Each bivalent vaccine candidate induced strong humoral immunity to RABV G and EBOV GP, and conferred protection from both lethal RABV and mouse-adapted EBOV challenge in mice [13]. Our primary focus is the development of an inactivated vaccine for use in humans based on the potential for superior safety and the history of the successful existing RABV vaccine that is widely used in humans, but we are also

pursuing the live attenuated vaccine candidates for use in nonhuman primate populations in Africa at risk for lethal EBOV infection [19] and [20]. Here, we expand our investigation of the immune response to the RABV vaccine candidates expressing EBOV GP. Three critical elements of an effective vaccine platform for the filoviruses were assessed: (a) the ability to induce EBOV-specific T-cell immunity, (b) coformulation of vaccine candidates to induce multivalent antibody responses,

BKM120 supplier and Birinapant concentration (c) induction of GP-specific immunity in the presence of pre-existing vector immunity to the RABV vaccine. The recovery and propagation of the vaccine candidates used in this study have been described previously [13] and [18]. The SADB19-derived BNSP333 virus serves as the parent rabies vaccine vector RVA (Fig. 1). RV-GP expresses the EBOV Mayinga GP ectodomain and transmembrane domain fused to the RABV G cytoplasmic domain. Inactivated RV-GP (INAC-RV-GP) was generated by treatment of sucrose purified virus all stocks with a

1:2000 dilution of beta-propiolactone (BPL) overnight at 4 °C followed by 30 min at 37 °C. RVΔG-GP expresses intact EBOV Mayinga GP and contains a deletion in the RABV G gene requiring propagation on complementary cells which express RABV G. BPL inactivated INAC-RV-HC50 expresses a chimeric protein composed of the heavy chain carboxyterminal half (HC50) of botulinum neurotoxin A fused with 30 amino acids of RABV G ectodomain (ED), transmembrane domain (TM) and cytoplasmic domain (CD) [18]. A recombinant vaccinia virus expressing EBOV Mayinga GP was constructed using published methods [21]. All mouse experiments were approved by the NIAID Division of Intramural Research Animal Care and Use Committee. Injections of 0.1 ml live or inactivated virus were administered via the intramuscular (i.m.) route, 0.05 ml in each hind leg. Live vaccines were delivered 1× at 5 × 105 FFU, and 10 μg of the killed vaccines, which are equivalent to approximately 109 FFU, were delivered on day 0 or on days 0 and 14. Groups of 10 Balb/c mice (Jackson Laboratories) were immunized with either vehicle, RVA (parent virus), RV-GP, RVΔG-GP, INAC-RV-GP or INAC-RV-GP with an additional dose at day 14. For analysis of primary T cell response, four mice per group were sacrificed at day 7 post-immunization, and splenocytes were assayed for each individual mouse by ELISPOT (Fig. 2A).