3A,

each vaccination approach induced strong antibody responses against RABV G as expected since RABV G was present in each immunogen. Either a single dose or two doses of INAC-RV-HC50 VEGFR inhibitor induced botulinum HC50-specific antibodies, and interestingly, combined administration with INAC-RV-GP resulted in a slightly stronger HC50-specific response (Fig. 3B). Finally, analysis of the GP-specific antibody response indicated that single or boosted immunization with INAC-RV-GP induced strong immunity as expected (Fig. 3C). Importantly, co-administration of INAC-RV-GP and INAC-RV-HC50 induced antibody levels that were nearly identical to INAC-RV-GP immunization. These results indicate that a potent multivalent response can be induced by this inactivated vaccination platform. Co-immunization with three antigens, RABV G, HC50, and ZEBOV GP resulted in no decrease in antibody response against each individual immunogen. There is a possibility that some members of the target population for an Ebola vaccine such as lab workers or first responders may be previously vaccinated with the currently approved RABV vaccine and thus have pre-existing immunity to RABV. This pre-existing immunity might interfere with induction of the EBOV GP-specific antibodies upon immunization with INAC-RV-GP.

Therefore, we sought to determine in the mouse model if prior vaccination with a RABV vaccine would inhibit the induction of GP-specific antibodies (Fig. 4). Groups of five mice

Vandetanib were immunized once on day Vasopressin Receptor 0 with vehicle, 10 μg INAC-RV-HC50 or INAC-RV-GP. A fourth group was immunized with 10 μg inactivated INAC-RV-HC50 on day 0 followed by 10 μg inactivated INAC-RV-GP on day 28. Four weeks after immunization, serum from each group was assayed by ELISA against (A) RABV G, (B) HC50, and (C) EBOV GP. As expected, each vaccination approach induced strong antibody responses against RABV G (Fig. 4A) and vaccination with INAC-RV-HC50 or INAC-RV-HC50 followed by INAC-RV-GP induced potent HC50-specific antibodies (Fig. 4B). Interestingly, vaccination with INAC-RV-HC50 followed by INAC-RV-GP induced similar levels of GP-specific antibodies to vaccination with INAC-GP alone (Fig. 4C). These results indicate that immunization with INAC-RV-GP can induce GP-specific antibodies in the presence of pre-existing RABV immunity. The presence of a potent RABV G-specific antibody response at day 28 prior to immunization with INAC-RV-GP was confirmed (data not shown). Several vaccination strategies have been demonstrated to confer protection from Ebola hemorrhagic fever in macaques, including DNA vaccines, virus-like particles, or recombinant viruses expressing GP including adenovirus, vesicular stomatitis virus, or paramyxoviruses [2], [4], [5], [6], [7], [8], [24], [25], [26], [27] and [28].