A Single-Dose Long-Lasting Live-Attenuated Chimeric Vaccine Candidate Against the Emerging Tick-Borne Kyasanur Forest Disease - PROJECT SUMMARY Kyasanur Forest disease virus (KFDV), an emerging tick-borne pathogen, is endemic in India and causes life- threatening KFD, characterized by hemorrhagic fever with neurological manifestations. In recent years, KFDV has spread beyond the endemic region, posing a growing threat to global public health. In the US, the virus is an NIAID category C priority pathogen and an HHS select agent because it poses potential dangers to public health and national security; however, neither an adequate vaccine nor a specific antiviral drug is available against the pathogen. Within the family Flaviviridae, KFDV belongs to the genus Flavivirus, which also includes Japanese encephalitis virus (JEV) and Zika virus (ZIKV). Recently, we developed a novel vaccine platform based on a full- length infectious cDNA clone of the live-attenuated JEV vaccine SA14-14-2 approved for human use. We then used our SA14-14-2-based vaccine platform to generate a recombinant chimeric rJEV/ZIKVprME virus as a live- attenuated vaccine candidate for ZIKV, by replacing the two viral envelope protein prM and E genes of JEV SA14- 14-2 with the corresponding genes of ZIKV P6-740. Our preclinical data showed that rJEV/ZIKVprME is fully attenuated and induces robust long-term protective immunity following a single immunization in mice, indicating that our SA14-14-2-based chimeric vaccine development strategy is applicable to other flaviviruses. The current proposal seeks to capitalize on our SA14-14-2-based chimeric vaccine development strategy, combined with our understanding of the structure and function of two neutralizing antibody-mediated protective immunity-inducing flavivirus envelope proteins (prM and E), to create and refine prM-E gene-replaced rJEV/KFDVprME viruses that are genetically stabilized, fully attenuated, strongly immunogenic, and highly efficacious against KFDV infection. Toward this goal, we will pursue the following specific aims: In Aim 1, we will examine the degree of attenuation and genetic stability of rJEV/KFDVprME variants, each adapted to growth in a cell line certified for human vaccine production, by determining their potential for lack of pathogenicity and transplacental transmissibility and by deep sequencing the replicating chimeric viruses in BALB/c mice, respectively. In Aim 2, we will assess the balance of attenuation and immunogenicity of the rJEV/KFDVprME variants by comparing their degree of attenuation to the levels of KFDV prM/E-specific humoral and cellular immune responses elicited in BALB/c mice after infection with each of the rJEV/KFDVprME variants. In Aim 3, we will analyze the impact of humoral immunity on the short- and long-term protective efficacy of rJEV/KFDVprME variants by performing (i) challenge experiments with KFDV in BALB/c mice at various time points after a single immunization with each of the two most promising rJEV/KFDVprME variants and (ii) passive transfer of IgG purified from the immune sera to naïve BALB/c mice prior to a challenge with KFDV. Collectively, our efforts will establish an optimal balance of attenuation, genetic stability, immunogenicity, and protective efficacy in one or more live-attenuated chimeric vaccine candidates for KFDV and elucidate the molecular basis for attenuation and the immunologic correlates of protection.
