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.

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.

Comparison of Zaire and Bundibugyo Ebolavirus Polymerase Complexes and Susceptibility to Antivirals through a Newly Developed Bundibugyo Minigenome System.

Members of the genus Ebolavirus cause lethal disease in humans, with Zaire ebolavirus (EBOV) being the most pathogenic (up to 90% morality) and Bundibugyo ebolavirus (BDBV) the least pathogenic (∼37% mortality). Historically, there has been a lack of research on BDBV, and there is no means to study BDBV outside of a high-containment laboratory. Here, we describe a minigenome replication system to study BDBV transcription and compare the efficacy of small-molecule inhibitors between EBOV and BDBV. Using this system, we examined the ability of the polymerase complex proteins from EBOV and BDBV to interact and form a functional unit as well as the impact of the genomic untranslated ends, known to contain important signals for transcription (3'-untranslated region) and replication (5'-untranslated region). Various levels of compatibility were observed between proteins of the polymerase complex from each ebolavirus, resulting in differences in genome transcription efficiency. Most pronounced was the effect of the nucleoprotein and the 3'-untranslated region. These data suggest that there are intrinsic specificities in the polymerase complex and untranslated signaling regions that could offer insight regarding observed pathogenic differences. Further adding to the differences in the polymerase complexes, posttransfection/infection treatment with the compound remdesivir (GS-5734) showed a greater inhibitory effect against BDBV than EBOV. The delayed growth kinetics of BDBV and the greater susceptibility to polymerase inhibitors indicate that disruption of the polymerase complex is a viable target for therapeutics. IMPORTANCE Ebolavirus disease is a viral infection and is fatal in 25 to 90% of cases, depending on the viral species and the amount of supportive care available. Two species have caused outbreaks in the Democratic Republic of the Congo, Zaire ebolavirus (EBOV) and Bundibugyo ebolavirus (BDBV). Pathogenesis and clinical outcome differ between these two species, but there is still limited information regarding the viral mechanism for these differences. Previous studies suggested that BDBV replicates slower than EBOV, but it is unknown if this is due to differences in the polymerase complex and its role in transcription and replication. This study details the construction of a minigenome replication system that can be used in a biosafety level 2 laboratory. This system will be important for studying the polymerase complex of BDBV and comparing it with other filoviruses and can be used as a tool for screening inhibitors of viral growth.

Single-dose attenuated Vesiculovax vaccines protect primates against Ebola Makona virus.

The family Filoviridae contains three genera, Ebolavirus (EBOV), Marburg virus, and Cuevavirus. Some members of the EBOV genus, including Zaire ebolavirus (ZEBOV), can cause lethal haemorrhagic fever in humans. During 2014 an unprecedented ZEBOV outbreak occurred in West Africa and is still ongoing, resulting in over 10,000 deaths, and causing global concern of uncontrolled disease. To meet this challenge a rapid-acting vaccine is needed. Many vaccine approaches have shown promise in being able to protect nonhuman primates against ZEBOV. In response to the current ZEBOV outbreak several of these vaccines have been fast tracked for human use. However, it is not known whether any of these vaccines can provide protection against the new outbreak Makona strain of ZEBOV. One of these approaches is a first-generation recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing the ZEBOV glycoprotein (GP) (rVSV/ZEBOV). To address safety concerns associated with this vector, we developed two candidate, further-attenuated rVSV/ZEBOV vaccines. Both attenuated vaccines produced an approximately tenfold lower vaccine-associated viraemia compared to the first-generation vaccine and both provided complete, single-dose protection of macaques from lethal challenge with the Makona outbreak strain of ZEBOV.

Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primates.

The current outbreak of Ebola virus in West Africa is unprecedented, causing more cases and fatalities than all previous outbreaks combined, and has yet to be controlled. Several post-exposure interventions have been employed under compassionate use to treat patients repatriated to Europe and the United States. However, the in vivo efficacy of these interventions against the new outbreak strain of Ebola virus is unknown. Here we show that lipid-nanoparticle-encapsulated short interfering RNAs (siRNAs) rapidly adapted to target the Makona outbreak strain of Ebola virus are able to protect 100% of rhesus monkeys against lethal challenge when treatment was initiated at 3 days after exposure while animals were viraemic and clinically ill. Although all infected animals showed evidence of advanced disease including abnormal haematology, blood chemistry and coagulopathy, siRNA-treated animals had milder clinical features and fully recovered, while the untreated control animals succumbed to the disease. These results represent the first, to our knowledge, successful demonstration of therapeutic anti-Ebola virus efficacy against the new outbreak strain in nonhuman primates and highlight the rapid development of lipid-nanoparticle-delivered siRNA as a countermeasure against this highly lethal human disease.

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.

Use of Single-Injection Recombinant Vesicular Stomatitis Virus Vaccine to Protect Nonhuman Primates Against Lethal Nipah Virus Disease.

Nipah virus (NiV) is a zoonotic pathogen that causes high case-fatality rates (CFRs) in humans. Two NiV strains have caused outbreaks: the Malaysia strain (NiVM), discovered in 1998-1999 in Malaysia and Singapore (≈40% CFR); and the Bangladesh strain (NiVB), discovered in Bangladesh and India in 2001 (≈80% CFR). Recently, NiVB in African green monkeys resulted in a more severe and lethal disease than NiVM. No NiV vaccines or treatments are licensed for human use. We assessed replication-restricted single-injection recombinant vesicular stomatitis vaccine NiV vaccine vectors expressing the NiV glycoproteins against NiVB challenge in African green monkeys. All vaccinated animals survived to the study endpoint without signs of NiV disease; all showed development of NiV F Ig, NiV G IgG, or both, as well as neutralizing antibody titers. These data show protective efficacy against a stringent and relevant NiVB model of human infection.

Single-Dose Trivalent VesiculoVax Vaccine Protects Macaques from Lethal Ebolavirus and Marburgvirus Challenge.

Previous studies demonstrated that a single intramuscular (i.m.) dose of an attenuated recombinant vesicular stomatitis virus (rVSV) vector (VesiculoVax vector platform; rVSV-N4CT1) expressing the glycoprotein (GP) from the Mayinga strain of Zaire ebolavirus (EBOV) protected nonhuman primates (NHPs) from lethal challenge with EBOV strains Kikwit and Makona. Here, we studied the immunogenicities of an expanded range of attenuated rVSV vectors expressing filovirus GP in mice. Based on data from those studies, an optimal attenuated trivalent rVSV vector formulation was identified that included rVSV vectors expressing EBOV, Sudan ebolavirus (SUDV), and the Angola strain of Marburg marburgvirus (MARV) GPs. NHPs were vaccinated with a single dose of the trivalent formulation, followed by lethal challenge 28 days later with each of the three corresponding filoviruses. At day 14 postvaccination, a serum IgG response specific for all three GPs was detected in all the vaccinated macaques. A modest and balanced cell-mediated immune response specific for each GP was also detected in a majority of the vaccinated macaques. No matter the level of total GP-specific immune response detected postvaccination, all the vaccinated macaques were protected from disease and death following lethal challenge with each of the three filoviruses. These findings indicate that vaccination with a single dose of attenuated rVSV-N4CT1 vectors each expressing a single filovirus GP may provide protection against the filoviruses most commonly responsible for outbreaks of hemorrhagic fever in sub-Saharan Africa.IMPORTANCE The West African Ebola virus Zaire outbreak in 2013 showed that the disease was not only a regional concern, but a worldwide problem, and highlighted the need for a safe and efficacious vaccine to be administered to the populace. However, other endemic pathogens, like Ebola virus Sudan and Marburg, also pose an important health risk to the public and therefore require development of a vaccine prior to the occurrence of an outbreak. The significance of our research was the development of a blended trivalent filovirus vaccine that elicited a balanced immune response when administered as a single dose and provided complete protection against a lethal challenge with all three filovirus pathogens.

Marburg and Ravn Viruses Fail to Cause Disease in the Domestic Ferret (Mustela putorius furo).

The domestic ferret was recently described as a uniformly lethal model for 3 species of Ebolavirus. More importantly, this new model utilizes nonadapted wild-type Ebolaviruses. Here, in a proof-of-concept study, we infected ferrets with different variants of the closely related Marburg and Ravn viruses using different doses and routes of exposure. Although ferrets produced a neutralizing humoral response to challenge, we did not observe disease or viremia in any animal. The lack of disease in ferrets underscores the notion that differential mechanisms to immunity among filoviruses exist and may provide a model to better understand how differences contribute to disease.

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.

Efficacy of Human Monoclonal Antibody Monotherapy Against Bundibugyo Virus Infection in Nonhuman Primates.

Background: The 2013-2016 Ebola virus disease (EVD) epidemics in West Africa highlighted a need for effective therapeutics for treatment of the disease caused by filoviruses. Monoclonal antibodies (mAbs) are promising therapeutic candidates for prophylaxis or treatment of virus infections. Data about efficacy of human mAb monotherapy against filovirus infections in preclinical nonhuman primate models are limited. Methods: Previously, we described a large panel of human mAbs derived from the circulating memory B cells from Bundibugyo virus (BDBV) infection survivors that bind to the surface glycoprotein (GP) of the virus. We tested one of these neutralizing mAbs that recognized the glycan cap of the GP, designated mAb BDBV289, as monotherapy in rhesus macaques. Results: We found that recombinant mAb BDBV289-N could confer up to 100% protection to BDBV-infected rhesus macaques when treatment was initiated as late as 8 days after virus challenge. Protection was associated with survival and decreased viremia levels in the blood of treated animals. Conclusions: These findings define the efficacy of monotherapy of lethal BDBV infection with a glycan cap-specific mAb and identify a candidate mAb therapeutic molecule that could be included in antibody cocktails for prevention or treatment of ebolavirus infections.

Nipah Virus C and W Proteins Contribute to Respiratory Disease in Ferrets.

UNLABELLED: Nipah virus (NiV) is a highly lethal paramyxovirus that recently emerged as a causative agent of febrile encephalitis and severe respiratory disease in humans. The ferret model has emerged as the preferred small-animal model with which to study NiV disease, but much is still unknown about the viral determinants of NiV pathogenesis, including the contribution of the C protein in ferrets. Additionally, studies have yet to examine the synergistic effects of the various P gene products on pathogenesis in animal models. Using recombinant NiVs (rNiVs), we examine the sole contribution of the NiV C protein and the combined contributions of the C and W proteins in the ferret model of NiV pathogenesis. We show that an rNiV void of C expression resulted in 100% mortality, though with limited respiratory disease, like our previously reported rNiV void of W expression; this finding is in stark contrast to the attenuated phenotype observed in previous hamster studies utilizing rNiVs void of C expression. We also observed that an rNiV void of both C and W expression resulted in limited respiratory disease; however, there was severe neurological disease leading to 60% mortality, and the surviving ferrets demonstrated sequelae similar to those for human survivors of NiV encephalitis. IMPORTANCE: Nipah virus (NiV) is a human pathogen capable of causing lethal respiratory and neurological disease. Many human survivors have long-lasting neurological impairment. Using a ferret model, this study demonstrated the roles of the NiV C and W proteins in pathogenesis, where lack of either the C or the W protein independently decreased the severity of clinical respiratory disease but did not decrease lethality. Abolishing both C and W expression, however, dramatically decreased the severity of respiratory disease and the level of destruction of splenic germinal centers. These ferrets still suffered severe neurological disease: 60% succumbed to disease, and the survivors experienced long-term neurological impairment, such as that seen in human survivors. This new ferret NiV C and W knockout model may allow, for the first time, the examination of interventions to prevent or mitigate the neurological damage and sequelae experienced by human survivors.

The Synthetic Antiviral Drug Arbidol Inhibits Globally Prevalent Pathogenic Viruses.

UNLABELLED: Arbidol (ARB) is a synthetic antiviral originally developed to combat influenza viruses. ARB is currently used clinically in several countries but not in North America. We have previously shown that ARB inhibits in vitro hepatitis C virus (HCV) by blocking HCV entry and replication. In this report, we expand the list of viruses that are inhibited by ARB and demonstrate that ARB suppresses in vitro infection of mammalian cells with Ebola virus (EBOV), Tacaribe arenavirus, and human herpesvirus 8 (HHV-8). We also confirm suppression of hepatitis B virus and poliovirus by ARB. ARB inhibited EBOV Zaire Kikwit infection when added before or at the same time as virus infection and was less effective when added 24 h after EBOV infection. Experiments with recombinant vesicular stomatitis virus (VSV) expressing the EBOV Zaire glycoprotein showed that infection was inhibited by ARB at early stages, most likely at the level of viral entry into host cells. ARB inhibited HHV-8 replication to a similar degree as cidofovir. Our data broaden the spectrum of antiviral efficacy of ARB to include globally prevalent viruses that cause significant morbidity and mortality. IMPORTANCE: There are many globally prevalent viruses for which there are no licensed vaccines or antiviral medicines. Some of these viruses, such as Ebola virus or members of the arenavirus family, rapidly cause severe hemorrhagic diseases that can be fatal. Other viruses, such as hepatitis B virus or human herpesvirus 8 (HHV-8), establish persistent infections that cause chronic illnesses, including cancer. Thus, finding an affordable, effective, and safe drug that blocks many viruses remains an unmet medical need. The antiviral drug arbidol (ARB), already in clinical use in several countries as an anti-influenza treatment, has been previously shown to suppress the growth of many viruses. In this report, we expand the list of viruses that are blocked by ARB in a laboratory setting to include Ebola virus, Tacaribe arenavirus, and HHV-8, and we propose ARB as a broad-spectrum antiviral drug that may be useful against hemorrhagic viruses.

Oral and Conjunctival Exposure of Nonhuman Primates to Low Doses of Ebola Makona Virus.

Nonhuman primate (NHP) models of Ebola virus (EBOV) infection primarily use parenteral or aerosol routes of exposure. Uniform lethality can be achieved in these models at low doses of EBOV (≤100 plaque-forming units [PFU]). Here, we exposed NHPs to low doses of EBOV (Makona strain) by the oral or conjunctival routes. Surprisingly, animals exposed to 10 PFU by either route showed no signs of disease. Exposure to 100 PFU resulted in illness and/or lethal infection. These results suggest that these more natural routes require higher doses of EBOV to produce disease or that there may be differences between Makona and historical strains.

The Domestic Ferret (Mustela putorius furo) as a Lethal Infection Model for 3 Species of Ebolavirus.

Small-animal models have been developed for several Filoviridae species; however, serial adaptation was required to produce lethal infection. These adapted viruses have sequence changes in several genes, including those that modulate the host immune response. Nonhuman primate models do not require adaptation of filoviruses. Here, we describe lethal models of disease for Bundibugyo, Sudan, and Zaire species of Ebolavirus in the domestic ferret, using wild-type nonadapted viruses. Pathologic features were consistent with disease in primates. Of particular importance, this is the only known small-animal model developed for Bundibugyo and the only uniformly lethal animal model for Bundibugyo.

Molecular Diagnostic Field Test for Point-of-Care Detection of Ebola Virus Directly From Blood.

A molecular diagnostic method for robust detection of Ebola virus (EBOV) at the point of care (POC) directly from blood samples is described. This assay is based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) of the glycoprotein gene of EBOV. Complete reaction formulations were lyophilized in 0.2-mL polymerase chain reaction tubes. RT-LAMP reactions were performed on a battery-operated isothermal instrument. Limit of detection of this RT-LAMP assay was 2.8 × 102 plaque-forming units (PFU)/test and 1 × 103 PFU/test within 40 minutes for EBOV-Kikwit and EBOV-Makona, respectively. This assay was found to be specific for the detection of EBOV, as no nonspecific amplification was detected in blood samples spiked with closely related viruses and other pathogens. These results showed that this diagnostic test can be used at the point of care for rapid and specific detection of EBOV directly from blood with high sensitivity within 40 minutes.

Passive Immunotherapy: Assessment of Convalescent Serum Against Ebola Virus Makona Infection in Nonhuman Primates.

BACKGROUND: Convalescent serum and blood were used to treat patients during outbreaks of Zaire ebolavirus (ZEBOV) infection in 1976 and 1995, with inconclusive results. During the recent 2013-2016 West African epidemic, serum/plasma from survivors of ZEBOV infection was used to treat patients in the affected countries and several repatriated patients. The effectiveness of this strategy remains unknown. METHODS: Nine rhesus monkeys were experimentally infected with ZEBOV-Makona. Beginning on day 3 after exposure (at the onset of viremia), 4 animals were treated with homologous ZEBOV-Makona convalescent macaque sera, 3 animals were treated in parallel with heterologous Sudan ebolavirus (SEBOV) convalescent macaque sera, and 2 animals served as positive controls and were not treated. Surviving animals received additional treatments on days 6 and 9. RESULTS: Both untreated control animals died on postinfection day 9. All 4 ZEBOV-Makona-infected macaques treated with homologous ZEBOV-Makona convalescent sera died on days 8-9. One macaque treated with heterologous SEBOV convalescent sera survived, while the other animals treated with the heterologous SEBOV sera died on days 7 and 9. CONCLUSIONS: The findings suggest that convalescent sera alone is not sufficient for providing 100% protection against lethal ZEBOV infection when administered at the onset of viremia.