A multivalent VLP vaccine protects against aerosol challenge of three lethal encephalitis viruses in mice and nonhuman primates.

There are no FDA licensed vaccines or therapeutics for Venezuelan equine encephalitis virus (VEEV) which causes a debilitating acute febrile illness in humans that can progress to encephalitis. Previous studies demonstrated that murine and macaque monoclonal antibodies (mAbs) provide prophylactic and therapeutic efficacy against VEEV peripheral and aerosol challenge in mice. Additionally, humanized versions of two neutralizing mAbs specific for the E2 glycoprotein, 1A3B-7 and 1A4A-1, administered singly protected mice against aerosolized VEEV. However, no studies have demonstrated protection in nonhuman primate (NHP) models of VEEV infection. Here, we evaluated a chimeric antibody 1A3B-7 (c1A3B-7) containing mouse variable regions on a human IgG framework and a humanized antibody 1A4A-1 containing a serum half-life extension modification (Hu-1A4A-1-YTE) for their post-exposure efficacy in NHPs exposed to aerosolized VEEV. Approximately 24 hours after exposure, NHPs were administered a single bolus intravenous mAb. Control NHPs had typical biomarkers of VEEV infection including measurable viremia, fever, and lymphopenia. In contrast, c1A3B-7 treated NHPs had significant reductions in viremia and lymphopenia and on average approximately 50% reduction in fever. Although not statistically significant, Hu-1A4A-1-YTE administration did result in reductions in viremia and fever duration. Delay of treatment with c1A3B-7 to 48 hours post-exposure still provided NHPs protection from severe VEE disease through reductions in viremia and fever. These results demonstrate that post-exposure administration of c1A3B-7 protected macaques from development of severe VEE disease even when administered 48 hours following aerosol exposure and describe the first evaluations of VEEV-specific mAbs for post-exposure prophylactic use in NHPs. Viral mutations were identified in one NHP after c1A3B-7 treatment administered 24 hrs after virus exposure. This suggests that a cocktail-based therapy, or an alternative mAb against an epitope that cannot mutate without resulting in loss of viral fitness may be necessary for a highly effective therapeutic. Endemic in the Americas, Venezuelan equine encephalitis virus (VEEV) can be transmitted to humans, horses, and other animals through the bite of a mosquito. Beyond its natural prevalence, VEEV was previously developed as a biological weapon making the development of vaccines and therapeutics of the upmost importance. Despite over 60 years of research to identify effective therapeutics for VEEV disease, to-date no anti-VEEV therapeutics have progressed beyond pre-clinical testing in a mouse model. Here, we present the first evaluation of an anti-VEEV therapeutic in a nonhuman primate (NHP). We found that a monoclonal antibody given either one or two days after an aerosol exposure to VEEV protected from severe VEE disease. We also found the level of in vitro virus neutralization by a given antibody did not predict efficacy in NHPs. Importantly, we identified viral escape mutations in one NHP after treatment, highlighting the need for development of novel antibodies for inclusion in cocktail-based therapy against VEEV.

Eastern equine encephalitis virus (EEEV) is an alphavirus with a case fatality rate estimated to be as high as 75 % in humans and 90 % in horses. Surviving patients often have long-lasting and severe neurological sequelae. At present, there is no licensed vaccine or therapeutic for EEEV infection. This study completes the clinical and pathological analysis of mice infected with a North American strain of EEEV by three different routes: aerosol, intranasal, and subcutaneous. Such an understanding is imperative for use of the mouse model in vaccine and antiviral drug development. Twelve-week-old female BALB/c mice were infected with EEEV strain FL93-939 by the intranasal, aerosol, or subcutaneous route. Mice were euthanized 6 hpi through 8 dpi and tissues were harvested for histopathological and immunohistochemical analysis. Viral antigen was detected in the olfactory bulb as early as 1–2 dpi in aerosol and intranasal infected mice. However, histologic lesions in the brain were evident about 24 hours earlier (3 dpi vs 4 dpi), and were more pronounced following aerosol infection relative to intranasal infection. Following subcutaneous infection, viral antigen was also detected in the olfactory bulb, though not as routinely or as early. Significant histologic lesions were not observed until 6 dpi. These pathologic studies suggest EEEV enters the brain through the olfactory system when mice are exposed via the intranasal and aerosol routes. In contrast, the histopathologic lesions were delayed in the subcutaneous group and it appears the virus may utilize both the vascular and olfactory routes to enter the brain when mice are exposed to EEEV subcutaneously.

Recent studies highlight that infection with Coxsackievirus B3, Venezuelan equine encephalitis virus (VEEV), Marburg virus, or stimulation using poly I:C (dsRNA), upregulates the signaling adaptor protein MyD88 and impairs the host antiviral type I interferon (IFN) responses. In contrast, MyD88 deficiency (MyD88^-/-) increases the type I IFN and survivability of mice implying that MyD88 up regulation limits the type I IFN response. Reasoning that MyD88 inhibition in a virus-like manner may increase type I IFN responses, our studies revealed lipopolysaccharide stimulation of U937 cells or poly I:C stimulation of HEK293-TLR3, THP1 or U87 cells in the presence of a previously reported MyD88 inhibitor (compound 4210) augmented IFN-β and RANTES production. Consistent with these results, overexpression of MyD88 decreased IFN-β, whereas MyD88 inhibition rescued IFN-β production concomitant with increased IRF3 phosphorylation, suggesting IRF-mediated downstream signaling to the IFN-β response. Further, compound 4210 treatment inhibited MyD88 interaction with IRF3/IRF7 indicating that MyD88 restricts type I IFN signaling through sequestration of IRF3/IRF7. In cell based infection assays, compound 4210 treatment suppressed replication of VEEV, Eastern equine encephalitis virus, Ebola virus (EBOV), Rift Valley Fever virus, Lassa virus, and Dengue virus with IC₅₀ values ranging from 11 to 42 μM. Notably, administration of compound 4210 improved survival, weight change, and clinical disease scores in mice following challenge with VEEV TC-83 and EBOV. Collectively, these results provide evidence that viral infections responsive to MyD88 inhibition lead to activation of IRF3/IRF7 and promoted a type I IFN response, thus, raising the prospect of an approach of host-directed antiviral therapy.

Western equine encephalitis virus (WEEV) causes symptoms in humans ranging from mild febrile illness to life-threatening encephalitis, and no human medical countermeasures are licensed. A previous study demonstrated that immune serum from vaccinated mice protected against lethal WEEV infection, suggesting the utility of antibodies for pre- and post-exposure treatment. Here, three neutralizing and one binding human-like monoclonal antibodies were evaluated against WEEV aerosol challenge. Dose-dependent protection was observed with two antibodies administered individually, ToR69-3A2 and ToR68-2C3. In vitro neutralization was not a critical factor for protection in this murine model, as ToR69-3A2 is a strong neutralizing antibody, and ToR68-2C3 is a non-neutralizing antibody. This result highlights the importance of both neutralizing and non-neutralizing antibodies in the protection of mice from WEEV lethality.

The purpose of this study was to evaluate the effects of the route of administration on the immunogenicity and efficacy of a combined western, eastern, and Venezuelan equine encephalitis (WEVEE) virus-like replicon particle (VRP) vaccine in cynomolgus macaques. The vaccine consisted of equal amounts of WEEV, EEEV, and VEEV VRPs. Thirty-three animals were randomly assigned to five treatment or control groups. Animals were vaccinated with two doses of WEVEE VRPs or the control 28 days apart. Blood was collected 28 days following primary vaccination and 21 days following boost vaccination for analysis of the immune response to the WEVEE VRP vaccine. NHPs were challenged by aerosol 28 or 29 days following second vaccination with WEEV CBA87. Vaccination with two doses of WEVEE VRP was immunogenic and resulted in neutralizing antibody responses specific for VEEV, EEEV and WEEV. None of the vaccinated animals met euthanasia criteria following aerosol exposure to WEEV CBA87. However, one NHP control (total of 11 controls) met euthanasia criteria after infection with WEEV CBA87. Statistically significant differences in median fever hours were noted in control NHPs compared to vaccinated NHPs, providing a quantitative measure of infection and efficacy of the vaccine against a WEEV challenge. Alterations in lymphocytes, monocytes, and neutrophils were observed. Lymphopenia was observed in control NHPs.