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.

An introduction to the Marburg virus vaccine consortium, MARVAC.

The emergence of Marburg virus (MARV) in Guinea and Ghana triggered the assembly of the MARV vaccine "MARVAC" consortium representing leaders in the field of vaccine research and development aiming to facilitate a rapid response to this infectious disease threat. Here, we discuss current progress, challenges, and future directions for MARV vaccines.

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.

Perspectives on Advancing Countermeasures for Filovirus Disease: Report From a Multisector Meeting.

Although there are now approved treatments and vaccines for Ebola virus disease, the case fatality rate remains unacceptably high even when patients are treated with the newly approved therapeutics. Furthermore, these countermeasures are not expected to be effective against disease caused by other filoviruses. A meeting of subject-matter experts was held during the 10th International Filovirus Symposium to discuss strategies to address these gaps. Several investigational therapeutics, vaccine candidates, and combination strategies were presented. The greatest challenge was identified to be the implementation of well-designed clinical trials of safety and efficacy during filovirus disease outbreaks. Preparing for this will require agreed-upon common protocols for trials intended to bridge multiple outbreaks across all at-risk countries. A multinational research consortium including at-risk countries would be an ideal mechanism to negotiate agreement on protocol design and coordinate preparation. Discussion participants recommended a follow-up meeting be held in Africa to establish such a consortium.

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.

Transcriptional Analysis of Lymphoid Tissues from Infected Nonhuman Primates Reveals the Basis for Attenuation and Immunogenicity of an Ebola Virus Encoding a Mutant VP35 Protein.

Infection with Zaire ebolavirus (EBOV), a member of the Filoviridae family, causes a disease characterized by high levels of viremia, aberrant inflammation, coagulopathy, and lymphopenia. EBOV initially replicates in lymphoid tissues and disseminates via dendritic cells (DCs) and monocytes to liver, spleen, adrenal gland, and other secondary organs. EBOV protein VP35 is a critical immune evasion factor that inhibits type I interferon signaling and DC maturation. Nonhuman primates (NHPs) immunized with a high dose (5 × 105 PFU) of recombinant EBOV containing a mutated VP35 (VP35m) are protected from challenge with wild-type EBOV (wtEBOV). This protection is accompanied by a transcriptional response in the peripheral blood reflecting a regulated innate immune response and a robust induction of adaptive immune genes. However, the host transcriptional response to VP35m in lymphoid tissues has not been evaluated. Therefore, we conducted a transcriptional analysis of axillary and inguinal lymph nodes and spleen tissues of NHPs infected with a low dose (2 × 104 PFU) of VP35m and then back-challenged with a lethal dose of wtEBOV. VP35m induced early transcriptional responses in lymphoid tissues that are distinct from those observed in wtEBOV challenge. Specifically, we detected robust antiviral innate and adaptive responses and fewer transcriptional changes in genes with roles in angiogenesis, apoptosis, and inflammation. Two of three macaques survived wtEBOV back-challenge, with only the nonsurvivor displaying a transcriptional response reflecting Ebola virus disease. These data suggest that VP35 is a key modulator of early host responses in lymphoid tissues, thereby regulating disease progression and severity following EBOV challenge.IMPORTANCE Zaire Ebola virus (EBOV) infection causes a severe and often fatal disease characterized by inflammation, coagulation defects, and organ failure driven by a defective host immune response. Lymphoid tissues are key sites of EBOV pathogenesis and the generation of an effective immune response to infection. A recent study demonstrated that infection with an EBOV encoding a mutant VP35, a viral protein that antagonizes host immunity, can protect nonhuman primates (NHPs) against lethal EBOV challenge. However, no studies have examined the response to this mutant EBOV in lymphoid tissues. Here, we characterize gene expression in lymphoid tissues from NHPs challenged with the mutant EBOV and subsequently with wild-type EBOV to identify signatures of a protective host response. Our findings are critical for elucidating viral pathogenesis, mechanisms of host antagonism, and the role of lymphoid organs in protective responses to EBOV to improve the development of antivirals and vaccines against EBOV.

Early Transcriptional Changes within Liver, Adrenal Gland, and Lymphoid Tissues Significantly Contribute to Ebola Virus Pathogenesis in Cynomolgus Macaques.

Ebola virus (EBOV) continues to pose a significant threat to human health, as evidenced by the 2013-2016 epidemic in West Africa and the ongoing outbreak in the Democratic Republic of the Congo. EBOV causes hemorrhagic fever, organ damage, and shock culminating in death, with case fatality rates as high as 90%. This high lethality combined with the paucity of licensed medical countermeasures makes EBOV a critical human pathogen. Although EBOV infection results in significant damage to the liver and the adrenal glands, little is known about the molecular signatures of injury in these organs. Moreover, while changes in peripheral blood cells are becoming increasingly understood, the host responses within organs and lymphoid tissues remain poorly characterized. To address this knowledge gap, we tracked longitudinal transcriptional changes in tissues collected from EBOV-Makona-infected cynomolgus macaques. Following infection, both liver and adrenal glands exhibited significant and early downregulation of genes involved in metabolism, coagulation, hormone synthesis, and angiogenesis; upregulated genes were associated with inflammation. Analysis of lymphoid tissues showed early upregulation of genes that play a role in innate immunity and inflammation and downregulation of genes associated with cell cycle and adaptive immunity. Moreover, transient activation of innate immune responses and downregulation of humoral immune responses in lymphoid tissues were confirmed with flow cytometry. Together, these data suggest that the liver, adrenal gland, and lymphatic organs are important sites of EBOV infection and that dysregulating the function of these vital organs contributes to the development of Ebola virus disease.IMPORTANCE Ebola virus (EBOV) remains a high-priority pathogen since it continues to cause outbreaks with high case fatality rates. Although it is well established that EBOV results in severe organ damage, our understanding of tissue injury in the liver, adrenal glands, and lymphoid tissues remains limited. We begin to address this knowledge gap by conducting longitudinal gene expression studies in these tissues, which were collected from EBOV-infected cynomolgus macaques. We report robust and early gene expression changes within these tissues, indicating they are primary sites of EBOV infection. Furthermore, genes involved in metabolism, coagulation, and adaptive immunity were downregulated, while inflammation-related genes were upregulated. These results indicate significant tissue damage consistent with the development of hemorrhagic fever and lymphopenia. Our study provides novel insight into EBOV-host interactions and elucidates how host responses within the liver, adrenal glands, and lymphoid tissues contribute to EBOV pathogenesis.

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.

Postexposure Efficacy of Recombinant Vesicular Stomatitis Virus Vectors Against High and Low Doses of Marburg Virus Variant Angola in Nonhuman Primates.

A recombinant vesicular stomatitis virus (rVSV) expressing the Marburg virus (MARV) Musoke variant glycoprotein fully protects macaques against 2 MARV variants and Ravn virus as a preventive vaccine and MARV variant Musoke as a postexposure treatment. To evaluate postexposure efficacy against the most pathogenic MARV variant, Angola, we engineered rVSVs expressing homologous Angola glycoprotein. Macaques were challenged with high or low doses of variant Angola and treated 20-30 minutes after exposure. A total of 25% and 60%-75% of treated macaques survived the high-dose and low-dose challenges, respectively. The more rapid disease progression of variant Angola versus variant Musoke may account for the incomplete protection observed.

A safe insect-based Chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques.

Chikungunya virus (CHIKV), which induces chikungunya fever and chronic arthralgia, is an emerging public health concern. Safe and efficient vaccination strategies are needed to prevent or mitigate virus-associated acute and chronic morbidities for preparation of future outbreaks. Eilat (EILV)/CHIKV, a chimeric alphavirus which contains the structural proteins of CHIKV and the non-structural proteins of EILV, does not replicate in vertebrate cells. The chimeric virus was previously reported to induce protective adaptive immunity in mice. Here, we assessed the capacity of the virus to induce quick and durable protection in cynomolgus macaques. EILV/CHIKV protected macaques from wild-type (WT) CHIKV infection one year after a single dose vaccination. Transcriptome and in vitro functional analyses reveal that the chimeric virus triggered toll-like receptor signaling and T cell, memory B cell and antibody responses in a dose-dependent manner. Notably, EILV/CHIKV preferentially induced more durable, robust, and broader repertoire of CHIKV-specific T cell responses, compared to a live attenuated CHIKV 181/25 vaccine strain. The insect-based chimeric virus did not cause skin hypersensitivity reactions in guinea pigs sensitized to mosquito bites. Furthermore, EILV/CHIKV induced strong neutralization antibodies and protected cynomolgus macaques from WT CHIKV infection within six days post vaccination. Transcriptome analysis also suggest that the chimeric virus induction of multiple innate immune pathways, including Toll-like receptor signaling, type I IFN and IL-12 signaling, antigen presenting cell activation, and NK receptor signaling. Our findings suggest that EILV/CHIKV is a safe, highly efficacious vaccine, and provides both rapid and long-lasting protection in cynomolgus macaques.

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.

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.