Project Summary/Abstract The NIH/NIAID 2017 Strategic Plan is specifically directed at promoting research that can provide solutions to mitigate the threat posed by emerging and re-emerging diseases. The tick-borne flavivirus (TBFV) group includes a number of important human pathogens that result in serious neurological or hemorrhagic diseases that are either Category B or C priority pathogens. The TBFVs are considered to be emerging or re-emerging pathogens due to increases in the number of human infections, the expansion of their geographic distribution, and emergence of new viruses. Additionally, the number of different viruses in the TBFV group poses challenges. There are two licensed Tick-borne encephalitis virus (TBEV) vaccines in Europe. These vaccines are based on the European strains of TBEV (TBEV-Eu), and while they provide some level of cross-neutralization, not all of the TBFV are covered. Furthermore, these vaccines are not licensed in the U.S. and are no longer available in Canada. The development of a multivalent vaccine that provides cross-protection against multiple pathogenic TBFVs would be of great value and is in line with the priorities of NIH/NIAID to develop multivalent and cross-protective technologies. We have demonstrated that two combinations of TBFV recombinant envelope subunit proteins (r80E) can provide protection against a diverse range of TBFV in a mouse challenge model. Additionally, passive transfer studies using serum from mice immunized with combinations of the r80E protein confirm the role of antibodies in protection against virus challenge. This Phase IIB application builds upon our initial efforts to develop a multivalent TBFV vaccine that provides broad cross-protection against viruses in this group. The proposed research aims to 1) optimizing the vaccine formulation; 2) produce pilot lots of the antigens; 3) conduct a pre-IND meeting with the FDA; and 4) conduct potency and toxicity testing to establish the suitability and safety of the final TBFV vaccine formulation. With the successful completion of these efforts, we will be positioned to advance a multicomponent TBFV vaccine into a cGMP manufacturing campaign and toward clinical evaluation.