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.

Potent Henipavirus Neutralization by Antibodies Recognizing Diverse Sites on Hendra and Nipah Virus Receptor Binding Protein.

Hendra (HeV) and Nipah (NiV) viruses are emerging zoonotic pathogens in the Henipavirus genus causing outbreaks of disease with very high case fatality rates. Here, we report the first naturally occurring human monoclonal antibodies (mAbs) against HeV receptor binding protein (RBP). All isolated mAbs neutralized HeV, and some also neutralized NiV. Epitope binning experiments identified five major antigenic sites on HeV-RBP. Animal studies demonstrated that the most potent cross-reactive neutralizing mAbs, HENV-26 and HENV-32, protected ferrets in lethal models of infection with NiV Bangladesh 3 days after exposure. We solved the crystal structures of mAb HENV-26 in complex with both HeV-RBP and NiV-RBP and of mAb HENV-32 in complex with HeV-RBP. The studies reveal diverse sites of vulnerability on RBP recognized by potent human mAbs that inhibit virus by multiple mechanisms. These studies identify promising prophylactic antibodies and define protective epitopes that can be used in rational vaccine design.

Establishment of an African green monkey model for COVID-19 and protection against re-infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for an unprecedented global pandemic of COVID-19. Animal models are urgently needed to study the pathogenesis of COVID-19 and to screen vaccines and treatments. We show that African green monkeys (AGMs) support robust SARS-CoV-2 replication and develop pronounced respiratory disease, which may more accurately reflect human COVID-19 cases than other nonhuman primate species. SARS-CoV-2 was detected in mucosal samples, including rectal swabs, as late as 15 days after exposure. Marked inflammation and coagulopathy in blood and tissues were prominent features. Transcriptome analysis demonstrated stimulation of interferon and interleukin-6 pathways in bronchoalveolar lavage samples and repression of natural killer cell- and T cell-associated transcripts in peripheral blood. Despite a slight waning in antibody titers after primary challenge, enhanced antibody and cellular responses contributed to rapid clearance after re-challenge with an identical strain. These data support the utility of AGM for studying COVID-19 pathogenesis and testing medical countermeasures.

A human monoclonal antibody combination rescues nonhuman primates from advanced disease caused by the major lineages of Lassa virus.

There are no approved treatments for Lassa fever (LF), which is responsible for thousands of deaths each year in West Africa. A major challenge in developing effective medical countermeasures against LF is the high diversity of circulating Lassa virus (LASV) strains with four recognized lineages and four proposed lineages. The recent resurgence of LASV in Nigeria caused by genetically distinct strains underscores this concern. Two LASV lineages (II and III) are dominant in Nigeria. Here, we show that combinations of two or three pan-lineage neutralizing human monoclonal antibodies (8.9F, 12.1F, 37.D) known as Arevirumab-2 or Arevirumab-3 can protect up to 100% of cynomolgus macaques against challenge with both lineage II and III LASV isolates when treatment is initiated at advanced stages of disease on day 8 after LASV exposure. This work demonstrates that it may be possible to develop postexposure interventions that can broadly protect against most strains of LASV.

A recombinant VSV-vectored vaccine rapidly protects nonhuman primates against lethal Nipah virus disease.

SignificanceConcern has increased about the pandemic potential of Nipah virus (NiV). Similar to SARS-CoV-2, NiV is an RNA virus that is transmitted by respiratory droplets. There are currently no NiV vaccines licensed for human use. While several preventive vaccines have shown promise in protecting animals against lethal NiV disease, most studies have assessed protection 1 mo after vaccination. However, in order to contain and control outbreaks, vaccines that can rapidly confer protection in days rather than months are needed. Here, we show that a recombinant vesicular stomatitis virus vector expressing the NiV glycoprotein can completely protect monkeys vaccinated 7 d prior to NiV exposure and 67% of animals vaccinated 3 d before NiV challenge.

Combination treatment of mannose and GalNAc conjugated small interfering RNA protects against lethal Marburg virus infection.

Marburg virus (MARV) infection results in severe viral hemorrhagic fever with mortalities up to 90%, and there is a pressing need for effective therapies. Here, we established a small interfering RNA (siRNA) conjugate platform that enabled successful subcutaneous delivery of siRNAs targeting the MARV nucleoprotein. We identified a hexavalent mannose ligand with high affinity to macrophages and dendritic cells, which are key cellular targets of MARV infection. This ligand enabled successful siRNA conjugate delivery to macrophages both in vitro and in vivo. The delivered hexa-mannose-siRNA conjugates rendered substantial target gene silencing in macrophages when supported by a mannose functionalized endosome release polymer. This hexa-mannose-siRNA conjugate was further evaluated alongside our hepatocyte-targeting GalNAc-siRNA conjugate, to expand targeting of infected liver cells. In MARV-Angola-infected guinea pigs, these platforms offered limited survival benefit when used as individual agents. However, in combination, they achieved up to 100% protection when dosed 24 h post infection. This novel approach, using two different ligands to simultaneously deliver siRNA to multiple cell types relevant to infection, provides a convenient subcutaneous route of administration for treating infection by these dangerous pathogens. The mannose conjugate platform has potential application to other diseases involving macrophages and dendritic cells.

Therapy for Argentine hemorrhagic fever in nonhuman primates with a humanized monoclonal antibody.

The COVID-19 pandemic has reemphasized the need to identify safe and scalable therapeutics to slow or reverse symptoms of disease caused by newly emerging and reemerging viral pathogens. Recent clinical successes of monoclonal antibodies (mAbs) in therapy for viral infections demonstrate that mAbs offer a solution for these emerging biothreats. We have explored this with respect to Junin virus (JUNV), an arenavirus classified as a category A high-priority agent and the causative agent of Argentine hemorrhagic fever (AHF). There are currently no Food and Drug Administration-approved drugs available for preventing or treating AHF, although immune plasma from convalescent patients is used routinely to treat active infections. However, immune plasma is severely limited in quantity, highly variable in quality, and poses significant safety risks including the transmission of transfusion-borne diseases. mAbs offer a highly specific and consistently potent alternative to immune plasma that can be manufactured at large scale. We previously described a chimeric mAb, cJ199, that provided protection in a guinea pig model of AHF. To adapt this mAb to a format more suitable for clinical use, we humanized the mAb (hu199) and evaluated it in a cynomolgus monkey model of AHF with two JUNV isolates, Romero and Espindola. While untreated control animals experienced 100% lethality, all animals treated with hu199 at 6 d postinoculation (dpi) survived, and 50% of animals treated at 8 dpi survived. mAbs like hu199 may offer a safer, scalable, and more reproducible alternative to immune plasma for rare viral diseases that have epidemic potential.

The Mucin-Like Domain of the Ebola Glycoprotein Does Not Impact Virulence or Pathogenicity in Ferrets.

BACKGROUND: Ebola virus (EBOV) is considered among the most dangerous viruses with case fatality rates approaching 90% depending on the outbreak. While several viral proteins (VPs) including VP24, VP35, and the soluble glycoprotein are understood to contribute to virulence, less is known of the contribution of the highly variable mucin-like domain (MLD) of EBOV. Early studies have defined a potential role in immune evasion of the MLD by providing a glycan shield to critical glycoprotein residues tied to viral entry. Nonetheless, little is known as to what direct role the MLD plays in acute EBOV disease (EVD). METHODS: We generated an infectious EBOV clone that lacks the MLD and assessed its virulence in ferrets compared with wild-type (WT) virus. RESULTS: No differences in growth kinetics were observed in vitro, nor were there any differences in time to death, viremia, or clinical picture in ferrets infected with recombinant EBOV (rEBOV)-WT or rEBOV-Δmucin. CONCLUSIONS: The EBOV MLD does not play a critical role in acute pathogenesis of EVD in ferrets.

A Recombinant Vesicular Stomatitis Virus-Based Vaccine Provides Postexposure Protection Against Bundibugyo Ebolavirus Infection.

BACKGROUND: The filovirus Bundibugyo virus (BDBV) causes severe disease with a mortality rate of approximately 20%-51%. The only licensed filovirus vaccine in the United States, Ervebo, consists of a recombinant vesicular stomatitis virus (rVSV) vector that expresses Ebola virus (EBOV) glycoprotein (GP). Ervebo was shown to rapidly protect against fatal Ebola disease in clinical trials; however, the vaccine is only indicated against EBOV. Recent outbreaks of other filoviruses underscore the need for additional vaccine candidates, particularly for BDBV infections. METHODS: To examine whether the rVSV vaccine candidate rVSVΔG/BDBV-GP could provide therapeutic protection against BDBV, we inoculated seven cynomolgus macaques with 1000 plaque-forming units of BDBV, administering rVSVΔG/BDBV-GP vaccine to 6 of them 20-23 minutes after infection. RESULTS: Five of the treated animals survived infection (83%) compared to an expected natural survival rate of 21% in this macaque model. All treated animals showed an early circulating immune response, while the untreated animal did not. Surviving animals showed evidence of both GP-specific IgM and IgG production, while animals that succumbed did not produce significant IgG. CONCLUSIONS: This small, proof-of-concept study demonstrated early treatment with rVSVΔG/BDBV-GP provides a survival benefit in this nonhuman primate model of BDBV infection, perhaps through earlier initiation of adaptive immunity.

Natural History of Nonhuman Primates After Oral Exposure to Ebola Virus Variant Makona.

BACKGROUND: The primary route of infection by Ebola virus (EBOV) is through contact of mucosal surfaces. Few studies have explored infection of nonhuman primates (NHPs) via the oral mucosa, which is a probable portal of natural infection in humans. METHODS: To further characterize the pathogenesis of EBOV infection via the oral exposure route, we challenged cohorts of cynomolgus monkeys with low doses of EBOV variant Makona. RESULTS: Infection with 100 or 50 PFU of EBOV Makona via the oral route resulted in 50% and 83% lethality, respectively. Animals that progressed to fatal disease exhibited lymphopenia, marked coagulopathy, high viral loads, and increased levels of serum markers of inflammation and hepatic/renal injury. Survival in these cohorts was associated with milder fluctuations in leukocyte populations, lack of coagulopathy, and reduced or absent serum markers of inflammation and/or hepatic/renal function. Surprisingly, 2 surviving animals from the 100- and 50-PFU cohorts developed transient low-level viremia in the absence of other clinical signs of disease. Conversely, all animals in the 10 PFU cohort remained disease free and survived to the study end point. CONCLUSIONS: Our observations highlight the susceptibility of NHPs, and by extension, likely humans, to relatively low doses of EBOV via the oral route.

Pathogenesis of Aerosolized Ebola Virus Variant Makona in Nonhuman Primates.

BACKGROUND: Highly pathogenic filoviruses such as Ebola virus (EBOV) hold capacity for delivery by artificial aerosols, and thus potential for intentional misuse. Previous studies have shown that high doses of EBOV delivered by small-particle aerosol cause uniform lethality in nonhuman primates (NHPs), whereas only a few small studies have assessed lower doses in NHPs. METHODS: To further characterize the pathogenesis of EBOV infection via small-particle aerosol, we challenged cohorts of cynomolgus monkeys with low doses of EBOV variant Makona, which may help define risks associated with small particle aerosol exposures. RESULTS: Despite using challenge doses orders of magnitude lower than previous studies, infection via this route was uniformly lethal across all cohorts. Time to death was delayed in a dose-dependent manner between aerosol-challenged cohorts, as well as in comparison to animals challenged via the intramuscular route. Here, we describe the observed clinical and pathological details including serum biomarkers, viral burden, and histopathological changes leading to death. CONCLUSIONS: Our observations in this model highlight the striking susceptibility of NHPs, and likely humans, via small-particle aerosol exposure to EBOV and emphasize the need for further development of diagnostics and postexposure prophylactics in the event of intentional release via deployment of an aerosol-producing device.

A Highly Attenuated Panfilovirus VesiculoVax Vaccine Rapidly Protects Nonhuman Primates Against Marburg Virus and 3 Species of Ebola Virus.

BACKGROUND: The family Filoviridae consists of several virus members known to cause significant mortality and disease in humans. Among these, Ebola virus (EBOV), Marburg virus (MARV), Sudan virus (SUDV), and Bundibugyo virus (BDBV) are considered the deadliest. The vaccine, Ervebo, was shown to rapidly protect humans against Ebola disease, but is indicated only for EBOV infections with limited cross-protection against other filoviruses. Whether multivalent formulations of similar recombinant vesicular stomatitis virus (rVSV)-based vaccines could likewise confer rapid protection is unclear. METHODS: Here, we tested the ability of an attenuated, quadrivalent panfilovirus VesiculoVax vaccine (rVSV-Filo) to elicit fast-acting protection against MARV, EBOV, SUDV, and BDBV. Groups of cynomolgus monkeys were vaccinated 7 days before exposure to each of the 4 viral pathogens. All subjects (100%) immunized 1 week earlier survived MARV, SUDV, and BDBV challenge; 80% survived EBOV challenge. Survival correlated with lower viral load, higher glycoprotein-specific immunoglobulin G titers, and the expression of B-cell-, cytotoxic cell-, and antigen presentation-associated transcripts. CONCLUSIONS: These results demonstrate multivalent VesiculoVax vaccines are suitable for filovirus outbreak management. The highly attenuated nature of the rVSV-Filo vaccine may be preferable to the Ervebo "delta G" platform, which induced adverse events in a subset of recipients.

Long-term Prophylaxis Against Aerosolized Marburg Virus in Nonhuman Primates With an Afucosylated Monoclonal Antibody.

Marburg virus (MARV) causes a hemorrhagic fever disease in human and nonhuman primates with high levels of morbidity and mortality. Concerns about weaponization of aerosolized MARV have spurred the development of nonhuman primate (NHP) models of aerosol exposure. To address the potential threat of aerosol exposure, a monoclonal antibody that binds MARV glycoprotein was tested, MR186YTE, for its efficacy as a prophylactic. MR186YTE was administered intramuscularly to NHPs at 15 or 5 mg/kg 1 month prior to MARV aerosol challenge. Seventy-five percent (3/4) of the 15 mg/kg dose group and 50% (2/4) of the 5 mg/kg dose group survived. Serum analyses showed that the NHP dosed with 15 mg/kg that succumbed to infection developed an antidrug antibody response and therefore had no detectable MR186YTE at the time of challenge. These results suggest that intramuscular dosing of mAbs may be a clinically useful prophylaxis for MARV aerosol exposure.

A Lethal Aerosol Exposure Model of Nipah Virus Strain Bangladesh in African Green Monkeys.

The high case-fatality rates and potential for use as a biological weapon make Nipah virus (NiV) a significant public health concern. Previous studies assessing the pathogenic potential of NiV delivered by the aerosol route in African green monkeys (AGMs) used the Malaysia strain (NiVM), which has caused lower instances of respiratory illness and person-to-person transmission during human outbreaks than the Bangladesh strain (NiVB). Accordingly, we developed a small particle aerosol model of NiVB infection in AGMs. Consistent with other mucosal AGM models of NiVB infection, we achieved uniform lethality and disease pathogenesis reflective of that observed in humans.

An Intranasal Exposure Model of Lethal Nipah Virus Infection in African Green Monkeys.

Due to the difficulty in conducting clinical trials for vaccines and treatments against Nipah virus (NiV), licensure will likely require animal models, most importantly non-human primates (NHPs). The NHP models of infection have primarily relied on intratracheal instillation or small particle aerosolization of NiV. However, neither of these routes adequately models natural mucosal exposure to NiV. To develop a more natural NHP model, we challenged African green monkeys with the Bangladesh strain of NiV by the intranasal route using the laryngeal mask airway (LMA) mucosal atomization device (MAD). LMA MAD exposure resulted in uniformly lethal disease that accurately reflected the human condition.

A Cross-Reactive Humanized Monoclonal Antibody Targeting Fusion Glycoprotein Function Protects Ferrets Against Lethal Nipah Virus and Hendra Virus Infection.

BACKGROUND: Nipah virus (NiV) and Hendra virus (HeV) are zoonotic paramyxoviruses that cause severe disease in both animals and humans. There are no approved vaccines or treatments for use in humans; however, therapeutic treatment of both NiV and HeV infection in ferrets and non-human primates with a cross-reactive, neutralizing human monoclonal antibody (mAb), m102.4, targeting the G glycoprotein has been demonstrated. In a previous study, we isolated, characterized, and humanized a cross-reactive, neutralizing anti-F mAb (h5B3.1). The mAb h5B3.1 blocks the required F conformational change needed to facilitate membrane fusion and virus infection, and the epitope recognized by h5B3.1 has been structurally defined; however, the efficacy of h5B3.1 in vivo is unknown. METHODS: The post-infection antiviral activity of h5B3.1 was evaluated in vivo by administration in ferrets after NiV and HeV virus challenge. RESULTS: All subjects that received h5B3.1 from 1 to several days after infection with a high-dose, oral-nasal virus challenge were protected from disease, whereas all controls died. CONCLUSIONS: This is the first successful post-exposure antibody therapy for NiV and HeV using a humanized cross-reactive mAb targeting the F glycoprotein, and the findings suggest that a combination therapy targeting both F and G should be evaluated as a therapy for NiV/HeV infection.

Resistance of Cynomolgus Monkeys to Nipah and Hendra Virus Disease Is Associated With Cell-Mediated and Humoral Immunity.

BACKGROUND: The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are capable of causing severe and often lethal respiratory and/or neurologic disease in animals and humans. Given the sporadic nature of henipavirus outbreaks, licensure of vaccines and therapeutics for human use will likely require demonstration of efficacy in animal models that faithfully reproduce the human condition. Currently, the African green monkey (AGM) best mimics human henipavirus-induced disease. METHODS: The pathogenic potential of HeV and both strains of NiV (Malaysia, Bangladesh) was assessed in cynomolgus monkeys and compared with henipavirus-infected historical control AGMs. Multiplex gene and protein expression assays were used to compare host responses. RESULTS: In contrast to AGMs, in which henipaviruses cause severe and usually lethal disease, HeV and NiVs caused only mild or asymptomatic infections in macaques. All henipaviruses replicated in macaques with similar kinetics as in AGMs. Infection in macaques was associated with activation and predicted recruitment of cytotoxic CD8+ T cells, Th1 cells, IgM+ B cells, and plasma cells. Conversely, fatal outcome in AGMs was associated with aberrant innate immune signaling, complement dysregulation, Th2 skewing, and increased secretion of MCP-1. CONCLUSION: The restriction factors identified in macaques can be harnessed for development of effective countermeasures against henipavirus disease.

Intranasal exposure of African green monkeys to SARS-CoV-2 results in acute phase pneumonia with shedding and lung injury still present in the early convalescence phase.

We recently reported the development of the first African green monkey (AGM) model for COVID-19 based on a combined liquid intranasal (i.n.) and intratracheal (i.t.) exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we followed up on this work by assessing an i.n. particle only route of exposure using the LMA mucosal atomization device (MAD). Six AGMs were infected with SARS-CoV-2; three animals were euthanized near the peak stage of virus replication (day 5) and three animals were euthanized during the early convalescence period (day 34). All six AGMs supported robust SARS-CoV-2 replication and developed respiratory disease. Evidence of coagulation dysfunction as noted by a transient increases in aPTT and circulating levels of fibrinogen was observed in all AGMs. The level of SARS-CoV-2 replication and lung pathology was not quite as pronounced as previously reported with AGMs exposed by the combined i.n. and i.t. routes; however, SARS-CoV-2 RNA was detected in nasal swabs of some animals as late as day 15 and rectal swabs as late as day 28 after virus challenge. Of particular importance to this study, all three AGMs that were followed until the early convalescence stage of COVID-19 showed substantial lung pathology at necropsy as evidenced by multifocal chronic interstitial pneumonia and increased collagen deposition in alveolar walls despite the absence of detectable SARS-CoV-2 in any of the lungs of these animals. These findings are consistent with human COVID-19 further demonstrating that the AGM faithfully reproduces the human condition.