A Monovalent and Trivalent MVA-Based Vaccine Completely Protects Mice Against Lethal Venezuelan, Western, and Eastern Equine Encephalitis Virus Aerosol Challenge.

Venezuelan, eastern and western equine encephalitis viruses (EEV) can cause severe disease of the central nervous system in humans, potentially leading to permanent damage or death. Yet, no licensed vaccine for human use is available to protect against these mosquito-borne pathogens, which can be aerosolized and therefore pose a bioterror threat in addition to the risk of natural outbreaks. Using the mouse aerosol challenge model, we evaluated the immunogenicity and efficacy of EEV vaccines that are based on the modified vaccinia Ankara-Bavarian Nordic (MVA-BN®) vaccine platform: three monovalent vaccines expressing the envelope polyproteins E3-E2-6K-E1 of the respective EEV virus, a mixture of these three monovalent EEV vaccines (Triple-Mix) as a first approach to generate a multivalent vaccine, and a true multivalent alphavirus vaccine (MVA-WEV, Trivalent) encoding the polyproteins of all three EEVs in a single non-replicating MVA viral vector. BALB/c mice were vaccinated twice in a four-week interval and samples were assessed for humoral and cellular immunogenicity. Two weeks after the second immunization, animals were exposed to aerosolized EEV. The majority of vaccinated animals exhibited VEEV, WEEV, and EEEV neutralizing antibodies two weeks post-second administration, whereby the average VEEV neutralizing antibodies induced by the monovalent and Trivalent vaccine were significantly higher compared to the Triple-Mix vaccine. The same statistical difference was observed for VEEV E1 specific T cell responses. However, all vaccinated mice developed comparable interferon gamma T cell responses to the VEEV E2 peptide pools. Complete protective efficacy as evaluated by the prevention of mortality and morbidity, lack of clinical signs and viremia, was demonstrated for the respective monovalent MVA-EEV vaccines, the Triple-Mix and the Trivalent single vector vaccine not only in the homologous VEEV Trinidad Donkey challenge model, but also against heterologous VEEV INH-9813, WEEV Fleming, and EEEV V105-00210 inhalational exposures. These EEV vaccines, based on the safe MVA vector platform, therefore represent promising human vaccine candidates. The trivalent MVA-WEV construct, which encodes antigens of all three EEVs in a single vector and can potentially protect against all three encephalitic viruses, is currently being evaluated in a human Phase 1 trial.

Common marmosets (Callithrix jacchus) as a nonhuman primate model to assess the virulence of eastern equine encephalitis virus strains.

Eastern equine encephalitis virus (EEEV) produces the most severe human arboviral disease in North America (NA) and is a potential biological weapon. However, genetically and antigenically distinct strains from South America (SA) have seldom been associated with human disease or mortality despite serological evidence of infection. Because mice and other small rodents do not respond differently to the NA versus SA viruses like humans, we tested common marmosets (Callithrix jacchus) by using intranasal infection and monitoring for weight loss, fever, anorexia, depression, and neurologic signs. The NA EEEV-infected animals either died or were euthanized on day 4 or 5 after infection due to anorexia and neurologic signs, but the SA EEEV-infected animals remained healthy and survived. The SA EEEV-infected animals developed peak viremia titers of 2.8 to 3.1 log(10) PFU/ml on day 2 or 4 after infection, but there was no detectable viremia in the NA EEEV-infected animals. In contrast, virus was detected in the brain, liver, and muscle of the NA EEEV-infected animals at the time of euthanasia or death. Similar to the brain lesions described for human EEE, the NA EEEV-infected animals developed meningoencephalitis in the cerebral cortex with some perivascular hemorrhages. The findings of this study identify the common marmoset as a useful model of human EEE for testing antiviral drugs and vaccine candidates and highlight their potential for corroborating epidemiological evidence that some, if not all, SA EEEV strains are attenuated for humans.

Venezuelan equine encephalitis virus: comparison of infectivity and virulence of strains V-38 and P676 in donkeys.

Two strains of Venezuelan equine encephalitis (VEE) virus were examined for the ability to replicate in, as well as to produce death among donkeys. One, a low passage strain known as strain P676 was originally isolated from mosquitos in Venezuela. The other, strain V-38 was isolated from a horse brain in 1938 and had undergone an unknown number of laboratory passages; it is used extensively for the preparation of inactivated VEE vaccine. Both strains were found to be approximately equal in their ability to infect donkeys. However, a quantity as small as 50% hamster intraperitoneal infectious units of strain V-38 resulted in fatal infection. On the other hand, as much as 631 million infectious units of strain P676 were nonfatal in one of two donkeys. It appears that strain V-38 is approximately 100 million times more virulent than strain P676 in equine species. One donkey which received strain P676 demonstrated a biphasic pattern of clinical illness and viremia, and there is suggestive evidence that another animal experienced a second and fatal clinical response 3 weeks after virus inoculation.

Venezuelan equine encephalomyelitis virus (VEEV):strain differentiation and specification of virulence markers.

Fourteen strains of VEEV of diverse origin and antigenicity were classified into five virulence categories according to their efficiency of infection and nature of response in mice. Mice of ages up to 30 days represented distinct levels of responsiveness and were suitable for the differentiation of VEEV strains of highest to lowest virulence. Stepwise changes with age of the susceptibility or responsiveness of mice appeared to determined the type of response as predominantly lethal (D), dual or intermediate (DP) or protective (P).

Correlates to increased lethality of attenuated Venezuelan encephalitis virus vaccine for immunosuppressed hamsters.

Splenectomy or pretreatment of adult hamsters with cyclophosphamide (Cytoxan) increased the lethality of the TC-83 vaccine strain of Venezuelan encephalitis virus (VEE), inoculated subcutaneously, from 12% for normal hamsters to 75 and 76%, respectively. Neither splenectomy nor cyclophosphamide treatment significantly increased the lethality of Pixuna virus. Cytoxantreated (Cy) hamsters developed and maintained levels of TC-83 virus higher than normal infected controls in blood, brain, spleen, and femoral bone marrow; splenectomy had a similar but less intense effect. A severe myeloid necrosis of femoral bone marrow developed 4 to 9 days after TC-83 virus inoculation in 78% of the Cy hamsters and in 48% of the splenectomized (Sx) hamsters. In contrast, only 13% of normal TC-83-infected hamsters developed this lesion. Extensive hemorrhagic lesions in the olfactory lobes and adjacent areas of the brain also developed more frequently in Cy or Sx hamsters than in normal infected controls. Lethally infected hamsters developed and maintained a severe thrombocytopenia, which may be related to the bone marrow lesion and to the hemorrhagic manifestations of lethal VEE infections.

Transplacental transmission of Venezuelan equine encephalomyelitis virus in mice.

Transplacental infection of mouse fetuses with Venezuelan equine encephalomyelitis was produced by intraperitoneal injection of dams at various stages of gestation with 10(3) suckling mouse intracerebral median lethal doses of an attenuated strain (TC-83). Virus inoculation, at times ranging from 6 days prior to mating to 9 days after conception, had no effect on conception rate, litter size, or survival of the newborn. Inoculation of the dam from the 10th to 13th days of gestation resulted in decreased litter size, an increase in stillbirths, and a decrease in birth-to-weaning survival. Inoculation of the dams later in gestation only decreased the birth-to-weaning survival. No evidence of morphologic abnormality was noted in any of the newborn.