from the subsets with all endpoint observations available from the 2 groups (n=17 for the favipiravir group, n=18 for the control group) showed a higher survival rate in the favipiravir group (64.8% vs. 27.8%) [29]. Improvement of disease symptoms was observable in the favipiravir group, combined with a significant reduction in viral RNA load (>100 fold) determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). These results indicated that favipiravir might be able to confer a survival benefit to EVD in humans [29].\nIn December 2014, a non-randomized, single-arm proof-of-concept clinical trial (the JIKI trial) was conducted to evaluate the safety and effectiveness of favipiravir at 4 treatment centers in Guinea (ClinicalTrials.gov identifier: NCT02329054) [28]. Among the EVD patients, 111 patients (99 aged 13 and older, 12 aged 6 and younger) received no other experimental therapies and completed the trial, and were thus included in the final analyses [28]. The primary outcome (Box 1) was mortality within a period of 14 days. Doses in\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 4\nadults were determined based on results from mouse studies [30], as well as on pharmacokinetic simulation and dosage tests in humans [28].\nAdult patients were given oral favipiravir at doses of 2400 mg, 2400 mg and 1200 mg every 8 h on day 0, and 1200 mg bid for the following 9 days (target time weighted average plasma concentration was 52 \u03bcg/ml) [31]. Dosages for children were adjusted based on body weight to reach similar drug concentrations as those in adults. Since age and viral load are associated with risk of EVD death [32\u201334], the patients were grouped according to age and baseline viral loads (determined as cycle threshold (Ct) by qRT-PCR) for analysis. The patients aged \u2265 13 years were divided into two subgroups: Group A of Ct \u2265 20 (Ct = 20 is 5 \u00d7 107 genome copies/ml) with lower viral load (n=55) and Group A of Ct < 20 with higher viral load (n=44). Twelve young children (\u2264 6 years old) were included in Group YC [28].\nBy the conclusion of the trial, 59 deaths had occurred within 10 days after the first dose, and 1 death at day 17. Mortality rates were 20% in Group A of Ct \u2265 20 (11 of 55, all with Ct < 25), 90.9% in Group A of Ct < 20 (40 of 44) and 75% in Group YC (9 of 12), all meeting the predefined target mortality (30% for Group A of Ct \u2265 20, 85% for Group A of Ct < 20, and 70% for Group YC). The fatality rates in Group A were consistent with a previously observed correlation between higher viral RNA load (Ct < 20) and higher patient mortality [32, 34]. The high mortality in young children was also consistent with previous observations from two of the four treatment centers [28]. Good tolerance to favipiravir was observed during treatment, whereas continuous monitoring of viremia showed reduction in viral loads in survivors but not in non-survivors [28]. Results of available biochemical tests showed more frequent elevation of creatinine, aspartate aminotransferase and creatine phosphokinase with death in Group A Ct < 20, suggesting high levels of renal and muscular damage [28, 35, 36]. Viral clearance in the three surviving children before discharge was also observed; however, the correlations between the secondary outcomes of this study and treatment (Box 1) were not as apparent as those observed in adults and adolescents. Overall, this study indicated that high doses of favipiravir could be tolerated in EVD patients with Ct \u2265 20, and furthermore, lower fatality rates observed in Group A Ct \u2265 20 suggested that favipiravir might be more beneficial during earlier stages of EVD relative to later stages. We posit that an important consideration will now be to compare patient data of Ct \u2265 20 from treatment centers with historical data, aiming to see if there is any survival advantage in such patients. This will indicate whether the enhanced survival is solely due to favipiravir treatment. If previous data is unavailable, the efficacy of favipiravir should be tested and compared in NHPs at Ct < 20 and Ct \u2265 20.\nOf note, a follow-up study reported that favipiravir plasma concentrations in 66 patients from the trial did not reach the the predefined target level 2 days after treatment initiation, the target decreasing to a median level of ~40% of the level that had actually been predicted by a pharmacokinetic model 4 days after treatment initiation [37]. In addition, no significant correlation was observed between plasma EBOV load reduction or mortality (20/66 died) and drug concentrations. This suggests that the study may have used insufficient favipiravir concentrations for the patients, and consequently, further dose studies will be needed.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 5\n\nNucleic Acid-Based Therapeutics Need Optimization\nNucleic acid-based compounds represent another category of small molecule therapeutics for EVD. Two classes of nucleic acid-based systems have been reported, including antisense phosphorodiamidate morpholino oligomers and siRNAs. Using short oligonucleotides[15, 16], both strategies focus on targeting either viral components responsible for transcription and replication of the viral genome such as EBOV RdRp (L polymerase), or targeting antigens involved with immune suppression, such as by VP35 and VP24 [5, 6]. However, only one siRNA-based treatment has been clinically investigated.\n\nTKM-100802 is a lyophilized nanoparticle siRNA formulation consisting of three siRNAs targeting EBOV VP24, VP35 and the L polymerase responsible for viral RNA transcription and replication [38]. In the NHP model, all four animals receiving seven doses of TKM-100802 via intravenous infusion survived challenge with a lethal dose of EBOV [39]. Following NHP studies, observations from a terminated trial in healthy adults identified an optimal dose of 0.3 mg/kg/day of TKM-100802 for safety and protective efficacy [40]. During the outbreak, it was administered to five EVD patients on compassionate grounds, but no safety and efficacy assessments could be made independently because the patients simultaneously received other treatments [41, 42].\n\nBecause the EBOV outbreak in West Africa was caused by the Makona variant of EBOV, which is distinct from the Mayinga and Kikwit variants in Central Africa, the existing product was reformulated to produce TKM-130803, with sequences specifically targeting this EBOV variant. This formulation demonstrated 100% survival (three of three) in NHPs when administered 72 h after challenge with EBOV Makona [43]. In response to the urgent need for EVD therapeutics, TKM-130803 was applied to a phase 2 trial through the Rapid Assessment of Potential Interventions and Drugs for Ebola (RAPIDE) clinical trial platform (Pan African Clinical Trials Registry PACTR201501000997429) [40]. In this nonrandomized, historically controlled trial, 17 EVD patients of 18 years or older were enrolled, with 3 participants in the observational cohort and the other 14 infused intravenously with TKM-130803 at 0.3 mg/kg/day for up to 7 days. During or following infusions, no obvious cytokine release-related adverse events were observed and no termination or infusion rate change was required [40]. One patient presented exacerbated tachypnoea 48 h after the second dose, but the association with infusion was unclear [40]. Overall, TKM-130803 infusion was well-tolerated and survival at day 14 following drug administration was the primary outcome. Amongst the 14 drug-treated patients, 11 died, with two deaths within 48 hours after admission, and only three patients who had received 7 doses of TKM-130803 survived [40]. In the observational cohort, two of the three participants died 3 days after admission. The endpoint survival probability was 0.27 (95% confidence interval 0.06\u20130.58), failing to reach the pre-specified threshold of 0.55, which indicates no improvement in patient survival compared to the control cohort [40]. A possible explanation could be disease severity, as TKM-130803-treated patients all exhibited high baseline viral RNA loads (\u2265 109 copies/ml plasma for the 11 who died) associated with fatality rates > 90% [32, 44]. In addition, 50% of the patients presented symptoms related to high fatality, including bleeding and diarrhea [45]. This suggested that there was an insufficient amount of time for the drug to take full effectiveness at the given dose and/or there was an insufficient drug\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 6\nconcentration in serum, even with extensive standards of care. It is possible that the potency of TKM-130803 might be improved if given at an earlier stage in EVD, but this has not been tested. Furthermore, the selected dose (0.3 mg/kg/d) may be sub-optimal for protection considering that 0.5 mg/kg/d for 7 days could only provide up to 67% protection against EBOV Makona in NHPs [40]. Moreover, the lipid formulation used in the trial was different from the one used in previous NHP studies, which may have negatively affected the efficacy of this drug in the trial. It is also not clear if siRNA uptake efficiency could have been affected due to damaged liver and/or renal functions and vascular leakage during advanced EVD [46, 47]. Of note, a study has shown that EBOV proteins VP30, VP35 and VP40 can inhibit siRNA function, possibly through interaction with the RNAi machinery and possibly blocking siRNA assembly [48]. However, whether the effect of TKM-130803 can be impeded by these viral proteins through the above mechanisms is currently unknown.\n\nEVD Immunotherapy\nIFN-\u03b2, a Proof-of-Concept Immunomodulation Trial\nIFNs are important components of innate immunity against viral infection and have been used as broad-spectrum antiviral therapies. Protective effects of IFN-\u03b1, -\u03b2 or -\u03b3 against EBOV have been tested in different animal models including mice [49, 50], guinea pigs [51], and NHPs [52, 53]. In one EVD patient, IFNs prepared from Sendai-virus-stimulated peripheral lymphocytes were administered intramuscularly in combination with\nconvalescent serum, and this patient survived [54]. However, IFN administration in combination with other experimental therapeutics has made it difficult to assess the effect of IFNs alone. Among the few available studies on IFN monotherapy, murine IFN-\u03b3 provided up to 100% protection against a recombinant vesicular stomatitis virus expressing EBOV glycoprotein (GP) in IFN-\u03b1/\u03b2 receptor-deficient mice [50]. In another study, six doses of human IFN-\u03b2 (10.5 ug/kg) administered subcutaneously (SC) extended the survival time of NHPs challenged with EBOV or Marburg virus, but did not improve the survival rate [53], suggesting that IFN treatments might be beneficial, but likely not fully protective by themselves. Based on the in vitro observation that IFN-\u03b2 could inhibit the replication of recombinant EBOV in HEK293 cells more strongly than IFN-\u03b1 could [55], a historically controlled clinical trial tested the efficacy of IFN-\u03b2-1a in nine EVD patients in Guinea [56]. Within 2 days following qRT-PCR-mediated confirmation of EVD, IFN-\u03b2-1a (30 ug/ day) was administered SC to patients daily, until patients were tested negative for EBOV, or perished [56]. Six of the patients survived in a 21-day observation window, with a survival rate of 67%, which was 2.5-fold higher than that of a control cohort treated with supportive care in the same time period at a treatment center nearby [56]. Comparison with another historical control cohort of matched age and baseline viremia showed slightly less than 2fold higher survival in the IFN group [56], suggesting potential treatment benefit. Rapid viral clearance and improvement of certain clinical symptoms including physical strength and gastrointestinal dysfunctions were observed with IFN-\u03b2-1a treatment [56], which again, suggested a potential treatment benefit.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 7\n\nInconclusive Results for Convalescent Plasma Therapy\nConvalescent whole blood (CWB) or plasma (CP) is taken from patients who have recovered from EBOV infection and carry specific anti-EBOV antibodies, which has been used as prophylactic and/or therapeutic against EVD [57, 58]. Furthermore, efforts have been made to collect blood donations from convalescent EVD patients since the first EBOV outbreak in Zaire (now Democratic Republic of the Congo) in 1976, but studies on the therapeutic effects of convalescent blood or plasma on EVD are very limited. In 1977, a researcher who was accidentally infected with EBOV received human IFNs, in conjunction with two infusions of convalescent serum and eventually recovered from the infection [54]. During the 1995 EBOV outbreak in Zaire, eight EVD patients received whole blood transfusion donated by surviving patients [57]. Each patient was given one blood transfusion of 150\u2013450 ml, 4 to 15 days following EVD onset and seven survived [57]. However, due to the combined use of other therapeutics [54], in addition to suggestions of virus attenuation late in an outbreak (which may have exaggerated any potential advantages gained from the treatment), the therapeutic benefit of convalescent blood has not been well-investigated so far and remains unclear.\nDuring the 2013\u201316 outbreak, the World Health Organization prioritized the use of CWB and CP to treat EVD patients. In February 2015, a non-randomized clinical trial was launched in Guinea to evaluate the safety and efficacy of CP (ClinicalTrials.gov identifier: NCT02342171) [58]. Ninety-nine patients received 2 transfusions of ABO blood groupcompatible CP (200\u2013250 ml/transfusion, or 10ml/kg body weight) with a 15-min interval [58]. The source of the CP for the two blood transfusions was from separate donors [58]. Eighty-four patients who met the screening criteria were included in the final analysis of mortality and other outcomes in comparison to a historical control group of 418 patients treated with supportive care in the 5 months prior to the trial [58]. Fourteen days after treatment, 26 patients in the CP group (31%) and 158 in the control group (38%) died, and these fatality rates did not reach a pre-determined 20% difference to achieve clinical relevance, even after statistical adjustments were made based on multiple factors such as age and Ct values [58]. However, serious adverse events were not observed among the 99 patients who received CP [58]. Nevertheless, due to the unavailability of on-site methods for determining the levels of specific antibodies in CP, the quality of each transfusion (and thus efficacy) was unknown during the trial [58]. Follow-up data from this study indicated that > 90% of the CP samples contained total anti-EBOV IgG titers > 1:1000, determined by ELISA; however, only 4% contained neutralizing antibody titers of 1:160 and 75% contained a titer < 1:40 [59]. Analysis based on age and baseline Ct values revealed lower mortality in patients receiving the highest IgG doses, but also higher mortality with higher doses of neutralizing antibodies. However, neither correlation between antibody doses and mortality was significant. Thus, the effectiveness of CWB or CP-based products against EVD remains inconclusive based on the currently available data.\n\nMAb-Based Therapeutics: A Potential for ZMapp\u2122\nOver 500 mAbs against EBOV have now been isolated from recovered patients [60\u201364] or developed from animal models, such as mice and NHPs [65\u201369]. Most antibodies are neutralizing in vitro and protective in vivo resulting in survival, but most have not yet been\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 8\ntested in clinical trials. ZMapp\u2122, a cocktail composed of three humanized mAbs targeting different sites on the surface GP of EBOV, has so far been the only one tested in clinical trials [70]. This antibody blend consists of an optimized combination of two mAb cocktails, ZMAb and MB-003, previously shown to be protective in NHPs, resulting in full survival when given at 24 h after infection, or partial survival even after the appearance of viremia [52, 71]. In a landmark study, ZMapp\u2122 was shown to reverse advanced EVD and provide 100% protection for rhesus macaques when given up to 5 days after challenge [72]. In March 2015, a phase 1a open-label trial was launched to evaluate the safety and pharmacokinetics of ZMapp\u2122 in healthy human adults (ClinicalTrials.gov identifier: NCT02389192). During the 2013\u201316 outbreak, ZMAb and ZMapp\u2122 were separately given to 25 patients on a compassionate basis [73]. Twenty-two patients survived without showing serious adverse events after receiving three doses of each antibody cocktail (50 mg/kg of body weight) [73]. However, the effectiveness of the cocktails could not be accessed because the patients had also received other treatments, including CP transfusion and intensive standards of supportive care [73].\nIn February 2015, a randomized and controlled phase 1/2 clinical trial, the Partnership for Research on Ebola Virus in Liberia II (PREVAIL II), was initiated to evaluate the efficacy and effectiveness of ZMapp\u2122 (ClinicalTrials.gov identifier: NCT02363322) [74]. The trial enrolled 72 patients (200 patients in the initial plan) from Liberia, Sierra Leone, Guinea, and the US, due to the substantial decline of EVD cases at the late stages of the outbreak [74]. The patients were randomized into either a control group receiving optimized standard of care only (oSOC, with aggressive fluid resuscitation, hemodynamic support, and other interventions available in an optimized care setting), or a treatment group receiving ZMapp\u2122 plus oSOC (n=36 per group). The time from onset of symptoms to treatment in all patients represented 4 to 7 days. After assignment, patients received the first intravenous infusion of ZMapp\u2122 (50 mg/kg of body weight) within 12\u201324 h, followed by two identical doses at every third day. The primary outcome was mortality at day 28 and data from 71 patients was included in the final analysis [74].\nThe fatality rates were 37% (13 of 35) in the control group and 22% (8 of 36) in the ZMapp\u2122 group, leading to a 91.2% posterior probability of superior protection from ZMapp\u2122, which did not reach the preset threshold of \u2265 97.5% [74]. Therefore, ZMapp\u2122 in combination with oSOC did not show a statistically significant decrease in fatality rate over oSOC, even though mortality was 40% lower in the ZMapp\u2122 group relative to the control group. Measurement of secondary outcomes revealed a shorter recovery period among subjects from the ZMapp\u2122 group and the absence in most of the patients, of major safety concerns associated with antibody infusions, such as headache, myalgia, fever and blood pressure changes. These findings have suggested potential safety and therapeutic benefit.\nHowever, for ZMapp\u2122, an insufficient number of available EVD patients may have affected the precision of statistical analyses. In addition, the therapeutic benefit of ZMapp\u2122 is likely underestimated, since seven of eight deaths in the ZMapp\u2122 group occurred before the second dose of antibodies was received, suggesting that these patients may have been near, or at the terminal stage of EVD at the start of the treatment. The fatality rate (1/29) in the subgroup of patients who had finished all three doses of ZMapp\u2122 was nearly eight times\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 9\nlower than among those who survived for > 3 days since admission into the control group [74]. This suggests that additional studies are clearly needed to properly evaluate the efficacy of ZMapp\u2122. Moreover, it is not clear whether sequence differences amongst the GPs of EBOV variants may have had any impact on the efficacy of the antibodies because of potential alterations of GP epitopes. Indeed, ZMapp\u2122 antibodies were developed against the GP of the Mayinga EBOV variant, and comparison of the genomic sequences of the Mayinga and the Makona variants has revealed considerable genetic variations [75], including a non-synonymous mutation in the binding site for mAb 13C6, one component in the formulation of ZMapp\u2122. Consequently, such variations might have affected the virus neutralizing effect of ZMapp\u2122 and thus the effectiveness of this therapeutic treatment. Nevertheless, ZMapp\u2122 might still be able to provide a survival benefit in EVD patients, but its clinical implementation warrants further investigation.\n\nConcluding Remarks\nDespite challenges of testing candidate therapeutics in the midst of EVD outbreaks, valuable experience has been gained in the design and conduct of expedited clinical trials. A common problem with the trials discussed here has been the relatively low enrollment of patients because trials may have been initiated late during an outbreak, rendering it difficult to conclude whether a specific treatment protocol presented any statistically significant benefits to patients. Non-randomized single-arm studies were conducted in most trials, where all EVD patients received experimental therapies. While ethically advantageous given that patients can receive a drug which might play a beneficial role in survival, the interpretation of perceived effects can be confounded by multiple factors, including the selection of historical controls, potential placebo-like effects, and spontaneous recovery (see Outstanding Questions and Box 1). To the best of our knowledge, the ZMapp\u2122 trial may be the only randomized and controlled EVD clinical study that has allowed testing of this compound independently from the current standard of care protocol [76]. The flexibility in its design might enable a promising therapeutic for patients before a trial ends. While the control treatment group did not receive a drug that could be effective, patients still received standard medical care, adding a safeguard mechanism, should any unforeseen negative effects stem from the administration of therapeutics. Nevertheless, there is currently a clear lack of harmonization between trial designs for the different candidates, which makes it difficult to compare the outcomes between treatments. It will be important for future trials to have similar design in order to allow better comparisons.\nAlthough no therapies from the trials discussed here have demonstrated statistical superiority over supportive care, it is worth noting that several EVD treatment candidates, such as favipiravir and ZMapp\u2122, have shown a beneficial trend; this effect may reach statistical significance with further enrollment of EVD patients, or perhaps considering the elimination of patients who died early following treatment, as in the case of the favipiravir study. We posit that the evaluation of a candidate therapeutic should be adjusted based on the different stages of EVD (as determined by Ct value), since the putative compounds may have higher efficacy rates earlier in EVD, although this has not yet been directly tested (see Outstanding Questions). Alternatively, combination therapy with these single agent\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 10\ntreatments may be more effective, but the efficacy should be evidently tested in animal models prior to clinical evaluation.\nMoreover, researchers will need to focus on how evolutionary changes in EBOV structure may affect efficacy of candidate targeting compounds. Indeed, genomic variations among EBOV variants from different outbreaks have been observed [70, 75], and genomic alterations in EBOV Makona GP and L genes have been shown to enhance viral transcription and replication [77], as demonstrated in luciferase reporter assays in which mutant Makona GP and L polymerase were shown to induce stronger activities in human Huh-7 cells, as well as procure a growth advantage over wild-type Makona in both Huh-7 and monkey Vero-E6 cells [77]. Furthermore, it has been proposed that EBOV genomic alterations may be associated with elevated pathogenicity and viral shedding in NHPs because the West African isolates causing the recent outbreak induced higher mortality, higher viremia level and more severe tissue injury compared to other isolates [78] (see Outstanding Questions). Consequently, surveillance of EBOV genetic variations and their impact on the efficacy of relevant therapies will be an important consideration to ensure optimized and successful therapeutic regimens for EVD patients in the future.\nOf clinical relevance, recently, mAbs isolated from human survivors or immunized animals, including mouse and monkey, have shown cross-recognition of and broad protection against multiple members of Ebolavirus in cell lines and animal models [61, 64, 69, 79\u201382]. Indeed, crossreactive mAbs represent a better choice for putative therapeutics since treatments against other members of Ebolavirus are less developed and currently, a large range of pretherapeutic candidates exist only for EBOV. Undoubtedly, the unpredictable nature of filovirus outbreaks highlights the importance of developing successful cross-reactive but efficacious therapeutic reagents to prevent and treat such fatal diseases associated with highly pathogenic viruses.\n\nAcknowledgments\nThis study was supported by the Public Health Agency of Canada, and partially supported by a NIH grant (U19 AI109762-1) to Gary Kobinger and Xiangguo Qiu, and the National Science and Technology Major Project (2016ZX10004222) to George F. Gao, Yuhai Bi and Gary Wong. The authors have no conflicts of interest to declare. Gary Wong, Gary Kobinger, and Xiangguo Qiu were involved in the development and characterization of ZMapp discussed in this review. Yuhai Bi is supported by the Youth Innovation Promotion Association of the Chinese Academy of Sciences (CAS) (2017122). Gary Wong is supported by a grant from the National Natural Science Foundation of China International Cooperation and Exchange Program (8161101193).\nGlossary\nConvalescent serum/plasma collected from convalescent patients who presumably carry specific antibodies against the pathogen causing the disease. Convalescent plasma, serum, or whole blood can be used as therapies for infectious diseases, particularly under circumstances of limited medical resources.\nCt cycle threshold. In real-time quantitative PCR reaction, Ct refers to the cycle at which fluorescent signals from PCR amplification exceed background signals. It is a measurement\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 11\nof the amount of PCR amplicon. The numerical value of Ct is inversely related to the amount of amplicons in a reaction; that is, the lower the Ct value, the higher the number of amplicons.\nEscape mutant a variant of a microorganism, such as a virus, arising through changes in genotype in response to an outside force, such as a host immune response or the effect of therapeutics.\nGP glycoprotein. For Ebola and Marburg viruses, GP is the only surface transmembrane (envelope) protein. The GP gene of Ebola virus is transcribed into two mRNAs, producing two soluble GPs (ssGP and sGP) and one full-length GP which is cleaved into structural GP1 and GP2 by cellular proteins. The Marburg virus GP gene encodes only a single GP protein. The surface GP for these two viruses play a central role in viral entry and fusion. The Ebola GP has been reported to contribute to viral pathogenesis.\nHistorically controlled clinical trial a type of clinical trial in which a treated group of patients is compared to a control group treated from a past outbreak, instead of a concurrent, independent group.\nL gene the gene encoding the RNA-dependent RNA polymerase of filoviruses including Ebola and Marburg viruses. The L polymerase is approx. 220\u2013250 kDa and is responsible for transcription and replication of the viral genome.\nMarburg virus member of the Filoviridae family; genus Marburgvirus. Similar to Ebola virus, Marburg virus is a highly infectious and fatal human pathogen. The virus was first identified in Germany in 1967 and has caused over 10 outbreaks since then. The fatality rate of Marburg virus disease can be up to 90%.\nPhosphorodiamidate morpholino oligomer (PMO) synthetic analogs of nucleic acids, approximately18\u201330 subunits long. PMOs can bind to complementary RNA and block processing, and thus, are used for inhibition of gene expression.\nPrimary outcome a variable that is monitored in a clinical study. Considered the most important or relevant variables to be examined in a clinical trial.\nReservoir hosts natural hosts of an infectious pathogen; can carry the pathogen with little to no disease symptoms.\nSecondary outcome additional variable that is related to a clinical study question, but is less important than primary outcome.\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 12\nSingle-arm clinical study contains only one group of participants receiving the same treatment.\nsiRNA small interfering RNA: short double-stranded RNA molecule (usually 21\u201323 nucleotides in length) produced by RNase III-cleavage and processing of long double-stranded RNA. siRNA is assembled into a protein-RNA complex, binds to homologous sequences in mRNA and guides sequence-specific cleavage and degradation of mRNA. Some siRNAs can mediate methylation of genomic DNA and histones at loci complementary to siRNA, leading to silencing of gene expression.\nSmall molecule inhibitors small chemical compounds or synthetic oligonucleotides with antiviral effects, through different mechanisms, such as interfering with the functions of viral proteins responsible for transcription and replication of a virus.\nSupportive treatment applied to manage symptoms of a disease, aiming to prevent, control or relieve symptoms or side-effects related to the treatment without targeting the underlying cause.\nTime weighted average plasma concentration the average concentration of a drug in plasma over a period of time.\nTachypnoea or tachypnea, refers to abnormally rapid breathing. It may be a sign of more severe or advanced EBOV infection.\n\nReferences\n1. Baseler L, et al. The Pathogenesis of Ebola Virus Disease. Annual review of pathology. 2017; 12:387\u2013418.\n2. West TE, von Saint Andre-von Arnim A. Clinical presentation and management of severe Ebola virus disease. Annals of the American Thoracic Society. 2014; 11:1341\u20131350. [PubMed: 25369317]\n3. Deen GF, et al. Ebola RNA Persistence in Semen of Ebola Virus Disease Survivors - Preliminary Report. The New England journal of medicine. 2015\n4. Thorson A, et al. Systematic review of the literature on viral persistence and sexual transmission from recovered Ebola survivors: evidence and recommendations. BMJ open. 2016; 6:e008859.\n5. Messaoudi I, et al. Filovirus pathogenesis and immune evasion: insights from Ebola virus and Marburg virus. Nature reviews. Microbiology. 2015; 13:663\u2013676. [PubMed: 26439085]\n6. Misasi J, Sullivan NJ. Camouflage and misdirection: the full-on assault of ebola virus disease. Cell. 2014; 159:477\u2013486. [PubMed: 25417101]\n7. Bray M, Geisbert TW. Ebola virus: the role of macrophages and dendritic cells in the pathogenesis of Ebola hemorrhagic fever. 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Clinical Features of and Risk Factors for Fatal Ebola Virus Disease, Moyamba District, Sierra Leone, December 2014-February 2015. Emerging infectious diseases. 2016; 22:1537\u20131544. [PubMed: 27268303]\n46. Wolf T, et al. Severe Ebola virus disease with vascular leakage and multiorgan failure: treatment of a patient in intensive care. Lancet. 2015; 385:1428\u20131435. [PubMed: 25534190]\n47. Wittrup A, Lieberman J. Knocking down disease: a progress report on siRNA therapeutics. Nature reviews. Genetics. 2015; 16:543\u2013552.\n48. Fabozzi G, et al. Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the mammalian RNA interference pathway. Journal of virology. 2011; 85:2512\u2013 2523. [PubMed: 21228243]\n49. Richardson JS, et al. Evaluation of Different Strategies for Post-Exposure Treatment of Ebola Virus Infection in Rodents. Journal of bioterrorism & biodefense. 2011\n50. Rhein BA, et al. Interferon-gamma Inhibits Ebola Virus Infection. PLoS pathogens. 2015; 11:e1005263. [PubMed: 26562011]\n51. Qiu X, et al. Monoclonal antibodies combined with adenovirus-vectored interferon significantly extend the treatment window in Ebola virus-infected guinea pigs. Journal of virology. 2013; 87:7754\u20137757. [PubMed: 23616649]\n52. Qiu X, et al. mAbs and Ad-vectored IFN-alpha therapy rescue Ebola-infected nonhuman primates when administered after the detection of viremia and symptoms. Science translational medicine. 2013; 5:207ra143.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 15\n53. Smith LM, et al. Interferon-beta therapy prolongs survival in rhesus macaque models of Ebola and Marburg hemorrhagic fever. The Journal of infectious diseases. 2013; 208:310\u2013318. [PubMed: 23255566]\n54. Emond RT, et al. A case of Ebola virus infection. British medical journal. 1977; 2:541\u2013544. [PubMed: 890413]\n55. McCarthy SD, et al. A Rapid Screening Assay Identifies Monotherapy with Interferon-\u03b2 and Combination Therapies with Nucleoside Analogs as Effective Inhibitors of Ebola Virus. PLoS neglected tropical diseases. 2016; 10:e0004364. [PubMed: 26752302]\n56. Konde MK, et al. Interferon beta-1a for the treatment of Ebola virus disease: A historically controlled, single-arm proof-of-concept trial. PloS one. 2017; 12:e0169255. [PubMed: 28225767]\n57. Mupapa K, et al. Treatment of Ebola hemorrhagic fever with blood transfusions from convalescent patients. International Scientific and Technical Committee. The Journal of infectious diseases. 1999; 179(Suppl 1):S18\u201323. [PubMed: 9988160]\n58. van Griensven J, et al. Evaluation of Convalescent Plasma for Ebola Virus Disease in Guinea. The New England journal of medicine. 2016; 374:33\u201342. [PubMed: 26735992]\n59. van Griensven J, et al. Efficacy of Convalescent Plasma in Relation to Dose of Ebola Virus Antibodies. The New England journal of medicine. 2016; 375:2307\u20132309.\n60. Maruyama T, et al. Ebola virus can be effectively neutralized by antibody produced in natural human infection. Journal of virology. 1999; 73:6024\u20136030. [PubMed: 10364354]\n61. Flyak AI, et al. Cross-Reactive and Potent Neutralizing Antibody Responses in Human Survivors of Natural Ebolavirus Infection. Cell. 2016; 164:392\u2013405. [PubMed: 26806128]\n62. Bornholdt ZA, et al. Isolation of potent neutralizing antibodies from a survivor of the 2014 Ebola virus outbreak. Science. 2016; 351:1078\u20131083. [PubMed: 26912366]\n63. Corti D, et al. Protective monotherapy against lethal Ebola virus infection by a potently neutralizing antibody. Science. 2016; 351:1339\u20131342. [PubMed: 26917593]\n64. Wec AZ, et al. Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses. Cell. 2017; 169:878\u2013890 e815. [PubMed: 28525755]\n65. Wilson JA, et al. Epitopes involved in antibody-mediated protection from Ebola virus. Science. 2000; 287:1664\u20131666. [PubMed: 10698744]\n66. Takada A, et al. Identification of protective epitopes on ebola virus glycoprotein at the single amino acid level by using recombinant vesicular stomatitis viruses. Journal of virology. 2003; 77:1069\u2013 1074. [PubMed: 12502822]\n67. Qiu X, et al. Characterization of Zaire ebolavirus glycoprotein-specific monoclonal antibodies. Clinical immunology. 2011; 141:218\u2013227. [PubMed: 21925951]\n68. Keck ZY, et al. Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein. Journal of virology. 2015; 90:279\u2013291. [PubMed: 26468532]\n69. Zhao X, et al. Immunization-Elicited Broadly Protective Antibody Reveals Ebolavirus Fusion Loop as a Site of Vulnerability. Cell. 2017; 169:891\u2013904 e815. [PubMed: 28525756]\n70. Davidson E, et al. Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies. Journal of virology. 2015; 89:10982\u201310992. [PubMed: 26311869]\n71. Pettitt J, et al. Therapeutic intervention of Ebola virus infection in rhesus macaques with the MB-003 monoclonal antibody cocktail. Science translational medicine. 2013; 5:199ra113.\n72. Qiu X, et al. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature. 2014; 514:47\u201353. [PubMed: 25171469]\n73. Mendoza EJ, et al. Progression of Ebola Therapeutics During the 2014\u20132015 Outbreak. Trends in molecular medicine. 2016; 22:164\u2013173. [PubMed: 26774636]\n74. Group, P.I.W. et al. A Randomized, Controlled Trial of ZMapp for Ebola Virus Infection. The New England journal of medicine. 2016; 375:1448\u20131456. [PubMed: 27732819]\n75. Kugelman JR, et al. Evaluation of the potential impact of Ebola virus genomic drift on the efficacy of sequence-based candidate therapeutics. mBio. 2015; 6\n76. Dodd LE, et al. Design of a Randomized Controlled Trial for Ebola Virus Disease Medical Countermeasures: PREVAIL II, the Ebola MCM Study. The Journal of infectious diseases. 2016; 213:1906\u20131913. [PubMed: 26908739]\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 16\n77. Dietzel E, et al. Functional Characterization of Adaptive Mutations during the West African Ebola Virus Outbreak. Journal of virology. 2017; 91\n78. Wong G, et al. Pathogenicity Comparison Between the Kikwit and Makona Ebola Virus Variants in Rhesus Macaques. The Journal of infectious diseases. 2016; 214:S281\u2013S289. [PubMed: 27651412]\n79. Howell KA, et al. Antibody Treatment of Ebola and Sudan Virus Infection via a Uniquely Exposed Epitope within the Glycoprotein Receptor-Binding Site. Cell reports. 2016; 15:1514\u20131526. [PubMed: 27160900]\n80. Hernandez H, et al. Development and Characterization of Broadly Cross-reactive Monoclonal Antibodies Against All Known Ebolavirus Species. The Journal of infectious diseases. 2015; 212(Suppl 2):S410\u2013413. [PubMed: 25999057]\n81. Holtsberg FW, et al. Pan-ebolavirus and Pan-filovirus Mouse Monoclonal Antibodies: Protection against Ebola and Sudan Viruses. Journal of virology. 2015; 90:266\u2013278. [PubMed: 26468533]\n82. Furuyama W, et al. Discovery of an antibody for pan-ebolavirus therapy. Scientific reports. 2016; 6:20514. [PubMed: 26861827]\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 17\n\nBox 1 \u2022 \u2022 \u2022 \u2022\n\nClinician\u2019s Corner\nSingle-arm trials are advantageous in terms of ethics since all patients receive the potentially life-saving drug, but are disadvantageous scientifically since the exact impact of the drug will be unknown without a proper control group.\nRandomized, controlled trials are advantageous scientifically since a control group exists to compare the efficacy of the treatment group, but are disadvantageous ethically since not all patients receive the experimental drug.\nThe primary outcome of a clinical trial for filovirus therapeutics will always be survival, since it is only possible to test efficacy on infected patients (i.e., during an outbreak).\nTwo key secondary outcomes of a clinical trial for filovirus therapeutics will be the consideration of changes in viremia (RNA and live virus level) following treatment, as well as adverse effects.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 18\nTRENDS BOX\n\u2022 Ebola virus disease (EVD) causes severe hemorrhagic fever in humans with high fatality rates, with no approved drugs for treatment. Several candidate therapeutics were clinically assessed during the recent 2013\u20132016 outbreak in West Africa.\n\u2022 Two small molecule inhibitors of viral replication and transcription, a nucleotide analog (favipiravir) and short interfering RNA, did not yield a survival benefit in clinical trials, though administration of favipiravir appeared to be more beneficial for patients with lower viral loads (i.e. in the earlier stages of EVD).\n\u2022 The survival benefit was inconclusive in clinical trials with immune productbased therapies, including interferon, convalescent plasma and a monoclonal antibody (mAb) cocktail. The data shows that the mAb-cocktail, ZMapp\u2122, may have the best potential for a substantial therapeutic benefit.\n\u2022 Further clinical investigations that could be rapidly initiated early during an outbreak will help conclusively evaluate the true effectiveness of available candidate therapeutics.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nLiu et al.\n\nPage 19\nOUTSTANDING QUESTIONS BOX\n\u2022 Have we gained sufficient knowledge from current clinical investigations into candidate therapeutics to save lives in the next outbreak?\n\u2022 Is there an optimal design to better balance study strength vs. ethical concerns for testing Ebola virus therapeutics?\n\u2022 Can more than one type of therapeutic be evaluated side-by-side in patients from the same trial during an outbreak?\n\u2022 Given that Ebola virus infections are aggressive and the timeframe for treatment is limited, can we develop better/more sensitive diagnostic systems for the early and rapid identification of EVD?\n\u2022 Can we identify markers that correlate with EVD severity so that therapeutic strategies can be personalized for more efficient treatments?\n\u2022 Are we sufficiently prepared for potential threats from other filovirus species, such as Sudan, Bundibugyo and Marburg viruses?\n\u2022 Do we have alternative methods for the treatment of Ebola virus escape mutants?\n\u2022 Is cross-protection possible across all filoviruses (especially between Marburg and Ebola)?\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nClinical Trials for Candidate EVD Therapeutics\n\nTable 1\n\nLiu et al.\n\neutics vir 30803 a scent plasma (CP) \u2122\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\nClinical trial\nNCT023 29054 Nonrandomized, multicenter,\nproof-ofconcept phase 2 trial\n[28]\nPACTR2 0150100 0997429\nNonrandomized phase 2 trial\n[40]\nISRCTN 1741494 6\nNonrandomized,\nproof-ofconcept phase 1/2 trial [56]\nNonrandomized phase 2/3\ntrial [58]\nNCT023 42171 Randomized, controlled, multicen ter\n\nTreated patients 111 14 9 84 36\n\nReference treatment window 6 d in mice [26] 3 d in NHPs [43] N/A N/A 5 d in NHPs [72]\n\nAverage time from symptom onset to admission\n1.5 d (\u2264 6 y), 3\u20134 d (\u2265 13 y)\n2 d\n2\u20133 d\nNR\n4\u20137 d\n\nMedian/a verage start\npoint of treatment\n4 d from symptom\nonset\n23 hrs post admission\n1 d upon baseline Ct determination\n48 hrs upon EVD\nconfirmation\n12 to 24 hrs following randomization\n\nRoute Oral Intravenous infusion Subcutaneous injection Transfusion Intravenous infusion\n\nDose and course\n6,000 mg on d 0; 2,400 mg/d, d 1~9\n0.3 mg/kg/d for 7 d\n30 ug/d\n2 transfusion s of 200\u2013250 ml of CP or 10 ml of CP/kg, 15-min interval\n3 doses of 50 mg/kg, 3-d\ninterval\n\nPrimary outcome\nMortality on-trial\nSurvival at d 14 Survival at d 21\nMortality from 3 to 16 d after diagnosis\nMortality at d 28\n\nEfficacy (# deaths and\n% mortality)\nand effectiveness\n60 and 54%, no difference\nfrom historical controls (58%); potential benefit to \u2265 13 y and Ct \u2265 20 (20%);\n9/12 and 75%;, no benefit\n3 and 33% vs 17 and 81% in control cohort; potentially beneficial\n26 and 31%, no difference\nfrom historical controls (38%); potential benefit to < 5-y (20%) or pregnancy\n(25%)\n8 (7 before the 2nd dose) and 22% vs 37% in the control arm;\n\nAdverse reactions\nNo severe adverse reactions\nOne case of worsened tachypnoea\nMainly flu-like symptoms\nNo severe adverse reactions\nOne case of hypertension\n\nPage 20\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nAuthor Manuscript\n\nLiu et al.\n\neutics te\n\nClinical trial\n\nTreated patients\n\nReference treatment window\n\nAverage time from symptom onset to admission\n\nMedian/a verage start\npoint of treatment\n\nphase 1/2 trial [74]\n\nRoute\n\nDose and course\n\nPrimary outcome\n\nEfficacy (# deaths and\n% mortality)\nand effectiveness\npotentially beneficial\n\nAdverse reactions\n\nTrends Mol Med. Author manuscript; available in PMC 2018 September 01.\n\npplicable ported\n\nPage 21\n\n\n",
    "authors": [
      "Guodong Liu",
      "Gary Wong",
      "Shuo Su",
      "Yuhai Bi",
      "Frank Plummer",
      "George F. Gao",
      "Gary Kobinger",
      "Xiangguo Qiu"
    ],
    "doi": "10.1016/j.molmed.2017.07.002",
    "date": "2017-09",
    "item_type": "journalArticle",
    "url": ""
  },
  {
    "key": "FTY7APMX",
    "title": "Will There Be a Cure for Ebola?",
    "abstract": "Despite the unprecedented Ebola virus outbreak response in West Africa, no Ebola medical countermeasures have been approved by the US Food and Drug  Administration. However, multiple valuable lessons have been learned about the  conduct of clinical research in a resource-poor, high risk-pathogen setting.  Numerous therapeutics were explored or developed during the outbreak, including  repurposed drugs, nucleoside and nucleotide analogues (BCX4430, brincidofovir,  favipiravir, and GS-5734), nucleic acid-based drugs (TKM-Ebola and AVI-7537), and  immunotherapeutics (convalescent plasma and ZMapp). We review Ebola therapeutics  progress in the aftermath of the West Africa Ebola virus outbreak and attempt to  offer a glimpse of a path forward.",
    "full_text": "PA57CH17-Cardile ARI 10 December 2016 10:54\n\nFurther ANNUAL\nREVIEWS\nClick here to view this article's online features:\n\u2022 Download \ufb01gures as PPT slides \u2022 Navigate linked references \u2022 Download citations \u2022 Explore related articles \u2022 Search keywords\n\nWill There Be a Cure for Ebola?\nAnthony P. Cardile, Travis K. Warren, Karen A. Martins, Ronald B. Reisler, and Sina Bavari\nUS Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702; email: anthony.p.cardile.mil@mail.mil\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nAnnu. Rev. Pharmacol. Toxicol. 2017. 57:329\u201348\nFirst published online as a Review in Advance on December 7, 2016\nThe Annual Review of Pharmacology and Toxicology is online at pharmtox.annualreviews.org\nThis article\u2019s doi: 10.1146/annurev-pharmtox-010716-105055\nCopyright c 2017 by Annual Reviews. All rights reserved\n\nKeywords\nBCX4430, brincidofovir, favipiravir, GS-5734, ZMapp, convalescent plasma\nAbstract\nDespite the unprecedented Ebola virus outbreak response in West Africa, no Ebola medical countermeasures have been approved by the US Food and Drug Administration. However, multiple valuable lessons have been learned about the conduct of clinical research in a resource-poor, high risk\u2013 pathogen setting. Numerous therapeutics were explored or developed during the outbreak, including repurposed drugs, nucleoside and nucleotide analogues (BCX4430, brincidofovir, favipiravir, and GS-5734), nucleic acid\u2013 based drugs (TKM-Ebola and AVI-7537), and immunotherapeutics (convalescent plasma and ZMapp). We review Ebola therapeutics progress in the aftermath of the West Africa Ebola virus outbreak and attempt to offer a glimpse of a path forward.\n\n329\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\nINTRODUCTION Despite the unprecedented response to the West Africa Ebola virus (EBOV) outbreak, there remain no US Food and Drug Administration (FDA)-approved Ebola medical countermeasures (MCMs). As a result, the treatment of EBOV infection remains limited to clinical supportive care, administration of investigational MCMs through expanded access, or enrollment into clinical treatment trials studying candidate MCMs. The following question or a similar variant was asked frequently during and following the West Africa EBOV outbreak: Will there be a cure for Ebola?\nTaken at face value, this seems to be a simple question. However, the meaning of cure can vary depending on context. The Merriam-Webster Online Dictionary de\ufb01nes a cure, in part, as (a) recovery or relief from a disease, (b) something (as a drug or treatment) that cures a disease, and (c) a complete or permanent solution or remedy (1). Identi\ufb01cation of a cure, therefore, may be approached from the perspective of prevention, treatment, or eradication. Only smallpox and rinderpest have been eradicated from nature (2, 3). The prospect of EBOV eradication is untenable at this time, given our incomplete understanding of potential reservoir hosts and subsequent initial transmission from reservoir hosts to humans. During the past two years, researchers have made tremendous strides in the development of EBOV MCMs for the prevention via vaccination or treatment of EBOV infection. EBOV vaccine development has been reviewed in detail recently by other sources (4, 5). The goal of this review is to summarize these advances, with a focus on therapeutics development and the challenges encountered during the recent West Africa EBOV outbreak, and to offer a perspective on the path forward. Tables 1 and 2 summarize relevant in vitro, nonhuman primate (NHP), and clinical studies of select MCMs and highlight data gaps that need to be \ufb01lled.\nREPURPOSED DRUGS Numerous drugs that are approved for other indications have been demonstrated to possess activity against EBOV. Researchers had thought that if already-approved drugs displayed in vitro or in vivo ef\ufb01cacy against EBOV, EBOV-infected patients could reasonably be treated with such therapies in an outbreak setting. Additionally, in the context of a highly lethal infection for which there were no known MCMs, some doctors and clinics used their best knowledge to select approved drugs in order to treat symptoms, despite a lack of data on EBOV infection. This approach was ineffective during the West Africa outbreak, however, and could have resulted in harm to patients, as certain drugs were given to patients outside the auspices of research protocols. For example, there were unsubstantiated claims that lamivudine, statins, and angiotensin receptor blockers (ARBs) were used successfully to treat EBOV-infected patients in West Africa (6). Similarly, a Liberian physician reported successful treatment of EBOV-infected patients with lamivudine (7). In subsequent studies, lamivudine was shown to have no in vitro activity against the 1995 isolate EBOV-Kikwit or EBOV-Makona (7). In Sierra Leone, local physicians reported treating EBOVinfected patients successfully at multiple sites with the statin atorvastatin (40 mg daily) and the ARB irbesartan (150 mg daily) (8\u201310). Fedson et al. (8\u201310) have suggested that statins and ARBs could treat the host response to Ebola infection owing to their potential to restore endothelial barrier integrity, but they can have adverse effects that could worsen EBOV disease (e.g., ARBs can cause acute kidney injury and hyperkalemia; statins can cause hepatotoxicity and myopathy). The use of lamivudine, atorvastatin, and irbesartan was not associated with approved clinical trial\n330 Cardile et al.\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nPA57CH17-Cardile ARI 10 December 2016 10:54 www.annualreviews.org \u2022 Cure for Ebola 331\n\nTable 1 Select in vitro and in vivo (NHP) characteristics of Ebola virus therapeutics\n\nTherapeutic name Amiodarone\nAmodiaquine\nBCX4430\n\nIn vitro EC50 (strain)\n0.25 \u03bcg/mL (Mayinga) (22)\n2.6 and 8.4 \u03bcM (pseudotyped virus) (14)\n11.8 \u03bcM (Kikwit), 3.4 \u03bcM (SUDV-Boniface) (28)\n\nIn vitro CC50 16.69 \u03bcg/mL\nNA\n>100 \u03bcM\n\nIn vitro therapeutic\nindex (CC50 /IC50 ) 67\nNA\n8 to >29\n\nNHP species NA NA Rhesus (31)\n\nBrincidofovir Favipiravir GS-5734\nsiEbola-3\n\n0.6\u20130.88 \u03bcM (Kikwit, Mayinga, Makona) (36)\n67 \u03bcM (Mayinga) (55)\n0.06\u20130.14 \u03bcM (Kikwit, Makona, Sudan, Bundibugyo) (67)\nNA\n\n>10 \u03bcM >1,000 \u03bcM 1.7 to >20 \u03bcM\nNA\n\n<1 to >15 >14 12 to >333\nNA\n\nNA NA Rhesus (67)\nRhesus (77)\n\nAVI-7537\n\n585 nM (Kikwit) (78)\n\nNA\n\nNA\n\nRhesus (80)\n\nZMapp\n\n0.015\u20130.04 \u03bcg/mL\n\nNA\n\nNA\n\nRhesus (96)\n\n(Kikwit); 0.004\u2013\n\n0.02 \u03bcg/mL (Makona)\n\n(96)\n\nInterferon-\u03b2\n\nNA\n\nNA\n\nNA\n\nRhesus (102)\n\nRoute of challenge and challenge virus\nNA\n\nDosing NA\n\nTime therapy was initiated postinfection\nNA\n\nNA\n\nNA\n\nNA\n\nIM Kikwit NA\n\nIM, twice daily at 1 h doses of 16 mg/kg or 25 mg/kg\n\nNA\n\nNA\n\nNA IM Kikwit IM Makona IM Kikwit IM Kikwit\nIM Kikwit\n\nNA 10 mg/kg IM\ndaily for 12 daysa 0.5 mg/kg IV for 7 days 40 mg/kg IV for 14 days 50 mg/kg IV\n10.5 \u03bcg/kg SQ\n\nNA 3 daysa\n3 days\n1h\n3, 6, and 9 days postinfection; 4, 7, and 10 days postinfection; or 5, 8, and 11 days postinfection\n18 h and 1, 3, 5, 7, and 9 days postinfection\n\nNHP survival in treatment arm NA\nNA\n66.7% (4 of 6) survival in the low-dose group and 100% (6 of 6) survival in the high-dose group\nNA\nNA 100% (6/6)a\n100% (3/3)\n75% (6/8)\n100% (18/18)\nNo survival bene\ufb01t; prolonged time to death 13.8 days versus 8.3 days for control (P = 0.0097)\n\nAbbreviations: CC50, 50% cytotoxic concentration; EC50, 50% effective concentration; IC50, 50% inhibitory concentration; IM, intramuscular; IV, intravenous; NA, not applicable; NHP, nonhuman primate; SQ, subcutaneous; SUDV, Sudan virus. aBest regimen of multiple regimens tested (67).\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nPA57CH17-Cardile ARI 10 December 2016 10:54 332 Cardile et al.\n\nTable 2 Clinically relevant characteristics of select EBOV therapeutics\n\nTherapeutic name(s)\nAmiodarone\n\nClinical studies\nAdministered as a compassionate therapy to approximately 65 patients hospitalized in Sierra Leone (retrospective analysis) (6, 24)\n\nRoute of administration\nIV or PO\n\nDosing\nDay 1: 5 mg/kg (\ufb01rst hour), total of 20 mg/kg (remaining 23 h) via IV\nDays 2\u20133: 20 mg/kg (24 h) via IV\nDays 4\u201310: 30 mg/kg via oral tablets given 3 times/day (97)\n\nAmodiaquine\nBCX4430 Brincidofovir\n\nRetrospective comparison\n\nPO\n\nof EBOV-infected\n\npatients who received\n\nartemether\u2013lumefantrine\n\nand artesunate\u2013\n\namodiaquine therapy for\n\nmalaria (31)\n\nPhase I clinical trials\n\nIM\n\nongoing (34)\n\nPhase II trial for Ebola\n\nPO\n\nterminated\n\n7.5\u201315.0 mg/kg for malaria (31)\nND Initial dose of 200 mg, then\n100 mg twice weekly for a total of 5 doses (97)\n\nPlasma half-life in humans\n20\u201347 days (28)\n211 h (31)\nND Dose-dependent,\nranging from 6.15 h at 0.025 mg/kg to 32.7 h at 1.5 mg/kg (41)\n\nClearance Hepatic (28)\nHepatic\nND Nonrenal; its\nmetabolite, cidofovir, was detected in the urine (41)\n\nAdverse drug reactions Infusion-related\nhypotension, sinus bradycardia, ventricular arrhythmias, hepatotoxicity, gastrointestinal problems (nausea, vomiting, anorexia, diarrhea, and constipation), and neurologic dysfunction; thyroid, ocular, cutaneous, and pulmonary toxicity from chronic exposure (25\u201328) Agranulocytosis, hepatotoxicity (31)\nND\nAbdominal pain, nausea, diarrhea,\naphthous stomatitis (41)\n\nClinical ef\ufb01cacy Reportedly reduced case\nfatality rates; however, the data are currently unavailable, and these claims should be interpreted with caution (6, 24).\nArtesunate\u2013amodiaquine group had a 31% lower risk of death than the artemether\u2013lumefantrine group (P = 0.004), with a stronger effect observed among patients without malaria (31).\nNo human ef\ufb01cacy data are available to date.\nClinical trials were terminated owing to slow enrollment and withdrawal of the drug for investigational use in Ebola patients by the company (11, 45).\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nPA57CH17-Cardile ARI 10 December 2016 10:54 www.annualreviews.org \u2022 Cure for Ebola 333\n\nFavipiravir\n\nSingle-arm,\n\nPO\n\nproof-of-concept trial for\n\nEbola (64)\n\nGS-5734\n\nPhase I clinical trials\n\nIV\n\nongoing (70, 71)\n\nLoading dose of 2,400 mg, followed by 1,200 mg every 8 h on day 1 (6,000 mg total) and a maintenance dose of 1,200 mg twice a day for 9 days (64, 66)\n\n4.5\u20135.8 h (67)\n\nND\n\nND\n\nTKM-Ebola-\n\nPhase I clinical trial\n\nIV\n\nND\n\nGuinea\n\ncompleted; Phase II trial\n\n(siEbola-3)\n\nterminated (87, 88)\n\nAVI-7537 ZMapp\n\nPhase I clinical trial completed (85)\nPhase I clinical trial ongoing; Phase II trial completed\n\nIV infusion in 150 mL normal saline over 30 min (85)\nIV\n\n4.5 mg/kg (85) 50 mg/kg\n\nND\n2\u20135 h (85) ND\n\nMetabolized by oxidases; metabolites excreted in the urine (67)\nND\nND\nMajority secreted in urine by 24 h (85)\n\nDuring in\ufb02uenza trials: mild to moderate diarrhea, asymptomatic increase of blood uric acid and transaminases, and decreases in neutrophil counts (67)\nND\nPostinfusion fever, rigors, dizziness, chest tightness, and tachycardia related to transient in\ufb02ammatory responses (beginning within 6 h of infusion and lasting up to 24 h) (67)\nHeadache; nausea; elevated AST, ALT, and amylase; uveitis (85)a\n\nPossible survival bene\ufb01t in patients with Ct values > 20, and no improvement in survival when Ct values are < 20 (64)\nCompassionate use in a case of EBOV recrudescence in a female nurse in the United Kingdom and a case of acute EBOV infection in a female infant in Guinea (72\u201373)\nPhase II trial terminated, as interim analysis indicated that continuing enrollment was not likely to demonstrate an overall therapeutic bene\ufb01t (87, 88)\nNo clinical data to date\n\nND\n\nCommon side effects\n\nNo statistically signi\ufb01cant\n\nreported from cases in the\n\nmortality bene\ufb01t, with\n\nliterature were\n\n37% mortality in the\n\ninfusion-related fever,\n\ncontrol arm and 22.2%\n\nhypotension, tachycardia,\n\nmortality in the treatment\n\nrash, and polypnea (67)\n\narm of the study.\n\nAbbreviations: Ct, cycle threshold; EBOV, Ebola virus; IV, intravenous; ND, not determined; PO, oral. aConsidered to be drug related; however, a retinal specialist suggested recurrent toxoplasmosis as the underlying cause of the uveitis based on the subject\u2019s region of origin (West Africa) and the presence of retinal scarring.\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\nprotocols and was not administered with adequate controls and oversight, and data have not been submitted for peer-reviewed publication to date. Claims of ef\ufb01cacy should be interpreted with caution until more data are available.\nPer World Health Organization (WHO) sources, preclinical data are available on azithromycin, erlotinib/sunitinib, atorvastatin, irbesartan, and sertraline, but these data were not available in the peer-reviewed literature at the time of writing (11). Other drugs with data for anti-EBOV activity include amitriptyline, amlodipine, azidothymidine, ba\ufb01lomycin A1, benztropine, bepridil, chloroquine, chlorpromazine, colchicine, cyproheptadine, despiramine, diltiazem, erythromycin, \ufb02uoxentine, heparin, imipiramine, nimodipine, nystatin, penbutolol, prochlorperazine, sertraline, and verapamil (12\u201320). Amiodarone and amodiaquine are discussed in more detail below, as they have more data available than many of the other drugs.\nAmiodarone (a multi-ion channel inhibitor and adrenoceptor antagonist) was found to inhibit EBOV cell entry in vitro (21). The proposed mechanism of action of amiodarone against EBOV is interference with the fusion of the viral envelope and endosomal membrane (22). The concentrations of amiodarone required for EBOV inhibition are within the range that is achieved in serum during antiarrhythmic therapy in humans (1.5\u20132.5 mg/mL) (21). Nonetheless, there are concerns that the high protein binding of the drug will result in ineffective serum concentrations. The drug was administered as a compassionate therapy to approximately 65 patients hospitalized in Sierra Leone and, in retrospective analysis, reportedly reduced case fatality rates; however, the data are currently unavailable, and these claims should be interpreted with caution (6, 23). Its use for treatment of EBOV patients could be problematic, given some of its serious side effects and drug interactions. Amiodarone can cause life-threatening ventricular arrhythmias, and in the setting of the severe electrolyte abnormalities observed during the recent outbreak, this side effect could be ampli\ufb01ed (24, 25). Amiodarone can also cause signi\ufb01cant gastrointestinal side effects (nausea, vomiting, and diarrhea) and hepatotoxicity, which could potentially exacerbate the EBOV disease process (26, 27).\nAmodiaquine is a 4-aminoquinoline antimalarial that has generated interest for the treatment of EBOV. Fifty percent inhibitory concentration (IC50) values for amodiaquine in vitro for EBOV entry and replication were lower than those for the similar antimalarial chloroquine (2.6 and 8.4 \u03bcM versus 4.7 and 16 \u03bcM, respectively) (14). At an Ebola treatment center in Liberia, the supply of artemether\u2013lumefantrine, the \ufb01rst-line antimalarial, ran out for a 12-day period in August 2014, and during that time patients received artesunate\u2013amodiaquine (28). After the in vitro EBOV activity of amodiaquine was published, a comparison of patients who received artemether-lumefantrine and artesunate-amodiaquine therapy for malaria was conducted. At admission, 194 patients were prescribed artemether\u2013lumefantrine and 71 were prescribed artesunate\u2013amodiaquine, and baseline characteristics of patients were similar between both groups and in a no-antimalarial group. A total of 125 of the 194 patients in the artemether\u2013lumefantrine group (64.4%) died, as compared with 36 of the 71 patients in the artesunate\u2013amodiaquine group (50.7%). In adjusted analyses, the artesunate\u2013amodiaquine group had a 31% lower risk of death than the artemether\u2013lumefantrine group (P = 0.004), with a stronger effect observed among patients without malaria. This information is compelling, but it is not known if this observed effect is de\ufb01nitively related to the ef\ufb01cacy of amodiaquine against EBOV, a lumefantrine-induced increased mortality in EBOV patients, or unmeasured patient characteristics that altered mortality risk (6, 28). This study is also inherently limited by its retrospective nature, and the medical record lacked information on completion of antimalarial courses of therapy (28). However, further research of amodiaquine as a therapeutic for EBOV may be warranted based on these observations.\n334 Cardile et al.\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\nDIRECT-ACTING ANTIVIRALS\nNucleoside and Nucleotide Analogues\nAntiviral nucleoside and nucleotide analogues are prodrugs that require activation by several successive phosphorylation steps catalyzed by different kinases that are present in the host cell or encoded by some of the viruses (29). These compounds can exert antiviral effects via inhibition of viral polymerases, other enzymes such as kinases, and/or incorporation into viral nucleic acids (30).\nBCX4430. BCX4430 is a novel nucleoside analogue that was synthesized as part of a smallmolecule library of inhibitors of viral RNA polymerase activity (31). BCX4430 was designed to inhibit viral RNA polymerase activity indirectly via RNA chain termination and is dependent on conversion of the parent compound to BCX4430-triphosphate (31). BCX4430 has displayed a broad antiviral spectrum in vitro, with activity against negative-sense RNA viruses (Filoviridae, Arenaviridae, Bunyaviridae, Orthomyxoviridae, Picornaviridae, and Paramyxoviridae) and positive-sense RNA viruses (Flaviviridae and Coronaviridae) (31). Of all the virus families, BCX4430 displayed the most potent in vitro activity against \ufb01loviruses (Marburg-Musoke, -Ci67, and -Angola; EBOVKikwit; and Sudan-Boniface) with 50% effective concentration (EC50) values ranging from 3.4 to 11.8 \u03bcM (31).\nIn a mouse model of EBOV disease, BCX4430 protected mice against an otherwise lethal challenge of mouse-adapted EBOV administered via intramuscular (IM) or oral routes at a dose of 150 mg/kg (the ef\ufb01cacies of lower doses were not evaluated) (31). In an as yet unpublished study to evaluate the ef\ufb01cacy of BCX4430 in a NHP EBOV disease model, BCX4430 was administered to rhesus monkeys by an IM route twice daily at doses of 16 or 25 mg/kg, beginning approximately 1 h after virus exposure (32). At both dose levels, BCX4430 conferred a statistically signi\ufb01cant survival bene\ufb01t compared to animals treated with vehicle alone, with 66.7% (4 of 6) survival in the low-dose group and 100% (6 of 6) survival in the high-dose group (32). Administration of BCX4430 also reduced plasma viral concentration in rhesus monkeys by nearly 3 log10 in both BCX4430 groups on day 8 during the most acute phase of disease (32). Finally, a two-part, doseranging study to evaluate the safety, tolerability, and pharmacokinetics of BCX4430 administered via IM injection in healthy subjects is currently recruiting patients (33). In part one, study subjects will receive a single dose of BCX4430, and in part two, subjects will receive BCX4430 for 7 days.\nBrincidofovir. Brincidofovir is a conjugate comprised of a lipid (1-0-hexadecyl-oxypropyl) covalently linked to the acyclic nucleotide analogue cidofovir (34, 35). This enables brincidofovir to be orally bioavailable and more active and reduces nephrotoxicity compared to cidofovir (34, 35). Brincidofovir has broad activity against DNA viruses, including poxviruses, herpesviruses, adenoviruses, and polyomaviruses (34).\nBrincidofovir was \ufb01rst considered as a possible EBOV therapy when in vitro data against EBOV were \ufb01rst presented in October 2014 (36). The FDA then made brincidofovir available for the treatment of EBOV patients through an Emergency Use Investigational New Drug (IND) application (37). Use of brincidofovir during the EBOV outbreak was rationalized on the basis that it had already been through Phase I studies and was in Phase II and III studies for other viral indications (cytomegalovirus and adenovirus infections) (38\u201340). At least four EBOV-infected patients may have been treated with brincidofovir (41\u201343). An open-label, multicenter study of brincidofovir against EBOV was set to commence, but in January 2015, clinical trials for\nwww.annualreviews.org \u2022 Cure for Ebola 335\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\nbrincidofovir for EBOV were terminated owing to slow enrollment and the withdrawal of the drug for investigational use in EBOV-infected patients by the company (11, 44).\nRecently, in vitro data have been published on the anti-EBOV activity of brincidofovir against wild-type EBOV and a strain of EBOV expressing green \ufb02uorescent protein (EBOV-GFP) (37). When cells were pretreated with brincidofovir and then infected with EBOV, the following variants were inhibited (EC50 values): EBOV-Mayinga (0.76 \u03bcM), EBOV-Makona (0.6\u20130.88 \u03bcM), and EBOV-Kikwit (0.66\u20130.79 \u03bcM) (37). Mechanistic studies of brincidofovir against EBOV demonstrated a different mechanism compared to its activity against DNA viruses. The precise mechanism remains unknown but has been speculated to be related to competition for phospholipases such as acid sphingomyelinase, which are required for ef\ufb01cient infection of cells by EBOV in vitro (15, 37). As opposed to DNA viruses, anti-EBOV activity required the lipid moiety, and in vitro activity against EBOV was observed for several nucleotide conjugates (37). In mouse models, brincidofovir was ineffective, and this may be related to the dose and dosing intervals used, as cidofovir diphosphate (CDV-PP) was assumed to be the active antiviral metabolite (11). Given that the half-life of intracellular CDV-PP is much longer than the plasma half-life of brincidofovir, higher doses, more frequent doses, or both would be required to show anti-EBOV activity in vivo (37). Complicating matters, in NHPs, which are the gold standard for testing the ef\ufb01cacy of EBOV therapeutics, brincidofovir is metabolized rapidly, making evaluation dif\ufb01cult (35, 37). Taken together, substantial barriers exist that make it impractical to develop brincidofovir as an EBOV MCM.\nFavipiravir. Favipiravir (T-705 or Avigan) is a pyrazinecarboxamide derivative that was developed initially as an orally active anti-in\ufb02uenza drug (45). T-705 is converted by host cells to T-705-ribofuranosyl-5 -triphosphate. It selectively inhibits viral RNA\u2013dependent RNA polymerase or causes lethal mutagenesis upon incorporation into viral RNA (46, 47). Favipiravir has broad-spectrum activity against RNA viruses, including bunyaviruses, arenaviruses, \ufb02aviviruses, noroviruses, alphaviruses, picornaviruses, and paramyxoviruses (48\u201355). Complete (100%) protection against aerosolized EBOV (E718) in interferon (IFN)-\u03b1/\u03b2 receptor knockout, immunode\ufb01cient mice was achieved when favipiravir was administered 1 h postchallenge followed by 14 days of twice-daily dosing of 150 mg/kg orally (55). In a similar study in an immunode\ufb01cient intranasal mouse model, favipiravir (300 mg/kg/day orally) was administered at six days postinfection (EBOV-Mayinga), with rapid viral clearance, reduced biochemical parameters of disease severity, and 100% survival of the exposed animals (56). However, when treatment was delayed to day 8 (peak viremia), favipiravir did not prevent death (56).\nFavipiravir had been evaluated in Phase II and III studies of in\ufb02uenza in Japan and the United States and was approved for use in in\ufb02uenza infection in Japan (57, 58). As a result, the drug was an attractive early candidate for clinical trials during the West Africa EBOV outbreak. Four case reports of patients treated with favipiravir for EBOV have been published (59\u201362). Additionally, there is one case report in which favipiravir was used to treat a patient who had recovered from acute EBOV infection and had postinfectious uveitis [vitreous \ufb02uid being positive for EBOV by polymerase chain reaction (PCR)] (61, 62).\nThe results of the JIKI (meaning hope in Kissi, a West African language) trial, a historically controlled, multisite, single-arm clinical trial to evaluate the ef\ufb01cacy of favipiravir in EBOVinfected patients in Guinea, were published recently (63). According to the authors of the study, a randomized, placebo-controlled study design was not chosen owing to ethical concerns (64). The authors argued that in the context of widespread distrust of Ebola treatment units, using a randomized design might have led more patients to avoid seeking care. Thus, the objectives of the study were to test the feasibility and acceptability of an emergency trial in the context of\n336 Cardile et al.\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\na large EBOV outbreak and to collect safety and ef\ufb01cacy data to optimize the design of future studies. All patients received favipiravir and standard care, which included oral or intravenous (IV) rehydration, electrolyte correction, empiric antimalarial and antibacterial therapies, analgesics, and antiemetic drugs. In vitro and mouse ef\ufb01cacy data for favipiravir against EBOV were combined with pharmacokinetic data from uninfected mice and humans to calculate a recommended dosing regimen for human patients infected with EBOV (65). This regimen consisted of a loading dose of 2,400 mg, followed by 1,200 mg every 8 h on day 1 (6,000 mg total) and then a maintenance dose of 1,200 mg twice a day for 9 days (63, 65). As a result, the dose proposed to treat EBOV in adults was 50% higher than the dose for in\ufb02uenza treatment (1,800 mg twice a day on day 1, 800 mg twice a day on days 2\u20135) (65). Calculations for weight-based dosing in children were also conducted (66).\nFor the primary statistical analysis, patients with cycle threshold (Ct) values >20 at admission were compared to those with Ct values <20 after analysis of recent historical data from the same Ebola treatment units revealed a strong association with mortality (63). Ct values of 20 were found to correspond to an RNA viral load of 5 \u00d7 107 genome copies/mL of plasma (7.7 log10 copies/mL). At entry into the trial, 55 subjects were enrolled with Ct >20, and 44 subjects had Ct < 20. Those with Ct values <20 had signi\ufb01cantly greater creatinine, blood urea nitrogen, aspartate aminotransferase, alanine aminotransferase, and creatine kinase levels at the time of admission than those with Ct >20. On-trial mortality was 20.0% in patients with Ct >20, which is 33% lower than the target value (30%); it was 90.9% in patients with Ct < 20, which was 7% higher than the target value (85%). Thus, there may be a survival bene\ufb01t in patients with Ct values >20 and no improvement in survival when Ct values are <20. No grade 3 or 4 clinical events were considered to be related to the drug by the investigators, and all deaths were associated with uncontrolled EBOV viremia and disease progression. There was no signi\ufb01cant difference in terms of mortality between adults who started favipiravir within 72 h and those who started favipiravir >72 h after symptoms \ufb01rst appeared [45.2% versus 54.4% (P = 0.5)].\nThe design of the JIKI trial has been criticized heavily, as there was low statistical power and no placebo arm, and as a result, the study authors admit that favipiravir ef\ufb01cacy was not proved (63). Further complicating the design, it used historical controls, which is a signi\ufb01cant confounder owing to changing case fatality rates and changing standards of care over time. Unfortunately, in this study blood samples were not able to be collected systematically to examine pharmacokinetics. Sissoko et al. (63) plan to examine plasma trough concentrations collected to update their dosing estimation model. It would be more helpful to conduct expanded safety and pharmacokinetics studies in African populations at risk for EBOV infection to determine optimal dosing for future studies.\nSissoko et al. (63) also state that their results suggest that trials of antiviral drugs in EBOV should be strati\ufb01ed by Ct value and that trials of antiviral monotherapy should primarily target patients with Ct > 20. This proposal ignores the fact that Ct values from site to site may not be generalizable owing to different methodologies and technicians (63); establishing a quali\ufb01ed, quantitative real time\u2013PCR assay will be critical to any cross-site analyses. Moreover, the proposal to stratify the analysis based on Ct value is an oversimpli\ufb01cation of the issues of protocol design. For favipiravir, researchers have not demonstrated that the dose administered was optimal: Higher doses or an alternate formulation (e.g., IV) of the drug may in fact result in ef\ufb01cacy in patients with Ct < 20. Thus, a revised conclusion could be that favipiravir as dosed in this study should not be used as a monotherapy in patients with Ct < 20. Additionally, other drugs may be more potent and have greater ef\ufb01cacy in EBOV patients with Ct < 20.\nOptimal Ebola therapy may need to parallel the treatment of HIV infection. In HIV treatment, combination therapy is critical to successful and sustained viral suppression. Furthermore, certain antiretroviral combinations (rilpivirine-based regimens, abacavir/lamivudine with efavirenz or\nwww.annualreviews.org \u2022 Cure for Ebola 337\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\natazanavir/ritonavir, and darunavir/ritonavir plus raltegrivir) have higher rates of virological failure than others when HIV viral RNA is >100,000 copies/mL (67). As in HIV antiviral therapy, therapy of EBOV may require more than one agent for ef\ufb01cacy, especially in individuals with high viral loads and more advanced disease.\nGS-5734. GS-5734 is a novel monophosphoramidate prodrug of an adenosine analogue, and data suggest that it selectively inhibits EBOV replication by targeting its RNA-dependent RNA polymerase and inhibiting viral RNA synthesis following ef\ufb01cient intracellular conversion to an active triphosphate nucleotide (68). GS-5734 has a 1 -cyano group, which provides potency and selectivity toward viral RNA polymerases. The drug was further modi\ufb01ed with a monophosphate promoiety to enhance intracellular metabolism into the active triphosphate metabolite. To demonstrate enhanced intracellular metabolism, human monocyte\u2013derived macrophages were incubated with GS-5734, with a rapid loading of cells with up to 30-fold higher levels of the active triphosphate metabolite compared to incubation with the parent 1 -cyano-substituted adenine C-nucleoside ribose analogue. GS-5734 displayed potent in vitro activity (EC50 values of 0.06\u2013 0.14 \u03bcM) against EBOV-Kikwit, EBOV-Makona, Sudan virus, Bundibugyo virus, and Marburg virus. GS-5734 also inhibited other RNA viruses, including respiratory syncytial virus, Jun\u00b4\u0131n virus, Lassa virus, and Middle East respiratory syndrome virus.\nPharmacokinetics were determined in NHPs and in rhesus macaques following IV infusion of 10 mg/kg; the half-life was 0.39 h with rapid systemic elimination and persistent levels of the active triphosphate metabolite (68). GS-5734 was distributed rapidly into peripheral blood mononuclear cells, was converted to active triphosphate metabolite within 2 h of administration, and maintained levels necessary for >50% viral inhibition for 24 h (68). In cynomolgus macaques that received 10 mg/kg IV doses of [14C] GS-5734, drug-derived material was distributed to testes, epididymis, eyes, and brain within 4 h of administration. Levels in brain at 4 h were low relative to other tissues but remained detectable above the drug plasma levels 168 h after dosing.\nThe ef\ufb01cacy of GS-5734 in EBOV-infected rhesus monkeys was assessed in the well-established EBOV-Kikwit IM infection model [1,000 PFU of 7U (7-uridylyl stretch, poly-U site in the glycoprotein sequence) virus] (68). The most effective dosing regimen of GS-5734 was 10 mg/kg administered via IM injection daily, initiated 3 days postinfection, with 100% survival. Compared to vehicle treatment, GS-5734 signi\ufb01cantly reduced plasma viral RNA by \u22651.7 log10 on day 4, and on days 5 and 7, levels were below the lower levels of quantitation (8 \u00d7 104 RNA copies/mL), whereas levels in animals receiving the vehicle exceeded 109 copies/mL. GS-5734 was associated with reduction in EBOV clinical disease scores, and mitigation of EBOV-induced laboratory abnormalities included platelet count, creatinine, transaminases, coagulation parameters, and creatinine kinase.\nIV GS-5734 is currently undergoing human Phase I safety and pharmacokinetic studies (68, 69). GS-5734 has been used as a compassionate therapy in two cases of EBOV patients who survived infection: a case of EBOV recrudescence in a female nurse in the United Kingdom and a case of acute EBOV infection in a female infant in Guinea (70, 71). GS-5734 could be a promising candidate for EBOV treatment given its potency and for eradication of persistently replicating virus in immunologically privileged sites given its tissue penetration. In addition, GS-5734 may be an attractive therapy for postexposure prophylaxis, as its active triphosphate form is rapidly accumulated and maintained in cells of the mononuclear lineage, which are important in EBOV pathogenesis.\nNucleic Acid\u2013Based Therapeutics\nTwo classes of nucleic acid\u2013based therapeutics have been studied to determine if they can treat EBOV infections: antisense phosphorodiamidate morpholino oligomers (PMOs) and small\n338 Cardile et al.\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\ninterfering RNAs (siRNAs). Both PMOs and siRNAs provided promising ef\ufb01cacy results in NHPs against EBOV, but because corporate support for further development of these products has been withdrawn, these are discussed only brie\ufb02y. PMOs are single-stranded deoxyribonucleotide oligomers that inhibit translation via steric hindrance (72, 73). siRNAs are doublestranded oligonucleotides that cause RNA interference (RNAi), silencing gene expression via mRNA degradation (72\u201374). Both oligonucleotides and naked RNAi molecules are unstable in vivo owing to nuclease digestion, are prone to poor intracellular uptake, and as a result require chemical modi\ufb01cations or additional formulation for in vivo administration (72, 73, 75, 76). The detailed development and chemical modi\ufb01cation of siRNAs and PMOs are described in detail in other sources (72, 73, 75).\nThe \ufb01rst-generation siRNA product, TKM-Ebola, was a combination of three siRNA molecules that targeted the EBOV RNA-dependent RNA polymerase L, virion protein 24 (VP24), and virion protein 35 (VP35) (77); in subsequent generations, only siRNAs designed to target L and VP35 were included (78). A PMO product, AVI-6002, is composed of AVI-7539 and AVI7537, which target VP35 and VP24, respectively (79, 80). TKM-Ebola (2 mg/kg total siRNA per dose, IV infusion) was administered to macaques 30 min after EBOV challenge, followed by doses on days 1\u20136, resulting in 100% survival (4/4) (77). Study of AVI-6002 in NHP models would eventually reveal that VP24 (AVI-7537) targeting was responsible for the observed activity, with 75% (6/8 NHPs) surviving with AVI-7537 therapy and none surviving with AVI-7539 therapy (81).\nWhen the West Africa EBOV outbreak strain was determined to be genetically unique (termed EBOV-Makona), there were concerns about the ef\ufb01cacy of siRNA and PMOs, as they are sequence-speci\ufb01c products targeting EBOV-Zaire. Researchers did not identify mutations that would be anticipated to impact AVI-7537 if used against EBOV-Makona (82). In regards to the siRNA product, mutations were detected in the binding sites of L and in VP35 targets (82). As a result, a new siRNA cocktail, siEbola-3, was designed to correct these mismatches to enable full complementarity to EBOV-Makona sequences (78). NHPs were infected with EBOV-Makona and administered siEbola-3 (0.5 mg/kg) beginning 72 h after infection, when animals were viremic and clinically ill, and had repeat daily treatments on days 4\u20139 post-challenge (78). All treated animals survived to study endpoint, whereas untreated control animals succumbed on days 8 and 9 (78).\nA Phase I clinical trial of the second-generation TKM-Ebola was initiated in January 2014, and in May 2014, the single-ascending dose portion of the study was completed. But in July 2014, it was put on clinical hold owing to concerns by the FDA regarding cytokine release, and the clinical hold has since been released (83). A Phase I clinical trial of AVI-6002 was completed in healthy adults (84). AVI-6002 was safe and well tolerated at the doses studied, with a maximum tolerated dose not observed (84). No further information on AVI-6002 or AVI-7537 has been published to date.\nAn ef\ufb01cacy study of siEbola-3 in Sierra Leone was initiated in March of 2015 (58, 85, 86). The study was an open-label, single-arm trial and was historically controlled but was terminated, as interim analysis indicated that continuing enrollment was not likely to demonstrate an overall therapeutic bene\ufb01t (85, 86). The product sponsor, Tekmira (now Arbutus Biopharma), has suspended its EBOV antiviral therapeutic program (87).\nIMMUNE THERAPEUTICS\nConvalescent Plasma and Hyperimmune Serum\nHistorically, clinical data have been limited on the bene\ufb01ts of convalescent blood products for EBOV therapy and were restricted to the case series from the 1995 EBOV-Kikwit outbreak in the Democratic Republic of the Congo and a few case reports (88). During the West Africa EBOV outbreak, the WHO had prioritized Ebola convalescent whole-blood and convalescent plasma\nwww.annualreviews.org \u2022 Cure for Ebola 339\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\ntransfusion for evaluation, with the rationale that this could be done quickly and, if proved to be safe and effective, could be implemented rapidly (89). Three convalescent plasma trials enrolled patients during the outbreak (89).\nTo date, the results of one nonrandomized, historically controlled study that was conducted in Guinea have been published (90). The authors determined that the randomization of patients was locally unacceptable in the setting of the EBOV outbreak (90). Patients of any age, including pregnant women who had symptomatic, laboratory-con\ufb01rmed EBOV, were enrolled (90). A total of 84 EBOV-infected patients were included in the primary analysis, and they were treated with two consecutive transfusions of 200\u2013250 mL of ABO-compatible convalescent plasma; levels of neutralizing antibodies were unknown and each unit of plasma was obtained from a separate convalescent donor (90). Small adults and children weighing less than 45 kg received two transfusions of 10 mL/kg of convalescent plasma (90). The transfusions were initiated on the day of diagnosis or up to 2 days later, and the level of neutralizing antibodies against EBOV in the plasma was not determined at the time of administration (90). The study was historically controlled with 418 patients who had been treated at the same center during the previous 5 months (90). From day 3 to day 16 after diagnosis, 26 of 84 patients (31%) in the convalescent-plasma group died, and 158 of 418 patients (38%) died in the control group. After adjustment for age and Ct values, mortality remained lower in the convalescent-plasma group, but the difference was not signi\ufb01cant (90). Eight patients (8%) had an adverse reaction during or soon after the transfusion with elevated temperature predominating, followed by pruritis or skin rash (90).\nThis trial had study design \ufb02aws similar to those in the JIKI trial that were dictated by the circumstances of the EBOV outbreak. The authors of this study admit that the inability to determine the level of neutralizing antibodies in the donor plasma was a limitation of the study (90). They state that the inability to conduct EBOV plaque-neutralization assays in a biosafety level 4 laboratory or to ship blood out of country was the limiting factor (90). Possibly, titers could have been conducted via ELISA or another method at the study site. If the use of plaque-neutralization titers is deemed necessary, then an assay based on an Ebola GP-pseudotyped virus or other test systems could be developed. Several in vivo studies have had varied results regarding the use of passive antibody therapy (91\u201394). Important themes that have emerged from these studies are that antibody titers in human convalescent plasma tend to be low, frequent dosing is needed to maintain antibody titers, survival is correlated in some studies with anti-EBOV IgG titers, and there is batch-to-batch titer variability in different plasma pools (91\u201394). Thus, in this clinical trial, the lack of a mortality bene\ufb01t may be related to the fact that the antibody titers were low in the transfused plasma and that plasma was not transfused frequently enough.\nThese issues could be overcome, as illustrated in an in vivo study with hyperimmune plasma. Polyclonal IgG was puri\ufb01ed from a large preparation of convalescent serum, which had been pooled from vaccinated macaques; these donor macaques subsequently survived challenge with a lethal dose of EBOV, suggesting that their antibody titers were adequate for protection (95). The fractionated IgG was evaluated and shown to have virus-neutralizing activity, and recipient NHPs were treated with 70\u2013100 mg/kg of fractionated IgG (95). Recipient NHPs were then infected with EBOV, and treatments were initiated 48 h postinfection, with additional treatments on days 4 and 8 postexposure (95). This regimen protected 100% of EBOV-challenged NHPs (95). The success with IgG treatments in this study was attributed to the polyclonal nature of the exogenous antibodies controlling viral infection and to the multiple treatments, which were thought to maintain suf\ufb01ciently high levels of IgG to permit the host\u2019s adaptive immune response to help clear the viral infection (95). However, the production of large quantities of hyperimmune plasma adequate to respond during an outbreak would be dif\ufb01cult, given that it would require a large supply of convalescent plasma or production in animals.\n340 Cardile et al.\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\nZMapp\nDuring the West Africa EBOV outbreak, ZMapp was lauded as a potential game changer, but it was available in exceedingly limited supply. The two US health-care workers who acquired Ebola in Liberia were treated with ZMapp, the \ufb01rst on days 9, 12, and 15 of illness and the second on days 10, 13, and 16, both without adverse effects (96). Both patients reported subjective improvement to varying degrees after receiving ZMapp, and both survived the infection (96). In these cases, it is not clear if ZMapp was one of the primary factors contributing to survival. Both patients were aggressively \ufb02uid resuscitated and had whole-blood transfusions in Liberia, including (in the case of Patient 1) blood from an Ebola survivor (96).\nZMapp is a mixture of MB-003 and ZMAb (97). ZMAb components were produced in Nicotiana benthamiana using the large-scale, current good manufacturing practice\u2013compatible rapid antibody production platform that was used for MB-003 (97). After a lethal IM challenge with EBOV, rhesus macaques were treated with ZMapp (50 mg/kg) at 3, 6, and 9 days postinfection; 4, 7, and 10 days postinfection; or 5, 8, and 11 days postinfection. All animals treated with ZMapp survived infection (97). Because ZMapp components were developed against EBOV-Kikwit, there were concerns that it would be ineffective or less effective during the West Africa outbreak. EBOV-Makona was evaluated via indirect comparison of published amino acid sequences, and the epitopes targeted by ZMapp were not mutated between the two virus variants (97).\nIn March 2015, a Phase I open-label study was launched to evaluate the safety and pharmacokinetics of a single dose of 50 mg/kg ZMapp in healthy adult volunteers (98). Recently, the long-awaited results of the multisite, randomized controlled trial of ZMapp in EBOV infection (PREVAILII) were presented at the Conference on Retroviruses and Opportunistic Infections (CROI) (99). Patients were randomized 1:1 to receive either the optimized standard of care (de\ufb01ned minimally as IV \ufb02uid resuscitation plus electrolyte monitoring) or the optimized standard of care plus three IV infusions of 50 mg/kg ZMapp three days apart (99). Seventy-two patients were strati\ufb01ed by baseline PCR Ct values (\u226422 versus >22) and by treatment site [United States versus Liberia or Sierra Leone versus Guinea (where favipiravir was part of the optimized standard of care)] (99). ZMapp did not confer a statistically signi\ufb01cant mortality bene\ufb01t, with 37% mortality in the control arm and 22.2% mortality in the treatment arm of the study (99). Even though the study was strati\ufb01ed, there are signi\ufb01cant confounders that may affect study results. The most concerning was that the 12 patients treated with ZMapp in Guinea were also treated with favipiravir, and it is questionable if these patients should be included in the analysis. One could easily argue that the patient treated in the United States should also be excluded from analysis given that the supportive care was likely signi\ufb01cantly different to that provided in Ebola treatment units.\nOther monoclonal antibodies and cocktails are under development, with most being in preclinical development (100\u2013104). MIL77 is the most advanced and is a collaboration between the Public Health Agency of Canada, Mapp Biopharmaceutical, and Beijing Mabworks (104). This drug is composed of ZMapp-like monoclonal antibodies in modi\ufb01ed Chinese hamster ovary cells and was administered to four patients during the West Africa outbreak (104). In a recent study, MIL77 was shown to be at least comparable to treatments using a similar formulation of the ZMapp antibodies in NHP models of EBOV-Makona disease (104).\nInterferon-\u03b2\nEBOV infection is associated with signi\ufb01cant IFN-\u03b1 production but little IFN-\u03b2 production; plasma concentrations of IFN-\u03b1 greatly exceed those seen in other viral infections (105). Early postexposure treatment with IFN-\u03b2 signi\ufb01cantly increased survival times of rhesus macaques infected with a lethal dose of EBOV, although it failed to alter mortality (105). Infected macaques\nwww.annualreviews.org \u2022 Cure for Ebola 341\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\nwere then treated with human recombinant IFN-\u03b2 (10.5 \u03bcg/kg) 18 h and 1, 3, 5, 7, and 9 days after EBOV infection, which signi\ufb01cantly prolonged the mean time to death (105). A historically controlled trial of IFN-\u03b2 was attempted in Guinea, with nine patients enrolling, but results are not currently available in the peer-reviewed literature (43).\nTHE PATH FORWARD\nThe net results of medical research that occurred during the West Africa EBOV outbreak have been described as a \u201cthin harvest\u201d in a recent article (43). Overall, the global health community was unprepared for a large EBOV outbreak and lacked the ability to initiate clinical trials rapidly in that setting. As a result of delays in implementation, many of the clinical trials discussed above were not initiated while cases existed in adequate numbers. In the future, deployable, mobile capabilities need to be developed to facilitate rapid conduct of clinical trials during an outbreak. This would require the following conditions be present in a country at risk for an outbreak: support of hostnation governments; prepositioned health care and clinical research infrastructure, including welltrained personnel, functioning laboratory support, logistical systems, human subjects research and regulatory oversight, and continuously exercised system function; trained and ready research teams; \ufb01led INDs; \ufb02exible and preapproved clinical research protocols; an established logistical tail, including available therapeutic products; and functional integration into host-nation and international response plans.\nIn addition, there were signi\ufb01cant ethical concerns about conducting blinded, randomized, placebo-controlled clinical trials in an outbreak setting with a virus with high mortality and transmission rates (106). Clinical researchers who support randomized, placebo-controlled trial designs in Ebola research argued that this design is the most ef\ufb01cient and powerful method for assessing the safety and effectiveness of available experimental interventions and that alternate trial designs are likely to lead to invalid results (107). Those against the use of placebo-controlled trials have argued that randomizing individuals in a treatment trial to the placebo-controlled arm, in which they receive supportive care only, denies them at least the possibility of bene\ufb01t that might result from an experimental treatment (107). In addition, patients that could potentially be enrolled in trials in an outbreak setting with high mortality could be considered a vulnerable population that cannot truly provide informed consent. There were also signi\ufb01cant concerns that in the context of widespread distrust of Ebola treatment units, using a randomized design could have led more patients to avoid seeking care. As a result of these concerns, only the ZMapp trial was conducted with a randomized, controlled design, whereas the other three trials (favipiravir, convalescent plasma, and TKM-Ebola) used an alternate design with historical controls. None of these trials showed ef\ufb01cacy of a product to support licensure at this time. Given that historically controlled trials were ineffective, randomized controlled trials need to be designed to mitigate ethical concerns. In addition, minimal standards for optimized supportive care and therapy for coinfections need to be adopted at all clinical trial treatment sites. One possible trial design that could facilitate this goal would be an adaptive trial, similar to that of the ZMapp trial (108). This design has been described in a recent publication and involved early, frequent interim analyses and a \u201cbarely Bayesian design\u201d (108, p. 6).\nIn conclusion, we return to the initial question posed: Will there be a cure for Ebola? At this time, despite signi\ufb01cant investments in clinical research during the West Africa EBOV outbreak, we are still a long way from a licensed MCM and thus a cure. In addition, researchers have learned that even in individuals considered to have been cured from Ebola, there can be recrudescence of the virus, and that a post-Ebola syndrome with chronic symptoms and viral persistence in immunologically privileged sites such as the eyes and gonads may occur more frequently than\n342 Cardile et al.\n\nDownloaded from www.annualreviews.org. Guest (guest) IP: 137.63.218.207 On: Tue, 20 Aug 2024 10:03:52\n\nPA57CH17-Cardile ARI 10 December 2016 10:54\npreviously thought. As a result, future therapeutics development should take into account a need for adequate tissue penetration into sites such as the eyes, gonads, and central nervous system. Multiple valuable lessons have been learned about the conduct of clinical research in a resource-poor, high risk\u2013pathogen setting. However, the timing and number of cases that will be associated with the next outbreak in which clinical candidates can be tested are unknown and cannot be predicted with accuracy. Until the next outbreak occurs with suf\ufb01cient patient numbers, further development of EBOV antiviral therapies and vaccines will be forced to rely on ef\ufb01cacy characterizations in animal models of the disease.\nDISCLOSURE STATEMENT The authors are not aware of any af\ufb01liations, memberships, funding, or \ufb01nancial holdings that might be perceived as affecting the objectivity of this review. The views expressed herein are those of the authors and do not re\ufb02ect the of\ufb01cial policy or position of the US Army Medical Research Institute of Infectious Diseases, US Army Medical Department, US Army Of\ufb01ce of the Surgeon General, Department of the Army, Department of Defense, or US Government. The authors are employees of the US government. This work was prepared as part of their of\ufb01cial duties. Mention of trade names, commercial products, or organizations does not imply endorsement by the US Government.\nACKNOWLEDGMENTS The authors would like to acknowledge funding from Medical Countermeasures Systems and the US Defense Threat Reduction Agency.\nLITERATURE CITED 1. Merriam-Webster.com. 2016. Cure. Accessed on Mar. 1. http://www.merriam-webster.com/ dictionary/cure 2. Greenwood B. 2014. The contribution of vaccination to global health: past, present and future. Philos. Trans. R. Soc. B 369:20130433 3. 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    "authors": [
      "Anthony P. Cardile",
      "Travis K. Warren",
      "Karen A. Martins",
      "Ronald B. Reisler",
      "Sina Bavari"
    ],
    "doi": "10.1146/annurev-pharmtox-010716-105055",
    "date": "2017-01-06",
    "item_type": "journalArticle",
    "url": ""
  }
]