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

Abstract

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.

Keywords: alphavirus; efficacy; equine encephalitis; modified vaccinia Ankara (MVA); vaccines.

Figure 1

Immune Response After Immunization with MVA-VEEV, Triple Mix, or Trivalent Vaccines. Mice were immunized (IM) twice (Day 0 and 28) with the respective vaccine or TBS. PRNT₅₀ (A) and Interferon gamma response (ELISpot) to the E1 peptide pool (B) and E2 peptide pool (C). The second vaccination on Day 28 (Day 42) resulted in a significant increase in the neutralizing antibody titers compared to Day 28 (p < 0.05) in all three vaccination groups. In addition, the monovalent MVA-VEEV and Trivalent IFNγ response was significantly greater (p < 0.05) than the IFNγ response measured at Day 28 (E1 and E2 peptide pool) and Day 42 for the Triple-Mix group (E1 peptide pool). Ten animals per group were evaluated at each time point. (A) horizontal lines represent group mean values. Standard error of the mean is included in (B, C).

Figure 2

Survival and Body Temperature Changes after VEEV TrD Aerosol Challenge of Immunized Mice. Mice were immunized (IM) twice (Day 0 and 28) with the respective vaccine or TBS and then exposed to an aerosolized dose of VEEV TrD on Day 42. Kaplan-Meier plots of survival results (A) and changes in body temperature during the post-challenge period (B). There was a significant difference in survival between the TBS group and all three vaccination groups (p < 0.01). In addition, there was a significant (p < 0.05) temperature decrease for the TBS control group (B) prior to succumbing to infection or meeting euthanasia criteria and a significant difference between the TBS control group and the vaccinated groups on Days 44–49 (p < 0.05). Ten animals per group were evaluated for survival and body temperature changes.

Figure 3

Immune Response After Immunization with Monovalent, Triple Mix, or Trivalent Vaccines. Mice were immunized (IM) twice (Day 0 and 28) with the respective vaccine or TBS. VEEV PRNT₅₀ (A), WEEV PRNT₅₀ (B), and EEEV PRNT₅₀ (C). On Day 28, the geometric mean MVA-WEEV PRNT₅₀ was significantly greater than the Trivalent and TBS control groups and on Day 42, the MVA-WEEV geometric mean PRNT₅₀ was significantly greater than the TBS group (p < 0.01). On Day 14, the geometric mean MVA-EEEV PRNT₅₀ was significantly greater than the Trivalent group and TBS control (p < 0.0001). On Day 42, the MVA-EEEV geometric mean PRNT₅₀ was significantly greater than the TBS control (p < 0.01). Five animals per group were evaluated at each time point for immune responses.

Figure 4

Survival and Body Temperature Changes after VEEV INH-9813, WEEV Fleming, or EEEV V105-00210 Aerosol Challenge of Immunized Mice. Mice were immunized (IM) twice (Day 0 and 28) with the respective vaccine or TBS and then exposed to an aerosolized dose of VEEV, WEEV, or EEEV on Day 42. Kaplan-Meier survival curves of VEEV (A), WEEV (B), and EEEV (C) challenge results are shown. There was a significant difference in survival between the TBS group and the vaccination groups (p < 0.01). Group mean changes (with 95% confidence intervals) in body temperature for VEEV (D), WEEV (E), and EEEV (F) challenge groups during the post-challenge period are shown. TBS control group mean body temperatures were significantly greater than baseline (p < 0.05) over the first week post-challenge. Ninety-five percent confidence intervals are not included when number of animals was less than three. Ten animals per group were evaluated for survival and body temperature changes.