7. Project Summary/Abstract The encephalitic New World alphaviruses (NWAVs), consisting of Eastern, Venezuelan and Western Equine Encephalitis Viruses (EEEV, VEEV and WEEV, respectively), are transmitted by mosquitoes through rodent or bird hosts and have caused significant periodic epizootic outbreaks in equines and humans in the Americas. NWAVs can cause severe neurological disease, with fatal encephalitis in up to 70% of cases, and significant long-term sequelae in survivors. With recent climate changes, geological redistribution of mosquitoes carrying NWAVs further enhances the potential for future outbreaks. Moreover, concern over their potential use as bioweapons is well-founded due to their ease of production, high infectivity, ability for aerosolization, and capacity to induce disease, resulting in select agent classification for E/VEEV. Despite awareness of these viruses for nearly 100 years, licensed human vaccines against E/V/WEEV remain unavailable for general use. The development of next-generation vaccines that can safely and effectively protect humans against these pathogenic alphavirus infections are urgently needed. This project seeks to develop a cross-protective recombinant subunit E/V/WEEV vaccine based on the linked ectodomain portions of Envelope 2 (E2) and E1 proteins of each NWAV adjuvanted with SLA-LSQ, a novel TLR4 agonist combined with the saponin QS-21 in a liposomal formulation. The proposed approach provides a means to deliver a safe and stable vaccine to protect against infection by all three NWAVs using a scalable manufacturing platform that, in combination with a proven Th1/Th2 balanced adjuvant, elicits a robust, efficacious and durable immune response through both neutralizing and non-neutralizing means. The proposed NWAV vaccine is based on our highly immunogenic and fully protective pre-clinical E2/E1 candidate vaccine for the closely related Chikungunya virus (CHIKV). New preliminary data demonstrates that mice immunized with the NWAV E2/E1 subunits generate high NAb titers to non-select agent strains of E/V/WEEV. The Specific Aims of this project are: 1) evaluate the immunogenicity and optimize formulations of individual and combined recombinant E/V/WEEV subunit proteins with SLA-LSQ adjuvant; 2) demonstrate the ability of the candidate vaccine to induce a durable immune response in mice; and 3) demonstrate the cross-protective efficacy of the candidate vaccine in mice upon NWAV challenge. Hawaii Biotech and the Baylor College of Medicine will collaborate to develop, evaluate and advance this novel trivalent NWAV vaccine candidate. The development of a cross-protective recombinant subunit vaccine to protect against all three pathogenic NWAVs would provide a valuable medical countermeasure to safeguard against the considerable threat posed by these encephalitic alphaviruses.

8. Project Narrative The proposed research is focused on the development of a cross-protective recombinant subunit candidate vaccine against Eastern, Venezuelan and Western Equine Encephalitis Viruses (EEEV, VEEV and WEEV, respectively) that are highly pathogenic in humans. The proposed vaccine combines recombinant glycoproteins from all three New World Alphaviruses into a single formulation. The use of a single vaccine candidate to protect against multiple related encephalitic Alphaviruses provides a valuable medical countermeasure for pandemic threats and a safeguard against biothreats. Validating the success of this vaccine candidate would have a significant impact on human health.