Asymmetric and non-stoichiometric glycoprotein recognition by two distinct antibodies results in broad protection against ebolaviruses.

Several ebolaviruses cause outbreaks of severe disease. Vaccines and monoclonal antibody cocktails are available to treat Ebola virus (EBOV) infections, but not Sudan virus (SUDV) or other ebolaviruses. Current cocktails contain antibodies that cross-react with the secreted soluble glycoprotein (sGP) that absorbs virus-neutralizing antibodies. By sorting memory B cells from EBOV infection survivors, we isolated two broadly reactive anti-GP monoclonal antibodies, 1C3 and 1C11, that potently neutralize, protect rodents from disease, and lack sGP cross-reactivity. Both antibodies recognize quaternary epitopes in trimeric ebolavirus GP. 1C11 bridges adjacent protomers via the fusion loop. 1C3 has a tripartite epitope in the center of the trimer apex. One 1C3 antigen-binding fragment anchors simultaneously to the three receptor-binding sites in the GP trimer, and separate 1C3 paratope regions interact differently with identical residues on the three protomers. A cocktail of both antibodies completely protected nonhuman primates from EBOV and SUDV infections, indicating their potential clinical value.

Pan-ebolavirus protective therapy by two multifunctional human antibodies.

Ebolaviruses cause a severe and often fatal illness with the potential for global spread. Monoclonal antibody-based treatments that have become available recently have a narrow therapeutic spectrum and are ineffective against ebolaviruses other than Ebola virus (EBOV), including medically important Bundibugyo (BDBV) and Sudan (SUDV) viruses. Here, we report the development of a therapeutic cocktail comprising two broadly neutralizing human antibodies, rEBOV-515 and rEBOV-442, that recognize non-overlapping sites on the ebolavirus glycoprotein (GP). Antibodies in the cocktail exhibited synergistic neutralizing activity, resisted viral escape, and possessed differing requirements for their Fc-regions for optimal in vivo activities. The cocktail protected non-human primates from ebolavirus disease caused by EBOV, BDBV, or SUDV with high therapeutic effectiveness. High-resolution structures of the cocktail antibodies in complex with GP revealed the molecular determinants for neutralization breadth and potency. This study provides advanced preclinical data to support clinical development of this cocktail for pan-ebolavirus therapy.

Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses.

Experimental monoclonal antibody (mAb) therapies have shown promise for treatment of lethal Ebola virus (EBOV) infections, but their species-specific recognition of the viral glycoprotein (GP) has limited their use against other divergent ebolaviruses associated with human disease. Here, we mined the human immune response to natural EBOV infection and identified mAbs with exceptionally potent pan-ebolavirus neutralizing activity and protective efficacy against three virulent ebolaviruses. These mAbs recognize an inter-protomer epitope in the GP fusion loop, a critical and conserved element of the viral membrane fusion machinery, and neutralize viral entry by targeting a proteolytically primed, fusion-competent GP intermediate (GPCL) generated in host cell endosomes. Only a few somatic hypermutations are required for broad antiviral activity, and germline-approximating variants display enhanced GPCL recognition, suggesting that such antibodies could be elicited more efficiently with suitably optimized GP immunogens. Our findings inform the development of both broadly effective immunotherapeutics and vaccines against filoviruses.

Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization.

Structural principles underlying the composition of protective antiviral monoclonal antibody (mAb) cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic mAb cocktail against Ebola virus. We systematically analyzed the antibody repertoire in human survivors and identified a pair of potently neutralizing mAbs that cooperatively bound to the ebolavirus glycoprotein (GP). High-resolution structures revealed that in a two-antibody cocktail, molecular mimicry was a major feature of mAb-GP interactions. Broadly neutralizing mAb rEBOV-520 targeted a conserved epitope on the GP base region. mAb rEBOV-548 bound to a glycan cap epitope, possessed neutralizing and Fc-mediated effector function activities, and potentiated neutralization by rEBOV-520. Remodeling of the glycan cap structures by the cocktail enabled enhanced GP binding and virus neutralization. The cocktail demonstrated resistance to virus escape and protected non-human primates (NHPs) against Ebola virus disease. These data illuminate structural principles of antibody cooperativity with implications for development of antiviral immunotherapeutics.

Multifunctional Pan-ebolavirus Antibody Recognizes a Site of Broad Vulnerability on the Ebolavirus Glycoprotein.

Ebolaviruses cause severe disease in humans, and identification of monoclonal antibodies (mAbs) that are effective against multiple ebolaviruses are important for therapeutics development. Here we describe a distinct class of broadly neutralizing human mAbs with protective capacity against three ebolaviruses infectious for humans: Ebola (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) viruses. We isolated mAbs from human survivors of ebolavirus disease and identified a potent mAb, EBOV-520, which bound to an epitope in the glycoprotein (GP) base region. EBOV-520 efficiently neutralized EBOV, BDBV, and SUDV and also showed protective capacity in relevant animal models of these infections. EBOV-520 mediated protection principally by direct virus neutralization and exhibited multifunctional properties. This study identified a potent naturally occurring mAb and defined key features of the human antibody response that may contribute to broad protection. This multifunctional mAb and related clones are promising candidates for development as broadly protective pan-ebolavirus therapeutic molecules.

High seroreactivities to orthoebolaviruses in rural Cameroon: a case-control study on non-human primate bites and a cross-sectional survey in rural population.

BACKGROUND: Ebola (EBOV) and Sudan (SUDV) orthoebolaviruses are responsible for lethal haemorrhagic fever outbreaks in humans in Central and West Africa, and in apes that can be at the source of human outbreaks for EBOV. METHODS: To assess the risk of exposure to orthoebolaviruses through contact with non-human primates (NHP), we tested the presence of antibodies against different viral proteins with a microsphere-based multiplex immunoassay in a case-control study on bites from NHPs in forest areas from Cameroon (n=795), and in cross-sectional surveys from other rural populations (n=622) of the same country. RESULTS: Seroreactivities against at least two viral proteins were detected in 13% and 12% of the samples for EBOV and SUDV, respectively. Probability of seroreactivity was not associated with history of NHP bites, but was three times higher in Pygmies compared to Bantus. Although no neutralizing antibodies to EBOV and SUDV were detected in a selected series of highly reactive samples, avidity results indicate strong affinity to SUDV antigens. CONCLUSION: The detection of high level of seroreactivities against orthoebolaviruses in rural Cameroon where no outbreaks have been reported, raises the possibilities of silent circulation of orthoebolavirus, or of other not yet documented filoviruses, in these forested regions.

Characterization of Ebola Virus Mucosal Challenge Routes in Cynomolgus Macaques.

Zaïre ebolavirus (EBOV) causes Ebola virus disease (EVD), a devastating viral hemorrhagic fever in humans. Nonhuman primate (NHP) models of EVD traditionally use intramuscular infection with higher case fatality rates and reduced mean time-to-death compared to contact transmission typical of human cases of EVD. A cynomolgus macaque model of oral and conjunctival EBOV was used to further characterize the more clinically relevant contact transmission of EVD. NHPs challenged via the oral route had an overall 50% survival rate. NHPs challenged with a target dose of 1 × 102 PFU or 1 × 104 PFU of EBOV via the conjunctival route had 40% and 100% mortality, respectively. Classic signs of lethal EVD-like disease were observed in all NHPs that succumbed to EBOV infection including viremia, hematological abnormalities, clinical chemistries indicative of hepatic and renal disease, and histopathological findings. Evidence of EBOV viral persistence in the eye was observed in NHPs challenged via the conjunctival route. IMPORTANCE This study is the first to examine the Kikwit strain of EBOV, the most commonly used strain, in the gold-standard macaque model of infection. Additionally, this is the first description of the detection of virus in the vitreous fluid, an immune privileged site that has been proposed as a viral reservoir, following conjunctival challenge. The oral and conjunctival macaque challenge model of EVD described here more faithfully recapitulates the prodrome that has been reported for human EVD. This work paves the way for more advanced studies to model contact transmission of EVD, including early events in mucosal infection and immunity, as well as the establishment of persistent viral infection and the emergence from these reservoirs.

Rapid detection of Ebolavirus using isothermal recombinase-aided amplification.

Ebolavirus disease (EVD) is an often-lethal disease caused by the genus Ebolavirus (EBOV). Although vaccines are being developed and recently used, outbreak control still relies on a combination of various factors, including rapid identification of EVD cases. This allows rapid patient isolation and control measure implementation. Ebolavirus diagnosis is performed in treatment centers or reference laboratories, which usually takes a few hours to days to confirm the outbreak or deliver a clear result. A fast and field-deployable molecular detection method, such as the isothermal amplification recombinase-aided amplification (RAA), could significantly reduce sample-to-result time. In this study, a RT-RAA assay was evaluated for EBOV detection. Various primer and probe combinations were screened; analytical sensitivity and cross-specificity were tested. A total of 40 archived samples from the 2014 to 2016 Ebola outbreak in West Africa were tested with both the reference method real-time RT-PCR and the established RT-RAA assay. The assay could detect down to 22.6 molecular copies per microliter. No other pathogens were detected with the Ebolavirus RT-RAA assay. Testing 40 samples yield clinical sensitivity and specificity of 100% each. This rapid isothermal RT-RAA assay can replace the previous RT-RPA and continue to offer rapid EBOV diagnostics.

Pathogenicity of Lloviu and Bombali Viruses in Type I Interferon Receptor Knockout Mice.

Type I interferon receptor knockout (IFNAR-/-) mice are not able to generate a complete innate immune response; therefore, these mice are often considered to assess the pathogenicity of emerging viruses. We infected IFNAR-/- mice with a low or high dose of Lloviu virus (LLOV) or Bombali virus (BOMV) by the intranasal (IN) or intraperitoneal (IP) route and compared virus loads at early and late time points after infection. No signs of disease and no viral RNA were detected after IN infection regardless of LLOV dose. In contrast, IP infections resulted in increased viral loads in the high-dose LLOV and BOMV groups at the early time point. The low-dose LLOV and BOMV groups achieved higher viral loads at the late time point. However, there was 100% survival in all groups and no signs of disease. In conclusion, our results indicate a limited value of the IFNAR-/- mouse model for investigation of the pathogenicity of LLOV and BOMV.

The Multiple Origins of Ebola Disease Outbreaks.

BACKGROUND: The origins of Ebola disease outbreaks remain enigmatic. Historically outbreaks have been attributed to spillover events from wildlife. However, recent data suggest that some outbreaks may originate from human-to-human transmission of prior outbreak strains instead of spillover. Clarifying the origins of Ebola disease outbreaks could improve detection and mitigation of future outbreaks. METHODS: We reviewed the origins of all Ebola disease outbreaks from 1976 to 2022 to analyze the earliest cases and characteristics of each outbreak. The epidemiology and phylogenetic relationships of outbreak strains were used to further identify the likely source of each outbreak. RESULTS: From 1976 to 2022 there were 35 Ebola disease outbreaks with 48 primary/index cases. While the majority of outbreaks were associated with wildlife spillover, resurgence of human-to-human transmission could account for roughly a quarter of outbreaks caused by Ebola virus. Larger outbreaks were more likely to lead to possible resurgence, and nosocomial transmission was associated with the majority of outbreaks. CONCLUSIONS: While spillover from wildlife has been a source for many Ebola disease outbreaks, multiple outbreaks may have originated from flare-ups of prior outbreak strains. Improving access to diagnostics as well as identifying groups at risk for resurgence of ebolaviruses will be crucial to preventing future outbreaks.

Structural and Energetic Basis for Differential Binding of Ebola and Marburg Virus Glycoproteins to a Bat-Derived Niemann-Pick C1 Protein.

BACKGROUND: Our previous study demonstrated that the fruit bat (Yaeyama flying fox)-derived cell line FBKT1 showed preferential susceptibility to Ebola virus (EBOV), whereas the human cell line HEK293T was similarly susceptible to EBOV and Marburg virus (MARV). This was due to 3 amino acid differences of the endosomal receptor Niemann-Pick C1 (NPC1) between FBKT1 and HEK293T (ie, TET and SGA, respectively, at positions 425-427), as well as 2 amino acid differences at positions 87 and 142 of the viral glycoprotein (GP) between EBOV and MARV. METHODS/RESULTS: To understand the contribution of these amino acid differences to interactions between NPC1 and GP, we performed molecular dynamics simulations and binding free energy calculations. The average binding free energies of human NPC1 (hNPC1) and its mutant having TET at positions 425-427 (hNPC1/TET) were similar for the interaction with EBOV GP. In contrast, hNPC1/TET had a weaker interaction with MARV GP than wild-type hNPC1. As expected, substitutions of amino acid residues at 87 or 142 in EBOV and MARV GPs converted the binding affinity to hNPC1/TET. CONCLUSIONS: Our data provide structural and energetic insights for understanding potential differences in the GP-NPC1 interaction, which could influence the host tropism of EBOV and MARV.

In silico analyses and experimental validation of the MHC class-I restricted epitopes of Ebolavirus GP.

Ebolavirus (EBOV) causes an extremely high mortality and prevalence disease called Ebola virus disease (EVD). There is only one glycoprotein (GP) on the virus particle surface, which mediates entry into the host cell. Major histocompatibility complex (MHC) class-I restricted cluster of differentiation 8 (CD8+) T cell responses are important antiviral immune responses. Therefore, it is of great importance to understand EBOV GP-specific MHC class-I restricted epitopes within immunogenicity. In this study, computational approaches were employed to predict the dominant MHC class-I molecule epitopes of EBOV GP for mouse H2 and major alleles of human leukocyte antigen (HLA) class-I supertypes. Our results yielded 42 dominant epitopes in H2 haplotypes and 301 dominant epitopes in HLA class-I haplotypes. After validation by enzyme-linked immunospot (ELISpot) assay, in-depth analyses to ascertain their nature of conservation, immunogenicity, and docking with the corresponding MHC class-I molecules were undertaken. Our study predicted MHC class-I restricted epitopes that may aid the advancement of anti-EBOV immune responses. An integrated strategy of epitope prediction, validation and comparative analyses was postulated, which is promising for epitope-based immunotherapy development and application to viral epidemics.

Development of an antibody cocktail for treatment of Sudan virus infection.

Antibody-based therapies are a promising treatment option for managing ebolavirus infections. Several Ebola virus (EBOV)-specific and, more recently, pan-ebolavirus antibody cocktails have been described. Here, we report the development and assessment of a Sudan virus (SUDV)-specific antibody cocktail. We produced a panel of SUDV glycoprotein (GP)-specific human chimeric monoclonal antibodies (mAbs) using both plant and mammalian expression systems and completed head-to-head in vitro and in vivo evaluations. Neutralizing activity, competitive binding groups, and epitope specificity of SUDV mAbs were defined before assessing protective efficacy of individual mAbs using a mouse model of SUDV infection. Of the mAbs tested, GP base-binding mAbs were more potent neutralizers and more protective than glycan cap- or mucin-like domain-binding mAbs. No significant difference was observed between plant and mammalian mAbs in any of our in vitro or in vivo evaluations. Based on in vitro and rodent testing, a combination of two SUDV-specific mAbs, one base binding (16F6) and one glycan cap binding (X10H2), was down-selected for assessment in a macaque model of SUDV infection. This cocktail, RIID F6-H2, provided protection from SUDV infection in rhesus macaques when administered at 50 mg/kg on days 4 and 6 postinfection. RIID F6-H2 is an effective postexposure SUDV therapy and provides a potential treatment option for managing human SUDV infection.

Bombali Ebolavirus in Mops condylurus Bats (Molossidae), Mozambique.

We detected Bombali ebolavirus RNA in 3 free-tailed bats (Mops condylurus, Molossidae) in Mozambique. Sequencing of the large protein gene revealed 98% identity with viruses previously detected in Sierra Leone, Kenya, and Guinea. Our findings further support the suspected role of Mops condylurus bats in maintaining Bombali ebolavirus.

Correlates of vaccine-induced protective immunity against Ebola virus disease.

Ebola virus disease is a deadly infection which occurs in sporadic outbreaks. Several vaccine candidates have been developed. The most advanced candidate is the recombinant VSVΔG-ZEBOV-GP vaccine, in which the Vesicular Stomatitis Virus (VSV) envelope glycoprotein is replaced by the Zaire strain Ebola virus (ZEBOV) glycoprotein (GP). This vaccine demonstrated 100% protection in a ring vaccination trial performed in Guinea in 2015, was granted "Breakthrough Therapy Designation" by the FDA and PRIority Medicines (PRIME), and is currently (June 2018) used to support outbreak control in Democratic Republic of Congo. rVSVΔG-ZEBOV-GP elicits a strong and durable antibody response in most vaccinees. This sustained Ebola GP-specific antibody response correlates with an early activation of innate immunity, especially of monocytes and of type-I interferon induced genes. Despite significant progress in the characterization of vaccine-induced immunity, human correlates of protection against Ebolavirus infection have not yet been fully established. A systems biology approach, integrating clinical, immunological, transcriptomic and metabolomic data from pre-clinical and clinical vaccine studies, together with data from disease survivors, will be instrumental to identify Ebola vaccine correlates of protection. The information generated for the rVSVΔG-ZEBOV-GP vaccine may also help identify the correlates of protection of the other Ebola vaccine candidates.

Ebola vaccination in the Democratic Republic of the Congo.

Following the April 2018 reemergence of Ebola in a rural region of the Democratic Republic of the Congo (DRC), the virus spread to an urban center by early May. Within 2 wk of the first case confirmation, a vaccination campaign was initiated in which 3,017 doses were administered to contacts of cases and frontline healthcare workers. To evaluate the spatial dynamics of Ebola transmission and quantify the impact of vaccination, we developed a geographically explicit model that incorporates high-resolution data on poverty and population density. We found that while Ebola risk was concentrated around sites initially reporting infections, longer-range dissemination also posed a risk to areas with high population density and poverty. We estimate that the vaccination program contracted the geographical area at risk for Ebola by up to 70.4% and reduced the level of risk within that region by up to 70.1%. The early implementation of vaccination was critical. A delay of even 1 wk would have reduced these effects to 33.3 and 44.8%, respectively. These results underscore the importance of the rapid deployment of Ebola vaccines during emerging outbreaks to containing transmission and preventing global spread. The spatiotemporal framework developed here provides a tool for identifying high-risk regions, in which surveillance can be intensified and preemptive control can be implemented during future outbreaks.

Ebolavirus polymerase uses an unconventional genome replication mechanism.

Most nonsegmented negative strand (NNS) RNA virus genomes have complementary 3' and 5' terminal nucleotides because the promoters at the 3' ends of the genomes and antigenomes are almost identical to each other. However, according to published sequences, both ends of ebolavirus genomes show a high degree of variability, and the 3' and 5' terminal nucleotides are not complementary. If correct, this would distinguish the ebolaviruses from other NNS RNA viruses. Therefore, we investigated the terminal genomic and antigenomic nucleotides of three different ebolavirus species, Ebola (EBOV), Sudan, and Reston viruses. Whereas the 5' ends of ebolavirus RNAs are highly conserved with the sequence ACAGG-5', the 3' termini are variable and are typically 3'-GCCUGU, ACCUGU, or CCUGU. A small fraction of analyzed RNAs had extended 3' ends. The majority of 3' terminal sequences are consistent with a mechanism of nucleotide addition by hairpin formation and back-priming. Using single-round replicating EBOV minigenomes, we investigated the effect of the 3' terminal nucleotide on viral replication and found that the EBOV polymerase initiates replication opposite the 3'-CCUGU motif regardless of the identity of the 3' terminal nucleotide(s) and of the position of this motif relative to the 3' end. Deletion or mutation of the first residue of the 3'-CCUGU motif completely abolished replication initiation, suggesting a crucial role of this nucleotide in directing initiation. Together, our data show that ebolaviruses have evolved a unique replication strategy among NNS RNA viruses resulting in 3' overhangs. This could be a mechanism to avoid antiviral recognition.

Remdesivir against COVID-19 and Other Viral Diseases.

Patients and physicians worldwide are facing tremendous health care hazards that are caused by the ongoing severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) pandemic. Remdesivir (GS-5734) is the first approved treatment for severe coronavirus disease 2019 (COVID-19). It is a novel nucleoside analog with a broad antiviral activity spectrum among RNA viruses, including ebolavirus (EBOV) and the respiratory pathogens Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, and SARS-CoV-2. First described in 2016, the drug was derived from an antiviral library of small molecules intended to target emerging pathogenic RNA viruses. In vivo, remdesivir showed therapeutic and prophylactic effects in animal models of EBOV, MERS-CoV, SARS-CoV, and SARS-CoV-2 infection. However, the substance failed in a clinical trial on ebolavirus disease (EVD), where it was inferior to investigational monoclonal antibodies in an interim analysis. As there was no placebo control in this study, no conclusions on its efficacy in EVD can be made. In contrast, data from a placebo-controlled trial show beneficial effects for patients with COVID-19. Remdesivir reduces the time to recovery of hospitalized patients who require supplemental oxygen and may have a positive impact on mortality outcomes while having a favorable safety profile. Although this is an important milestone in the fight against COVID-19, approval of this drug will not be sufficient to solve the public health issues caused by the ongoing pandemic. Further scientific efforts are needed to evaluate the full potential of nucleoside analogs as treatment or prophylaxis of viral respiratory infections and to develop effective antivirals that are orally bioavailable.

Novel Use of Capture-Recapture Methods to Estimate Completeness of Contact Tracing during an Ebola Outbreak, Democratic Republic of the Congo, 2018-2020.

Despite its critical role in containing outbreaks, the efficacy of contact tracing, measured as the sensitivity of case detection, remains an elusive metric. We estimated the sensitivity of contact tracing by applying unilist capture-recapture methods on data from the 2018-2020 outbreak of Ebola virus disease in the Democratic Republic of the Congo. To compute sensitivity, we applied different distributional assumptions to the zero-truncated count data to estimate the number of unobserved case-patients with any contacts and infected contacts. Geometric distributions were the best-fitting models. Our results indicate that contact tracing efforts identified almost all (n = 792, 99%) of case-patients with any contacts but only half (n = 207, 48%) of case-patients with infected contacts, suggesting that contact tracing efforts performed well at identifying contacts during the listing stage but performed poorly during the contact follow-up stage. We discuss extensions to our work and potential applications for the ongoing coronavirus pandemic.

Increased Likelihood of Detecting Ebola Virus RNA in Semen by Using Sample Pelleting.

Ebola virus RNA can reside for months or years in semen of survivors of Ebola virus disease and is probably associated with increased risk for cryptic sexual transmission of the virus. A modified protocol resulted in increased detection of Ebola virus RNA in semen and improved disease surveillance.