Cynomolgus macaques exposed to an aerosol containing a virulent strain of eastern equine encephalitis (EEE) virus developed neurological signs indicating encephalitis that corresponded with the onset of fever and an elevated heart rate. Viremia was either transient or undetectable even in animals that succumbed to the illness. The onset of illness was dose dependent, but once a febrile response was observed, macaques were moribund within 36 h. Simultaneously, a prominent leukocytosis was seen; 1 day before being moribund, macaques had a white blood cell count 120,000 cells/mL. The leukocytes were predominantly granulocytes. Increases in serum levels of blood urea nitrogen, sodium, and alkaline phosphatase were also seen. The rapid onset and severity of neurological signs mirror what has been reported for human cases of disease caused by EEE.
Because Venezuelan equine encephalitis viruses (VEEVs) are infectious by aerosol, they are considered to be a biological-weapons threat. Nonhuman-primate models are needed to evaluate the efficacy of candidate vaccines. In the present study, cynomolgus macaques, after aerosol exposure to either VEEV-IE or VEEV-IIIA, developed fever, viremia, and lymphopenia; the severity of the fever response, viremia, and lymphopenia correlated with the inhaled dose of VEEV. Of the 10 macaques in our study, 7 developed clinical signs indicative of encephalitis, including loss of balance and hypothermia. In the macaque, the enzootic strains used are infectious by aerosol and lead to disease, including clinical encephalitis.
Cynomolgus macaques were exposed by aerosol to a virulent strain of western equine encephalitis virus (WEEV). Between 4 and 6 days after exposure, macaques had a significantly elevated temperature that lasted for 3-4 days. Clinical signs of encephalitis began as the body temperature decreased, and then they rapidly increased in severity. Cynomolgus macaques with clinical signs of encephalitis had elevated white cell counts in the blood caused mostly by increased numbers of segmented neutrophils and monocytes. Elevated serum glucose levels also correlated with the severity of the clinical signs of encephalitis. Three cynomolgus macaques died; immunohistochemical evidence of viral antigen was present in the brain and central nervous system (CNS). Microscopic analysis also revealed a marked lymphocytic infiltrate in the CNS. Cynomolgus macaques will serve as a useful model of aerosol exposure to WEEV for the evaluation of potential vaccine candidates.
Alphavirus replicons were evaluated as potential vaccine candidates for Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), or eastern equine encephalitis virus (EEEV) when given individually or in combination (V/W/E) to mice or cynomolgus macaques. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in mice to their respective alphavirus. Protection from either subcutaneous or aerosol challenge with VEEV, WEEV, or EEEV was demonstrated out to 12 months after vaccination in mice. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in macaques and demonstrated good protection against aerosol challenge with an epizootic VEEV-IAB virus, Trinidad donkey. Similarly, the EEEV replicon and V/W/E combination vaccine elicited neutralizing antibodies against EEEV and protected against aerosol exposure to a North American variety of EEEV. Both the WEEV replicon and combination V/W/E vaccination, however, elicited poor neutralizing antibodies to WEEV in macaques, and the protection conferred was not as strong. These results demonstrate that a combination V/W/E vaccine is possible for protection against aerosol challenge and that cross-interference between the vaccines is minimal.
IMPORTANCE
Three related viruses belonging to the genus Alphavirus cause severe encephalitis in humans: Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), and eastern equine encephalitis virus (EEEV).Normally transmitted by mosquitoes, these viruses can cause disease when inhaled, so there is concern that these viruses could be used as biological weapons. Prior reports have suggested that vaccines for these three viruses might interfere with one another. We have developed a combined vaccine for Venezuelan equine encephalitis, western equine encephalitis, and eastern equine encephalitis expressing the surface proteins of all three viruses. In this report we demonstrate in both mice and macaques that this combined vaccine is safe, generates a strong immune response, and protects against aerosol challenge with the viruses that cause Venezuelan equine encephalitis, western equine encephalitis, and eastern equine encephalitis.
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