Therapeutic administration of a cross-reactive mAb targeting the fusion glycoprotein of Nipah virus protects nonhuman primates.

No licensed vaccines or therapies exist for patients infected with Nipah virus (NiV), although an experimental human monoclonal antibody (mAb) cross-reactive to the NiV and Hendra virus (HeV) G glycoprotein, m102.4, has been tested in a phase 1 trial and has been provided under compassionate use for both HeV and NiV exposures. NiV is a highly pathogenic zoonotic paramyxovirus causing regular outbreaks in humans and animals in South and Southeast Asia. The mortality rate of NiV infection in humans ranges from 40% to more than 90%, making it a substantial public health concern. The NiV G glycoprotein mediates host cell attachment, and the F glycoprotein facilitates membrane fusion and infection. We hypothesized that a mAb against the prefusion conformation of the F glycoprotein may confer better protection than m102.4. To test this, two potent neutralizing mAbs against NiV F protein, hu1F5 and hu12B2, were compared in a hamster model. Hu1F5 provided superior protection to hu12B2 and was selected for comparison with m102.4 for the ability to protect African green monkeys (AGMs) from a stringent NiV challenge. AGMs were exposed intranasally to the Bangladesh strain of NiV and treated 5 days after exposure with either mAb (25 milligrams per kilogram). Whereas only one of six AGMs treated with m102.4 survived until the study end point, all six AGMs treated with hu1F5 were protected. Furthermore, a reduced 10 milligrams per kilogram dose of hu1F5 also provided complete protection against NiV challenge, supporting the upcoming clinical advancement of this mAb for postexposure prophylaxis and therapy.

Oral administration of obeldesivir protects nonhuman primates against Sudan ebolavirus.

Obeldesivir (ODV, GS-5245) is an orally administered prodrug of the parent nucleoside of remdesivir (RDV) and is presently in phase 3 trials for COVID-19 treatment. In this work, we show that ODV and its circulating parent nucleoside metabolite, GS-441524, have similar in vitro antiviral activity against filoviruses, including Marburg virus, Ebola virus, and Sudan virus (SUDV). We also report that once-daily oral ODV treatment of cynomolgus monkeys for 10 days beginning 24 hours after SUDV exposure confers 100% protection against lethal infection. Transcriptomics data show that ODV treatment delayed the onset of inflammation and correlated with antigen presentation and lymphocyte activation. Our results offer promise for the further development of ODV to control outbreaks of filovirus disease more rapidly.

Monoclonal antibody therapy protects nonhuman primates against mucosal exposure to Lassa virus.

Lassa fever (LF) is an acute viral illness that causes thousands of deaths annually in West Africa. There are currently no Lassa virus (LASV) vaccines or antivirals approved for human use. Recently, we showed that combinations of broadly neutralizing human monoclonal antibodies (BNhuMAbs) known as Arevirumab-2 or Arevirumab-3 protected up to 100% of cynomolgus macaques against challenge with diverse lineages of LASV when treatment was initiated at advanced stages of disease. This previous work assessed efficacy against parenteral exposure. However, transmission of LASV to humans occurs primarily by mucosal exposure to virus shed from Mastomys rodents. Here, we describe the development of a lethal intranasal exposure macaque model of LF. This model is employed to show that Arevirumab cocktails rescue 100% of macaques from lethal LASV infection when treatment is initiated 8 days after LASV exposure. Our work demonstrates BNhuMAbs have utility in treating LASV infection acquired through mucosal exposure.

Monoclonal antibody therapy demonstrates increased virulence of a lineage VII strain of Lassa virus in nonhuman primates.

Lassa virus (LASV) is a World Health Organization (WHO) priority pathogen that causes high morbidity and mortality. Recently, we showed that a combination of three broadly neutralizing human monoclonal antibodies known as Arevirumab-3 (8.9F, 12.1F, 37.2D) based on the lineage IV Josiah strain protected 100% of cynomolgus macaques against heterologous challenge with lineage II and III strains of LASV when therapy was initiated beginning at day 8 after challenge. LASV strains from Benin and Togo represent a new lineage VII that are more genetically diverse from lineage IV than strains from lineages II and III. Here, we tested the ability of Arevirumab-3 to protect macaques against a LASV lineage VII Togo isolate when treatment was administered beginning 8 days after exposure. Unexpectedly, only 40% of treated animals survived challenge. In a subsequent study we showed that Arevirumab-3 protected 100% of macaques from lethal challenge when treatment was initiated 7 days after LASV Togo exposure. Based on our transcriptomics data, successful Arevirumab-3 treatment correlated with diminished neutrophil signatures and the predicted development of T cell responses. As the in vitro antiviral activity of Arevirumab-3 against LASV Togo was equivalent to lineage II and III strains, the reduced protection in macaques against Togo likely reflects the faster disease course of LASV Togo in macaques than other strains. This data causes concern regarding the ability of heterologous vaccines and treatments to provide cross protection against lineage VII LASV isolates.

Thermostable bivalent filovirus vaccine protects against severe and lethal Sudan ebolavirus and marburgvirus infection.

Although two vaccines for Zaire ebolavirus (EBOV) have been licensed and deployed successfully to combat recurring outbreaks of Ebolavirus Disease in West Africa, there are no vaccines for two other highly pathogenic members of the Filoviridae, Sudan ebolavirus (SUDV) and Marburg marburgvirus (MARV). The results described herein document the immunogenicity and protective efficacy in cynomolgus macaques of a single-vial, thermostabilized (lyophilized) monovalent (SUDV) and bivalent (SUDV & MARV) protein vaccines consisting of recombinant glycoproteins (GP) formulated with a clinical-grade oil-in-water nanoemulsion adjuvant (CoVaccine HT™). Lyophilized formulations of the vaccines were reconstituted with Water for Injection and used to immunize groups of cynomolgus macaques before challenge with a lethal dose of a human SUDV or MARV isolate. Sera collected after each of the three immunizations showed near maximal GP-binding IgG concentrations starting as early as the second dose. Most importantly, the vaccine candidates (monovalent or bivalent) provided 100% protection against severe and lethal filovirus disease after either SUDV or MARV infection. Although mild, subclinical infection was observed in a few macaques, all vaccinated animals remained healthy and survived the filovirus challenge. These results demonstrate the value that thermostabilized protein vaccines could provide for addressing an important gap in preparedness for future filovirus outbreaks.

The Therapeutic Monoclonal Antibody Bamlanivimab Does Not Enhance SARS-CoV-2 Infection by FcR-Mediated Mechanisms.

As part of the non-clinical safety package characterizing bamlanivimab (SARS-CoV-2 neutralizing monoclonal antibody), the risk profile for antibody-dependent enhancement of infection (ADE) was evaluated in vitro and in an African green monkey (AGM) model of COVID-19. In vitro ADE assays in primary human macrophage, Raji, or THP-1 cells were used to evaluate enhancement of viral infection. Bamlanivimab binding to C1q, FcR, and cell-based effector activity was also assessed. In AGMs, the impact of bamlanivimab pretreatment on viral loads and clinical and histological pathology was assessed to evaluate enhanced SARS-CoV-2 replication or pathology. Bamlanivimab did not increase viral replication in vitro, despite a demonstrated effector function. In vivo, no significant differences were found among the AGM groups for weight, temperature, or food intake. Treatment with bamlanivimab reduced viral loads in nasal and oral swabs and BAL fluid relative to control groups. Viral antigen was not detected in lung tissue from animals treated with the highest dose of bamlanivimab. Bamlanivimab did not induce ADE of SARS-CoV-2 infection in vitro or in an AGM model of infection at any dose evaluated. The findings suggest that high-affinity monoclonal antibodies pose a low risk of mediating ADE in patients and support their safety profile as a treatment of COVID-19 disease.

The Arenaviridae Family: Knowledge Gaps, Animal Models, Countermeasures, and Prototype Pathogens.

Lassa virus (LASV), Junin virus (JUNV), and several other members of the Arenaviridae family are capable of zoonotic transfer to humans and induction of severe viral hemorrhagic fevers. Despite the importance of arenaviruses as potential pandemic pathogens, numerous gaps exist in scientific knowledge pertaining to this diverse family, including gaps in understanding replication, immunosuppression, receptor usage, and elicitation of neutralizing antibody responses, that in turn complicates development of medical countermeasures. A further challenge to the development of medical countermeasures for arenaviruses is the requirement for use of animal models at high levels of biocontainment, where each model has distinct advantages and limitations depending on, availability of space, animals species-specific reagents, and most importantly the ability of the model to faithfully recapitulate human disease. Designation of LASV and JUNV as prototype pathogens can facilitate progress in addressing the public health challenges posed by members of this important virus family.

Modelling Marburg Virus Disease in Syrian Golden Hamsters: Contrasted Virulence Between Angola and Ci67 Strains.

BACKGROUND: Marburg virus (MARV) has caused numerous sporadic outbreaks of severe hemorrhagic fever in humans. Human case fatality rates of Marburg virus disease (MVD) outbreaks range from 20% to 90%. Viral genotypes of MARV can differ by over 20%, suggesting variable virulence between lineages may accompany this genetic divergence. Comparison of existing animal models of MVD employing different strains of MARV support differences in virulence across MARV genetic lineages; however, there are few systematic comparisons in models that recapitulate human disease available. METHODS: We compared features of disease pathogenesis in uniformly lethal hamster models of MVD made possible through serial adaptation in rodents. RESULTS: No further adaptation from a previously reported guinea pig-adapted (GPA) isolate of MARV-Angola was necessary to achieve uniform lethality in hamsters. Three passages of GPA MARV-Ci67 resulted in uniform lethality, where 4 passages of a GPA Ravn virus was 75% lethal. Hamster-adapted MARV-Ci67 demonstrated delayed time to death, protracted weight loss, lower viral burden, and slower histologic alteration compared to GPA MARV-Angola. CONCLUSIONS: These data suggest isolate-dependent virulence differences are maintained even after serial adaptation in rodents and may serve to guide choice of variant and model used for development of vaccines or therapeutics for MVD.

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.

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 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.

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.

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.

A Recombinant Chimeric Cedar Virus-Based Surrogate Neutralization Assay Platform for Pathogenic Henipaviruses.

The henipaviruses, Nipah virus (NiV), and Hendra virus (HeV) can cause fatal diseases in humans and animals, whereas Cedar virus is a nonpathogenic henipavirus. Here, using a recombinant Cedar virus (rCedV) reverse genetics platform, the fusion (F) and attachment (G) glycoprotein genes of rCedV were replaced with those of NiV-Bangladesh (NiV-B) or HeV, generating replication-competent chimeric viruses (rCedV-NiV-B and rCedV-HeV), both with and without green fluorescent protein (GFP) or luciferase protein genes. The rCedV chimeras induced a Type I interferon response and utilized only ephrin-B2 and ephrin-B3 as entry receptors compared to rCedV. The neutralizing potencies of well-characterized cross-reactive NiV/HeV F and G specific monoclonal antibodies against rCedV-NiV-B-GFP and rCedV-HeV-GFP highly correlated with measurements obtained using authentic NiV-B and HeV when tested in parallel by plaque reduction neutralization tests (PRNT). A rapid, high-throughput, and quantitative fluorescence reduction neutralization test (FRNT) using the GFP-encoding chimeras was established, and monoclonal antibody neutralization data derived by FRNT highly correlated with data derived by PRNT. The FRNT assay could also measure serum neutralization titers from henipavirus G glycoprotein immunized animals. These rCedV chimeras are an authentic henipavirus-based surrogate neutralization assay that is rapid, cost-effective, and can be utilized outside high containment.

Natural history of nonhuman primates after conjunctival exposure to Ebola virus.

Transmission of Ebola virus (EBOV) primarily occurs via contact exposure of mucosal surfaces with infected body fluids. Historically, nonhuman primate (NHP) challenge studies have employed intramuscular (i.m.) or small particle aerosol exposure, which are largely lethal routes of infection, but mimic worst-case scenarios such as a needlestick or intentional release, respectively. When exposed by more likely routes of natural infection, limited NHP studies have shown delayed onset of disease and reduced mortality. Here, we performed a series of systematic natural history studies in cynomolgus macaques with a range of conjunctival exposure doses. Challenge with 10,000 plaque forming units (PFU) of EBOV was uniformly lethal, whereas 5/6 subjects survived lower dose challenges (100 or 500 PFU). Conjunctival challenge resulted in a protracted time-to death compared to i.m. Asymptomatic infection was observed in survivors with limited detection of EBOV replication. Inconsistent seropositivity in survivors may suggest physical or natural immunological barriers are sufficient to prevent widespread viral dissemination.

Novel Tools for Lassa Virus Surveillance in Peri-domestic Rodents.

Background: Lassa fever (LF) is a rodent-borne disease endemic to West Africa. In the absence of licensed therapeutics or vaccines, rodent exclusion from living spaces remains the primary method of preventing LF. Zoonotic surveillance of Lassa virus (LASV), the etiologic agent of LF, can assess the burden of LASV in a region and guide public health measures against LF. Methods: In this study, we adapted commercially available LASV human diagnostics to assess the prevalence of LASV in peri-domestic rodents in Eastern Sierra Leone. Small mammal trapping was conducted in Kenema district, Sierra Leone between November 2018-July 2019. LASV antigen was detected using a commercially available LASV NP antigen rapid diagnostic test. LASV IgG antibodies against LASV nucleoprotein (NP) and glycoprotein (GP) were tested by adapting a commercially available semi-quantitative enzyme linked immunosorbent assay (ELISA) for detection of mouse-related and rat-related species IgG. Findings: Of the 373 tested specimens, 74 (20%) tested positive for LASV antigen. 40 (11%) specimens tested positive for LASV NP IgG, while an additional 12 (3%) specimens only tested positive for LASV GP IgG. Simultaneous antigen presence and IgG antibody presence was linked in Mastomys sp. specimens (p < 0.01), but not Rattus sp. specimens (p = 1). Despite the link between antigen presence and IgG antibody presence in Mastomys sp., the strength of antigen response did not correlate with the strength of IgG response to either GP IgG or NP IgG. Interpretation: The tools developed in this study can aid in the generation of valuable public health data for rapid field assessment of LASV burden during outbreak investigations and general LASV surveillance. Funding: Funding for this work was supported by the National Institute of Allergy and Infectious Diseases National Institute of Health, Department of Health and Human Services under the following grants: International Collaboration in Infectious Disease Research on Lassa fever and Ebola - ICIDR - U19 AI115589, Consortium for Viral Systems Biology - CViSB - 5U19AI135995, West African Emerging Infectious Disease Research Center - WARN-ID - U01AI151812, West African Center for Emerging Infectious Diseases: U01AI151801.

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.