Zaire Ebolavirus Glycoprotein Research Articles

Antibody responses to recombinant vesicular stomatitis virus-Zaire Ebolavirus vaccination for Ebola virus disease across doses and continents: 5-year durability

ObjectivesTo report 5-year persistence and avidity of antibodies produced by the live-attenuated recombinant vesicular stomatitis virus (rVSV) expressing the Zaire Ebolavirus (ZEBOV) glycoprotein (GP), known as rVSV-ZEBOV (Ervebo®). MethodsHealthy adults vaccinated with 300,000 or 10–50 million plaque-forming units of rVSV-ZEBOV in the WHO-coordinated trials of 2014–2015 were followed for up to 4 (Lambaréné, Gabon) and 5 (Geneva, Switzerland) years. We report seropositivity rates, geometric mean titres (GMTs), and population distribution of ZEBOV-GP ELISA IgG antibodies, neutralizing antibodies (pseudovirus and live-virus neutralization) and antibody avidity; the primary outcome was ZEBOV-GP ELISA IgG GMTs at 4 or 5 years compared with 1 year (Y1) after immunization. ResultsAmong the 168 eligible vaccinees (Geneva: 97 and Lambaréné: 71) enrolled 1 year post-immunization, 146 (87%) remained enrolled at 4 years (Geneva: n = 88, Lambaréné: n = 58), and 84 (87%, Geneva) at 5 years post-vaccination. ZEBOV-GP ELISA IgG GMTs plateaued, with no declining trend from 1 year through the last time point assessed (1147.8 [95% CI 874.3–1507.0] at Y1 versus 1548.1 [95% CI 1136.6–2108.5] at Y5 in Geneva volunteers receiving ≥10 million plaque-forming units of rVSV-ZEBOV), their avidity matching that of ZEBOV convalescents. Live-virus neutralizing antibodies were detected for shorter periods and in fewer vaccinees (53/95 [56%] at Y1 versus 35/84 [42%] at Y5 in Geneva volunteers, all dose levels). DiscussionTitres at Y1 emerged as a correlate of antibody persistence at Y5. The findings of persistent ZEBOV-GP ELISA IgG titres yet shorter-lasting, lower titres of live-virus neutralizing antibodies suggest the contribution of antibody-mediated protective mechanisms other than neutralization. Long-term clinical efficacy of rVSV-ZEBOV, however, requires further study.

RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy

Virus infection affects cellular proteostasis and provides an opportunity to study this cellular process under perturbation. The proteostasis network in the endoplasmic reticulum (ER) is composed of the calnexin cycle, and the two protein degradation pathways ER-associated protein degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD/ER-phagy/reticulophagy). Here we show that calnexin and calreticulin trigger Zaire Ebolavirus (EBOV) glycoprotein GP1,2 misfolding. Misfolded EBOV-GP1,2 is targeted by ERAD machinery, but this results in lysosomal instead of proteasomal degradation. Moreover, the ER Ub ligase RNF185, usually associated with ERAD, polyubiquitinates EBOV-GP1,2 on lysine 673 via ubiquitin K27-linkage. Polyubiquinated GP1,2 is subsequently recruited into autophagosomes by the soluble autophagy receptor sequestosome 1 (SQSTM1/p62), in an ATG3- and ATG5-dependent manner. We conclude that EBOV hijacks all three proteostasis mechanisms in the ER to downregulate GP1,2 via polyubiquitination and show that this increases viral fitness. This study identifies linkages among proteostasis network components previously thought to function independently.

Immunogenicity of rVSVΔG-ZEBOV-GP Ebola vaccine (ERVEBO®) in African clinical trial participants by age, sex, and baseline GP-ELISA titer: A post hoc analysis of three Phase 2/3 trials

BackgroundERVEBO®, a live recombinant vesicular stomatitis virus (VSV) vaccine containing the Zaire ebolavirus glycoprotein (GP) in place of the VSV GP (rVSVΔG-ZEBOV-GP), was advanced through clinical development by Merck & Co., Inc., Rahway, NJ, USA in collaboration with multiple partners to prevent Ebola virus disease (EVD) and has been approved for human use in several countries. MethodsWe evaluated data from three Phase 2/3 clinical trials conducted in Liberia (PREVAIL), Guinea (FLW), and Sierra Leone (STRIVE) during the 2013–2016 West African EVD outbreak to assess immune responses using validated assays. We performed a post hoc analysis of the association of vaccine response with sex, age (18–50 yrs & >50 yrs), and baseline (BL) GP-enzyme-linked immunosorbent assay (ELISA) titer (<200 & ≥200 EU/mL), including individual study (PREVAIL, FLW, or STRIVE) data and pooled data from all 3 studies. The endpoints were total IgG antibody response (EU/mL) measured by the GP-ELISA and neutralizing antibody response measured by the plaque reduction neutralization test (PRNT) to rVSVΔG-ZEBOV-GP at Days 28, 180, and 365 postvaccination. ResultsIn the overall pooled population, in all subgroups, and in each trial independently, GP-ELISA and PRNT geometric mean titers increased from BL, generally peaking at Day 28 and persisting through Day 365. Immune responses were greater in women and participants with BL GP-ELISA ≥ 200 EU/mL, but did not differ across age groups. ConclusionThese data demonstrate that rVSVΔG-ZEBOV-GP elicits a robust and durable immune response through 12 months postvaccination in participants regardless of age, sex, or BL GP-ELISA titer. The higher immune responses observed in women and participants with pre-existing immunity are consistent with those described previously and for other vaccines.Trials were registered as follows: PREVAIL: ClinicalTrials.gov NCT02344407; FLW: Pan African Clinical Trials Registry PACTR201503001057193; STRIVE: ClinicalTrials.gov NCT02378753. Protocols V920-009, 011, and 018.

Inmazeb: new hope for Zaire Ebola virus disease

Ebola virus disease first appeared in 1976 in Zaire (now democratic republic of Congo). Since then virus outbreaks occurred periodically in African countries. The cases notified in March 2014 in west Africa was largest outbreak till now. In 2020 there is ongoing outbreak of Zaire Ebola virus in democratic republic of Congo. Ebola virus is single stranded RNA virus which causes viral hemorrhagic fever in humans presenting as high fever, chills, loss of appetite, myalgia, headache. Till now there was no specific treatment, symptomatic treatment methods including infusion of electrolyte and/or antibiotics were mainly used. In October 2020 FDA approved the first treatment for Zaire Ebola virus disease in adult and pediatric patients, including neonates born to a mother who is RT-PCR positive for Zaire ebolavirus infection. The treatment is called Inmazeb, combination of three recombinant human IgG1κ monoclonal antibodies (Atoltivimab, Maftivimab, and Odesivimab-ebgn) each targeting the Zaire ebolavirus glycoprotein.

Transcriptomic signatures induced by the Ebola virus vaccine rVSVΔG-ZEBOV-GP in adult cohorts in Europe, Africa, and North America: a molecular biomarker study

SummaryBackgroundA recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP) vaccine has been reported as safe, immunogenic, and highly protective in a ring vaccination trial. We aimed to identify transcriptomic immune response biomarker signatures induced by vaccination and associated signatures with its immunogenicity and reactogenicity to better understand the potential mechanisms of action of the vaccine.Methods354 healthy adult volunteers were vaccinated in randomised, double-blind, placebo-controlled trials in Europe (Geneva, Switzerland [November, 2014, to January, 2015]) and North America (USA [Dec 5, 2014, to June 23, 2015]), and dose-escalation trials in Africa (Lambaréné, Gabon [November, 2014, to January, 2015], and Kilifi, Kenya [December, 2014, to January, 2015]) using different doses of the recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP; 3 × 105 to 1 × 108 plaque-forming units [pfu]). Longitudinal transcriptomic responses (days 0, 1, 2, 3, 7, 14, and 28) were measured in whole blood using a targeted gene expression profiling platform (dual-colour reverse-transcriptase multiplex ligation-dependent probe amplification) focusing on 144 immune-related genes. The effect of time and dose on transcriptomic response was also assessed. Logistic regression with lasso regularisation was applied to identify host signatures with optimal discriminatory capability of vaccination at day 1 or day 7 versus baseline, whereas random-effects models and recursive feature elimination combined with regularised logistic regression were used to associate signatures with immunogenicity and reactogenicity.FindingsOur results indicated that perturbation of gene expression peaked on day 1 and returned to baseline levels between day 7 and day 28. The magnitude of the response was dose-dependent, with vaccinees receiving a high dose (≥9 × 106 pfu) of rVSVΔG-ZEBOV-GP exhibiting the largest amplitude. The most differentially expressed genes that were significantly upregulated following vaccination consisted of type I and II interferon-related genes and myeloid cell-associated markers, whereas T cell, natural killer cell, and cytotoxicity-associated genes were downregulated. A gene signature associated with immunogenicity (common to all four cohorts) was identified correlating gene expression profiles with ZEBOV-GP antibody titres and a gene signatures associated with reactogenicity (Geneva cohort) was identified correlating gene expression profiles with an adverse event (ie, arthritis).InterpretationCollectively, our results identify and cross-validate immune-related transcriptomic signatures induced by rVSVΔG-ZEBOV-GP vaccination in four cohorts of adult participants from different genetic and geographical backgrounds. These signatures will aid in the rational development, testing, and evaluation of novel vaccines and will allow evaluation of the effect of host factors such as age, co-infection, and comorbidity on responses to vaccines.FundingInnovative Medicines Initiative 2 Joint Undertaking.

Ebola vaccines for mass immunisation in affected regions

After the resurgence of Ebola virus disease in Guinea, 5 years after it became the epicentre of the 2014–16 epidemic in west Africa that claimed more than 11 000 lives, and following successive outbreaks in the Democratic Republic of the Congo, the need for safe and effective vaccines to curb disease transmission remains as urgent as ever. In May, 2020, the Janssen Vaccines & Prevention heterologous primary and booster Ebola vaccine regimen, comprising the adenovirus type 26 vector-based vaccine expressing Zaire Ebola virus glycoprotein (Ad26.ZEBOV) and the modified vaccinia Ankara vector-based vaccine, encoding glycoproteins from Zaire Ebola virus, Sudan virus, and Marburg virus, and nucleoprotein from the Tai Forest virus (MVA-BN-Filo), administered 8 weeks apart, was granted marketing authorisation by the European Commission for the prevention of Ebola virus disease in adults and children aged 1 years or older. 1 Johnson & JohnsonJohnson & Johnson announces European Commission approval for Janssen's preventive Ebola vaccine. https://www.jnj.com/johnson-johnson-announces-european-commission-approval-for-janssens-preventive-ebola-vaccineDate: 2020 Date accessed: March 17, 2021 Google Scholar Clinical development of the Ad26.ZEBOV and MVA-BN-Filo vaccine regimen included several phase 1 clinical trials done simultaneously at various sites: in Uganda, which is endemic for Ebola virus disease and at risk of an outbreak, with reported cases of Ebola virus disease; in Tanzania and Kenya, which are also at risk of an outbreak, but have no previously reported cases of Ebola virus disease; and in the UK and the USA, which are only at risk of imported cases of Ebola virus disease. 2 Pigott DM Golding N Mylne A et al. Mapping the zoonotic niche of Ebola virus disease in Africa. Elife. 2014; 3e04395 Crossref PubMed Scopus (247) Google Scholar These trials allowed the collection of safety, tolerability, and immunogenicity data 3 Milligan ID Gibani MM Sewell R et al. Safety and immunogenicity of novel adenovirus type 26- and modified vaccinia ankara-vectored Ebola vaccines: a randomised clinical trial. JAMA. 2016; 315: 1610-1623 Crossref PubMed Scopus (187) Google Scholar , 4 Goldstein N Bockstal V Bart S et al. Safety and immunogenicity of heterologous and homologous two dose regimens of Ad26- and MVA-vectored Ebola vaccines: a randomised, controlled phase 1 study. J Infect Dis. 2020; (published online Sept 16.)https://doi.org/10.1093/infdis/jiaa586 Crossref Google Scholar , 5 Mutua G Anzala O Luhn K et al. Safety and immunogenicity of a 2-dose heterologous vaccine regimen with Ad26.ZEBOV and MVA-BN-Filo Ebola vaccines: 12-month data from a phase 1 randomized clinical trial in Nairobi, Kenya. J Infect Dis. 2019; 220: 57-67 Crossref PubMed Scopus (42) Google Scholar , 6 Anywaine Z Whitworth H Kaleebu P et al. Safety and immunogenicity of a 2-dose heterologous vaccination regimen with Ad26.ZEBOV and MVA-BN-Filo Ebola vaccines: 12-month data from a phase 1 randomized clinical trial in Uganda and Tanzania. J Infect Dis. 2019; 220: 46-56 Crossref PubMed Scopus (75) Google Scholar across various epidemiological settings. Additionally, African participants included malaria-naive individuals from a high-altitude setting (Nairobi, Kenya) and individuals with partial immunity to malaria (Mwanza, Tanzania, and Masaka, Uganda). This strategic approach led to the generation of locally relevant data and provided a robust evidence base to inform the selection of Ad26.ZEBOV as a prime and MVA-BN-Filo as a boost for phase 2 clinical trials. Safety and immunogenicity of the two-dose heterologous Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen in children in Sierra Leone: a randomised, double-blind, controlled trialThe Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen was well tolerated with no safety concerns in children aged 1–17 years, and induced robust humoral immune responses, suggesting suitability of this regimen for Ebola virus disease prophylaxis in children. Full-Text PDF Safety and long-term immunogenicity of the two-dose heterologous Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen in adults in Sierra Leone: a combined open-label, non-randomised stage 1, and a randomised, double-blind, controlled stage 2 trialThe Ad26.ZEBOV and MVA-BN-Filo vaccine regimen was well tolerated and immunogenic, with persistent humoral immune responses. These data support the use of this vaccine regimen for Ebola virus disease prophylaxis in adults. Full-Text PDF

Zoonotic risk factors associated with seroprevalence of Ebola virus GP antibodies in the absence of diagnosed Ebola virus disease in the Democratic Republic of Congo.

BackgroundEbola virus (EBOV) is a zoonotic filovirus spread through exposure to infected bodily fluids of a human or animal. Though EBOV is capable of causing severe disease, referred to as Ebola Virus Disease (EVD), individuals who have never been diagnosed with confirmed, probable or suspected EVD can have detectable EBOV antigen-specific antibodies in their blood. This study aims to identify risk factors associated with detectable antibody levels in the absence of an EVD diagnosis.MethodologyData was collected from September 2015 to August 2017 from 1,366 consenting individuals across four study sites in the DRC (Boende, Kabondo-Dianda, Kikwit, and Yambuku). Seroreactivity was determined to EBOV GP IgG using Zaire Ebola Virus Glycoprotein (EBOV GP antigen) ELISA kits (Alpha Diagnostic International, Inc.) in Kinshasa, DRC; any result above 4.7 units/mL was considered seroreactive. Among the respondents, 113 (8.3%) were considered seroreactive. Several zoonotic exposures were associated with EBOV seroreactivity after controlling for age, sex, healthcare worker status, location, and history of contact with an EVD case, namely: ever having contact with bats, ever having contact with rodents, and ever eating non-human primate meat. Contact with monkeys or non-human primates was not associated with seroreactivity.ConclusionsThis analysis suggests that some zoonotic exposures that have been linked to EVD outbreaks can also be associated with EBOV GP seroreactivity in the absence of diagnosed EVD. Future investigations should seek to clarify the relationships between zoonotic exposures, seroreactivity, asymptomatic infection, and EVD.

A Naturally Occurring Polymorphism in the Base of Sudan Virus Glycoprotein Decreases Glycoprotein Stability in a Species-Dependent Manner.

Sudan virus (SUDV) is one of five filoviruses that compose the genus Ebolavirus that has been responsible for episodic outbreaks in Central Africa. While the SUDV glycoprotein (GP) structure has been solved, GP residues that affect SUDV entry have not been extensively examined; many of the entry characteristics of SUDV GP are inferred from studies with the Zaire Ebola virus (EBOV) GP. Here, we investigate the effect on virus entry of a naturally occurring polymorphism in SUDV GP. Two of the earliest SUDV isolates contain glutamine at residue 95 (Q95) within the base region of GP1, whereas more recent SUDV isolates and GPs from all other ebolaviruses carry lysine at this position (K95). A K95Q change dramatically decreased titers of pseudovirions bearing SUDV GP, whereas the K95Q substitution in EBOV GP had no effect on titer. We evaluated virus entry to identify SUDV GP Q95-specific entry defects. The presence of Q95 in either EBOV or SUDV GP resulted in enhanced sensitivity of GP to proteolytic processing, yet this could not account for the SUDV-specific decrease in GP Q95 infectivity. We found that SUDV GP Q95 pseudovirions were more sensitive to imipramine, a GP-destabilizing antiviral. In contrast, SUDV GP K95 was more stable, requiring elevated temperatures to inhibit virus infection. Thus, the residue present at GP 95 has a critical role in stabilizing the SUDV glycoprotein, whereas this polymorphism has no effect on EBOV GP stability. These results provide novel insights into filovirus species-specific GP structure that affects virus infectivity. IMPORTANCE Filovirus outbreaks are associated with significant morbidity and mortality. Understanding the structural constraints of filoviral GPs that control virus entry into cells is critical for rational development of novel antivirals to block infection. Here, we identify a naturally occurring glutamine (Q) to lysine (K) polymorphism at residue 95 as a critical determinant of Sudan virus GP stability but not Zaire Ebola virus GP stability. We propose that glutamine at residue 95 in Sudan virus GP mediates decreased virus entry, thereby reducing infectivity. Our findings highlight a unique structural characteristic of Sudan virus GP that affects GP-mediated functionality. Further, it provides a cautionary note for the development of future broad-spectrum filovirus antivirals.

10061 Assessing immunogenicity of an Ebola vaccine in humans using a systems biology approach

ABSTRACT IMPACT: Understanding gene expression changes after viral vaccination and booster may help predict vaccine efficacy. OBJECTIVES/GOALS: Utilize a systems biology approach to identify gene expression changes after administration of Zaire Ebola virus glycoprotein expressed in a Chimp Adeno3 vector (ChAd3-EBOZ) and either boosted ˜7 weeks later with modified vaccinia Ankara MVA expressing Zaire and Marburg GPs plus Tai forest NP (MVA-BN ®Filo) or given saline (placebo). METHODS/STUDY POPULATION: As part of the phase 1b, open-label vaccination trial of ChAd3-EBO-Z in Mali, West Africa, peripheral blood mononuclear cells were isolated from eight volunteers for whole genome transcriptomics analysis. Four subjects received the MVA-BN ®-Filo booster and four received saline. Samples were taken prior to receipt of the booster or placebo, as well as 1, 7, and 14 days afterwards. Significant differentially expressed genes were identified using RNA-seq between baseline and post-MVA-BN ®Filo. Functional enrichment analysis against the GO Ontology Database and the Immune Signatures C7 collection of MSigDB (ImmuneSigDB) was performed. These differentially expressed genes were also examined for associations with Ebola antibody titers and cell-mediated immune responses. RESULTS/ANTICIPATED RESULTS: The majority of gene expression changes occurred on day 1 post-MVA-BN ®-Filo administration. 870 genes had significantly different expression when day 1 samples were compared to pre-booster baseline (791 upregulated/79 downregulated). Those upregulated genes are mainly involved type I interferon and regulation of viral life cycle pathways. The downregulated genes are involved in regulation of cellular defense response, lymphocyte mediated immunity. Comparing to the C7 Immune Signatures collection datasets, we identified more than 100 upregulated genes from 6 studies of yellow fever vaccination that were also significantly upregulated in our study. The top enriched ontological pathway of those genes is cellular response to type I Interferon. DISCUSSION/SIGNIFICANCE OF FINDINGS: The use of a systems biology approach to compare gene expression changes among vaccine studies utilizing whole genome transcriptomics data allows the identification of genes involved in the immune response to vaccination and might aid in predicting vaccine efficacy.

Estimation of the correlates of protection of the rVSVΔG-ZEBOV-GP Zaire ebolavirus vaccine: a post-hoc analysis of data from phase 2/3 clinical trials

Establishment of immune correlates of protection can provide a measurable criterion for assessing protection against infection or disease. For some vaccines, such as the measles vaccine, antibodies serve as the correlate of protection, but for others, such as human papillomavirus, the correlate of protection remains unknown. Merck & Co, Kenilworth, NJ, USA, in collaboration with multiple partners, developed a live recombinant vesicular stomatitis virus vaccine (rVSVΔG-ZEBOV-GP [ERVEBO]) containing the Zaire ebolavirus glycoprotein (GP) in place of the recombinant vesicular stomatitis virus GP to prevent Ebola virus disease. Seroresponse, defined as post-vaccination GP-ELISA of 200 ELISA units (EU) per mL or higher and two-times or more above baseline, was proposed; however, correlates of protection have not been determined. The objective of this post-hoc analysis was to infer possible correlates of protection for rVSVΔG-ZEBOV-GP. In this post-hoc analysis we included vaccinated participants with serology data from three phase 2/3 immunogenicity trials in Guinea, Sierra Leone, and Liberia (n=2199). Two of the trials were open-label, single-arm trials (one randomised [STRIVE], one non-randomised [FLW]); and one trial was randomised, placebo-controlled with two vaccine comparators (PREVAIL). Endpoints were total IgG antibody response (EU per mL) to rVSVΔG-ZEBOV-GP measured by GP-ELISA and neutralising antibody response to rVSVΔG-ZEBOV-GP measured by plaque reduction neutralisation test at days 14, 28, 180, and 365 after vaccination. Reverse cumulative distribution curves of the antibody concentrations were used to estimate statistical correlates of protection between 70% and 100% that might be applied to vaccine efficacy and effectiveness estimates. Although GP-ELISA and plaque reduction neutralisation tests showed similar response patterns, GP-ELISA provided a wider range of measurable titres and better differentiation for estimating correlates of protection compared with the plaque reduction neutralisation test. At day 14 after vaccination in the FLW trial, 1060 (100%) of 1060 participants had GP-ELISA levels at or above 68 EU per mL and 742 (70%) of 1060 had levels at or above 313 EU per mL. At day 28 after vaccination in the pooled population, 1953 (100%) of 1953 participants had levels at or above 73 EU per mL and 1368 (70%) of 1953 participants had levels at or above 735 EU per mL. GP-ELISA seroresponse 200 EU per mL or higher and two-times or more increase in antibody level from baseline occurred in 80% or higher of participants at each assessment and in 94% or higher of participants at any time after vaccination. Our results are consistent with previous work suggesting that seroresponse defined as GP-ELISA of 200 EU per mL or higher and two-times or more from baseline associated with vaccination might be the most appropriate dichotomous correlate of protection and falls within the seroprotective threshold range described herein. Merck Sharp & Dohme, Biomedical Advanced Research and Development Authority, Office of the Assistant Secretary for Preparedness and Response, US Department of Health and Human Services.

28. Immunogenicity of rVSVΔG-ZEBOV-GP Ebola Vaccine (ERVEBO™) in Participants by Age, Sex, and Baseline GP-ELISA Titer: A Post Hoc Analysis of Three Phase 2/3 Trials

BackgroundThe recent Ebola virus disease (EVD) outbreak in the Democratic Republic of the Congo highlights the sustained threat of EVD morbidity and mortality where healthcare and vaccine delivery are challenging. ERVEBO®, a live recombinant vesicular stomatitis virus (VSV) vaccine containing the Zaire ebolavirus glycoprotein (GP) in place of the VSV GP (rVSVΔG-ZEBOV-GP), was developed by Merck & Co., Inc., Kenilworth, NJ, USA in collaboration with multiple partners to prevent EVD and has been approved for human use in several countries.MethodsWe pooled data from three Phase 2/3 clinical trials conducted in Guinea (FLW), Sierra Leone (STRIVE), and Liberia (PREVAIL) during the 2013–2016 West African outbreak to assess immune responses using a validated assay in each of the three studies and performed a post hoc analysis by sex, age (18–50 yrs & >50 yrs) and baseline (BL) GP-enzyme-linked immunosorbent assay (ELISA) titer (< 200 & ≥200 EU/ml). The full analysis set (FAS) population included the primary immunogenicity populations (all vaccinated participants with serology data collected within an acceptable day range) from all three trials. The endpoints were total IgG antibody response (EU/mL) measured by the GP-ELISA and neutralizing antibody response measured by the plaque reduction neutralization test (PRNT) to rVSVΔG-ZEBOV-GP at Days 14, 28, 180, and 365 postvaccination.ResultsIn the overall population and in all subgroups, GP-ELISA and PRNT geometric mean titers increased from BL, with most peaking at Day 28 and persisting through Day 365. There were differences between males and females and between participants with BL GP-ELISA < 200 & ≥200 EU/ml. There did not appear to be a difference between age groups.ConclusionThese data demonstrate that rVSVΔG-ZEBOV-GP elicits a robust and durable immune response up to 12 months in participants regardless of age, sex, or BL GP-ELISA titer. The higher immune responses observed in females and participants with preexisting immunity are consistent with those described in published literature for other vaccines.DisclosuresJakub Simon, MD, MS, Merck Sharp & Dohme Corp, a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Employee, Shareholder) Stephen Kennedy, MD, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Scientific Research Study Investigator) Barbara Mahon, MD, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Employee, Shareholder) Sheri Dubey, MS, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Employee, Shareholder) Rebecca Grant-Klein, PhD, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Employee, Shareholder) Ken Liu, PhD, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Employee, Shareholder) Jonathan Hartzel, PhD, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Employee, Shareholder) Beth-Ann Coller, PhD, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Employee, Shareholder) Carolee Welebob, PhD, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Employee, Shareholder) Mary Hanson, PhD, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Employee, Shareholder) Rebecca Grais, PhD, Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (Scientific Research Study Investigator)

Safety and immunogenicity of a two-dose heterologous Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen in adults in Europe (EBOVAC2): a randomised, observer-blind, participant-blind, placebo-controlled, phase 2 trial

To address the unmet medical need for an effective prophylactic vaccine against Ebola virus we assessed the safety and immunogenicity of three different two-dose heterologous vaccination regimens with a replication-deficient adenovirus type 26 vector-based vaccine (Ad26.ZEBOV), expressing Zaire Ebola virus glycoprotein, and a non-replicating, recombinant, modified vaccinia Ankara (MVA) vector-based vaccine, encoding glycoproteins from Zaire Ebola virus, Sudan virus, and Marburg virus, and nucleoprotein from the Tai Forest virus. This randomised, observer-blind, placebo-controlled, phase 2 trial was done at seven hospitals in France and two research centres in the UK. Healthy adults (aged 18-65 years) with no history of Ebola vaccination were enrolled into four cohorts. Participants in cohorts I-III were randomly assigned (1:1:1) using computer-generated randomisation codes into three parallel groups (randomisation for cohorts II and III was stratified by country and age), in which participants were to receive an intramuscular injection of Ad26.ZEBOV on day 1, followed by intramuscular injection of MVA-BN-Filo at either 28 days (28-day interval group), 56 days (56-day interval group), or 84 days (84-day interval group) after the first vaccine. Within these three groups, participants in cohort II (14:1) and cohort III (10:3) were further randomly assigned to receive either Ad26.ZEBOV or placebo on day 1, followed by either MVA-BN-Filo or placebo on days 28, 56, or 84. Participants in cohort IV were randomly assigned (5:1) to receive one dose of either Ad26.ZEBOV or placebo on day 1 for vector shedding assessments. For cohorts II and III, study site personnel, sponsor personnel, and participants were masked to vaccine allocation until all participants in these cohorts had completed the post-MVA-BN-Filo vaccination visit at 6 months or had discontinued the trial, whereas cohort I was open-label. For cohort IV, study site personnel and participants were masked to vaccine allocation until all participants in this cohort had completed the post-vaccination visit at 28 days or had discontinued the trial. The primary outcome, analysed in all participants who had received at least one dose of vaccine or placebo (full analysis set), was the safety and tolerability of the three vaccination regimens, as assessed by participant-reported solicited local and systemic adverse events within 7 days of receiving both vaccines, unsolicited adverse events within 42 days of receiving the MVA-BN-Filo vaccine, and serious adverse events over 365 days of follow-up. The secondary outcome was humoral immunogenicity, as measured by the concentration of Ebola virus glycoprotein-binding antibodies at 21 days after receiving the MVA-BN-Filo vaccine. The secondary outcome was assessed in the per-protocol analysis set. This study is registered at ClinicalTrials.gov, NCT02416453, and EudraCT, 2015-000596-27. Between June 23, 2015, and April 27, 2016, 423 participants were enrolled: 408 in cohorts I-III were randomly assigned to the 28-day interval group (123 to receive Ad26.ZEBOV and MVA-BN-Filo, and 13 to receive placebo), the 56-day interval group (124 to receive Ad26.ZEBOV and MVA-BN-Filo, and 13 to receive placebo), and the 84-day interval group (117 to receive Ad26.ZEBOV and MVA-BN-Filo, and 18 to receive placebo), and 15 participants in cohort IV were assigned to receive Ad26.ZEBOV and MVA-BN-Filo (n=13) or to receive placebo (n=2). 421 (99·5%) participants received at least one dose of vaccine or placebo. The trial was temporarily suspended after two serious neurological adverse events were reported, one of which was considered as possibly related to vaccination, and per-protocol vaccination was disrupted for some participants. Vaccinations were generally well tolerated. Mild or moderate local adverse events (mostly pain) were reported after 206 (62%) of 332 Ad26.ZEBOV vaccinations, 136 (58%) of 236 MVA-BN-Filo vaccinations, and 11 (15%) of 72 placebo injections. Systemic adverse events were reported after 255 (77%) Ad26.ZEBOV vaccinations, 116 (49%) MVA-BN-Filo vaccinations, and 33 (46%) placebo injections, and included mostly mild or moderate fatigue, headache, or myalgia. Unsolicited adverse events occurred after 115 (35%) of 332 Ad26.ZEBOV vaccinations, 81 (34%) of 236 MVA-BN-Filo vaccinations, and 24 (33%) of 72 placebo injections. At 21 days after receiving the MVA-BN-Filo vaccine, geometric mean concentrations of Ebola virus glycoprotein-binding antibodies were 4627 ELISA units (EU)/mL (95% CI 3649-5867) in the 28-day interval group, 10 131 EU/mL (8554-11 999) in the 56-day interval group, and 11 312 mL (9072-14106) in the 84-day interval group, with antibody concentrations persisting at 1149-1205 EU/mL up to day 365. The two-dose heterologous regimen with Ad26.ZEBOV and MVA-BN-Filo was safe, well tolerated, and immunogenic, with humoral and cellular immune responses persisting for 1 year after vaccination. Taken together, these data support the intended prophylactic indication for the vaccine regimen. Innovative Medicines Initiative and Janssen Vaccines & Prevention BV. For the French translation of the abstract see Supplementary Materials section.

Characterization of rVSVΔG-ZEBOV-GP glycoproteins using automated capillary western blotting

Ebolavirus (EBOV) entry to host cells requires membrane-associated glycoprotein (GP). A recombinant vesicular stomatitis virus vector carrying Zaire Ebola virus glycoprotein (rVSV-ZEBOV) was developed as a vaccine against ebolaviruses. The VSV glycoprotein gene was deleted (rVSVΔG) and ZEBOV glycoprotein (GP) was inserted into the deleted VSV glycoprotein open reading frame (ORF) resulting in a live, replication-competent vector (rVSVΔG-ZEBOV-GP). Automated capillary westerns were used to characterize the rVSVΔG-ZEBOV-GP vaccine (ERVEBO®) manufacturing process with regards to glycoprotein (GP) structure and variants. The method shows a unique electropherogram profile for each process step which could be used to monitor process robustness. rVSVΔG-ZEBOV-GP encodes GP (GP1-GP2), secreted GP (sGP), and small secreted GP (ssGP) variants. Furthermore, a TACE-like activity was observed indirectly by detecting soluble GP2Δ after virus precipitation by ultracentrifugation. Capillary western blotting techniques can guide process development by evaluating process steps such as enzyme treatment. In addition, the technique can assess GP stability and process lot-to-lot consistency. Finally, capillary western-based technology was used to identify a unique biochemical profile of the rVSVΔG-ZEBOV-GP vaccine strain in final product. Virion membrane-bound GP1-GP2 is critical to vaccine-elicited protection by providing both neutralizing antibodies and T-cell response.

Immunogenicity and Safety of Ebola Virus Vaccines in Healthy Adults’ Volunteers: A Systematic Review and Network Meta-Analysis

Background: Clinical development of Ebola virus vaccines was accelerated by the West African Ebola virus epidemic which remain the deadliest in history ever witnessed. We aimed to compare and rank Ebola virus vaccines according to their immunogenicity and safety. Methods: We conducted a network meta-analysis using random-effect model within a frequentist setting to obtain direct and indirect comparison of trials. We searched Medline, Embase, and the Cochrane library (CENTRAL) until March 31, 2019, for published phase I or II randomized controlled trials (RCTs) vaccine against Ebola virus in healthy adult’s volunteers. We used the Cochrane Risk of Bias Tools to evaluate RCTs methods, and we extracted the proportion or number of participants who seroconverted and reported adverse events. The primary outcome was immunogenicity assessed by the seroconversion rates, defined as number of participants in each group who showed at least a four-fold increase in antibody titer for enzyme-linked immunosorbent assay (ELISA) 28 days after vaccination. Findings: A total of 18 RCTs, which assessed 18 different treatments involving 58 comparisons and 3,633 participants were included in the analysis. The mean age of participants was between 18 and 65 years, and 59% of them were male. The overall quality of evidence was rated as moderate to low. For immunogenicity, 2,198 (61%) participants had seroconversion at 28-days post-vaccination, and the recombinant vesicular virus-based vaccine expressing a Zaire Ebolavirus glycoprotein (rVSVΔ-ZEBOV-GP) at dose of 2 × 107 was significantly the better vaccine (P-score 0·78). Among the 4,746 reported adverse event (AEs), 97 were severe. For the injection-site pain, a total of 1,222 AEs was reported, and Placebo was associated with few events (P-score 0·93). For fatigue (805 reported AEs) and for headache (1,103 reported AEs), the DNA-EBOV vaccine at dose of ≤ 4 mg was the best one with a P-score of 0·96 and 0·85 respectively. For myalgia, 759 AEs were reported, and the ChAd3 vaccine at dose of 1010 was associated with a lower risk (P-score 0·95). Among the 760 AEs reported for fever, the rVSV-ZEBOV vaccine at dose of ≤ 5 × 105 was ranked as the best one (P-score 0·78). Except for the fatigue AEs, no significant global heterogeneity was found. Interpretation: To prevent the next outbreak of Ebola virus disease, the rVSVΔ-ZEBOV-GP vaccine at dose of 2 × 107 appears as the best available option for sufficiently high immunogenicity. Ad5.ZEBOV (1·6 × 1011) might be a possible alternative for immunogenicity. For AEs, no evidence of the best one vaccine is emerged· Larger RCTs are need to clarify the best candidate vaccine in terms of immunogenicity and safety. Funding Statement: None. Declaration of Interests: The authors declare no competing interests in relation with this work. Ethics Approval Statement: The study protocol number registered in the International Register of Systematic Reviews (PROSPERO) was: CRD42018109473.

Structural Basis for a Convergent Immune Response against Ebola Virus

Ebola virus disease is a severe health problem in Africa. Vaccines that display the Zaire ebolavirus glycoprotein spike complex are a prime component for the effort to combat it. The VH3-15/Vλ1-40-based class of antibodies was recently discovered to be a common response in individuals who received the Ebola virus vaccines. These antibodies display attractive properties, and thus likely contribute to the efficacy of the vaccines. Here, we use cryo-EM to elucidate how three VH3-15/Vλ1-40 antibodies from different individuals target the virus and found a convergent mechanism against a partially conserved site on the spike complex. Our study rationalizes the selection of the VH3-15/Vλ1-40 germline genes for specifically targeting this site and highlights Ebolavirus species-specific sequence divergences that may restrict breadth of VH3-15/Vλ1-40-based humoral response. The results from this study could help develop improved immunization schemes and further enable the design of immunogens that would be efficacious against a broader set of Ebolavirus species.

Effect of Gamma Irradiation on the Antibody Response Measured in Human Serum from Subjects Vaccinated with Recombinant Vesicular Stomatitis Virus-Zaire Ebola Virus Envelope Glycoprotein Vaccine.

.rVSVΔG-ZEBOV-GP vaccine is a live recombinant (r) vesicular stomatitis virus (VSV), where the VSV G protein is replaced with the Zaire Ebola virus (ZEBOV) glycoprotein (GP). For vaccine immunogenicity testing, clinical trial sera collected during an active ZEBOV outbreak underwent gamma irradiation (GI) before testing in biosafety level 2 laboratories to inactivate possible wild-type ZEBOV. Before irradiating pivotal trial samples, two independent studies evaluated the impact of GI (50 kGy) on binding ZEBOV-GP (ELISA) antibodies against rVSVΔG-ZEBOV-GP, using sera from a North American phase 1 study. Gamma irradiation was associated with slightly higher antibody concentrations in pre-vaccination samples and slightly lower concentrations postvaccination. Results indicate that GI is a viable method for treating samples from regions where filoviruses are endemic, with minor effects on antibody titers. The impact of GI on immunogenicity analyses should be considered when interpreting data from irradiated specimens.

Development and characterization of a Zaire Ebola (ZEBOV) specific IgM ELISA

Immunoglobulin M (IgM) is the first antibody induced after the onset of an adaptive immune response against a pathogen or vaccine. Serological assays play a central role in evaluating these adaptive immunological responses. Such assays are not only crucial for the assessment of vaccine immunogenicity, but also inform on exposure to pathogens and cross-reactivity with other viruses. To date, there is no ELISA-based assay available that measures IgM responses against Zaire Ebola virus (ZEBOV). To address this critical need, our laboratory has developed a novel immunoassay capable of detecting total IgM against ZEBOV glycoprotein in serum samples from individuals exposed to the antigen through infection or vaccination. Here, we describe a sensitive, high-throughput, and inexpensive assay that can be performed in any laboratory. The performance criteria of the newly developed ZEBOV glycoprotein-based IgM ELISA were assessed using antisera collected from human patients immunized with the rVSVΔG-ZEBOV-GP vaccine being tested in a phase 1 clinical trial. This assay demonstrates high specificity and sensitivity and will also be a valuable tool in the mission to find immune correlates of protection for a successful Ebola vaccine.

Expanding the watch list for potential Ebola virus antibody escape mutations.

The 2014 outbreak of Ebola virus disease (EVD) in Western Africa is the largest recorded filovirus disease outbreak and led to the death of over 11,000 people. The recent EVD outbreaks (since May 2018) in the Democratic Republic of the Congo has already claimed the lives of over 250 people. Tackling Ebola virus (EBOV) outbreaks remains a challenge. Over the years, significant efforts have been put into developing vaccines or antibody therapies which rely on an envelope glycoprotein (GP) of Zaire ebolavirus (strain Mayinga-76). Therefore, one key approach for combating EVD epidemics is to predict mutations that may diminish the effectiveness of the treatment. In a previous study we generated a watch list of potential antibody escape mutations of EBOV GP against the monoclonal antibody KZ52. Molecular modeling methods were applied to the three-dimensional experimental structure of EBOV GP bound to KZ52 to predict the effect of every possible single mutation in EBOV GP. The final watch list contained 34 mutations that were predicted to destabilize binding of KZ52 to EBOV GP but did not affect EBOV GP folding and its ability to form trimers. In this study, we expand our watch list by including three more monoclonal antibodies with distinct epitopes on GP, namely Antibody 100 (Ab100), Antibody 114 (Ab114) and 13F6-1-2. Our updated watch list contains 127 mutations, three of which have been seen in humans or are experimentally associated with reduced efficacy of antibody treatment. We believe mutations on this watch list require attention since they provide information about circumstances in which interventions could lose the effectiveness.

Glycoprotein GP as a basis for the universal vaccine against Ebola virus disease

Ebola virus disease (EVD) is one of the deadliest viral infections affecting humans and nonhuman primates. Of 6 known representatives of the Ebolavirus genus responsible for the disease, 3 can infect humans, causing acute highly contagious fever characterized by up to 90% fatality. These include Bundibugyo ebolavirus (BDBV), Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SUDV). The majority of the reported EVD cases are caused by ZEBOV. Vaccine development against the virus started in 1976, immediately after the causative agent of the infection was identified. So far, 4 vaccines have been approved. All of them are based on the protective epitope of the ZEBOV glycoprotein GP. Because SUDV and BDBV can also cause outbreaks and epidemics, it is vital to design a vaccine capable of conferring protection against all known ebolaviruses posing a threat to the human population. This article presents systematized data on the structure, immunogenicity and protective properties of ebolavirus glycoprotein GP, looks closely at the immunodominant epitopes of ZEBOV, SUDV and BDBV glycoprotein GP required to elicit a protective immune response, and offers a rational perspective on the development of a universal vaccine against EVD that relies on the use of vectors expressing two variants of GP represented by ZEBOV and SUDV.

Prevalence of infection among asymptomatic and paucisymptomatic contact persons exposed to Ebola virus in Guinea: a retrospective, cross-sectional observational study

The prevalence of Ebola virus infection among people who have been in contact with patients with Ebola virus disease remains unclear, but is essential to understand the dynamics of transmission. This study aimed to identify risk factors for seropositivity and to estimate the prevalence of Ebola virus infection in unvaccinated contact persons. In this retrospective, cross-sectional observational study, we recruited individuals between May 12, 2016, and Sept 8, 2017, who had been in physical contact with a patient with Ebola virus disease, from four medical centres in Guinea (Conakry, Macenta, N'zérékoré, and Forécariah). Contact persons had to be 7 years or older and not diagnosed with Ebola virus disease. Participants were selected through the Postebogui survivors' cohort. We collected self-reported information on exposure and occurrence of symptoms after exposure using a questionnaire, and tested antibody response against glycoprotein, nucleoprotein, and 40-kDa viral protein of Zaire Ebola virus by taking a blood sample. The prevalence of Ebola virus infection was estimated with a latent class model. 1721 contact persons were interviewed and given blood tests, 331 of whom reported a history of vaccination so were excluded, resulting in a study population of 1390. Symptoms were reported by 216 (16%) contact persons. The median age of participants was 26 years (range 7-88) and 682 (49%) were male. Seropositivity was identified in 18 (8·33%, 95% CI 5·01-12·80) of 216 paucisymptomatic contact persons and 39 (3·32%, 5·01-12·80) of 1174 (2-4) asymptomatic individuals (p=0·0021). Seropositivity increased with participation in burial rituals (adjusted odds ratio [aOR] 2·30, 95% CI 1·21-4·17; p=0·0079) and exposure to blood or vomit (aOR 2·15, 1·23-3·91; p=0·0090). Frequency of Ebola virus infection varied from 3·06% (95% CI 1·84-5·05) in asymptomatic contact persons who did not participate in burial rituals to 5·98% (2·81-8·18) in those who did, and from 7·17% (3·94-9·09) in paucisymptomatic contact persons who did not participate in burial rituals to 17·16% (12·42-22·31) among those who did. This study provides a new assessment of the prevalence of Ebola virus infection among contact persons according to exposure, provides evidence for the occurrence of paucisymptomatic cases, and reinforces the importance of closely monitoring at-risk contact persons. Institut National de la Santé et de la Recherche Médicale, Reacting, the French Ebola Task Force, Institut de Recherche pour le Développement, and Montpellier University Of Excellence-University of Montpellier.