PROJECT SUMMARY Current influenza vaccination inducing strain-specific immunity against variable hemagglutinin (HA) proteins is not effective in providing cross protection against antigenically different epidemic strains and unanticipated new pandemic viruses. Development of universal vaccines improving cross protection against antigenically different influenza viruses is a high priority. The fusion peptide (FP) located in the N-terminus of the stalk domain of HA is universally conserved in both influenza (flu) A and B viruses. Monoclonal and polyclonal antibodies against FP were shown to exhibit broad neutralizing activity. In preliminary data, we discovered a new HA2 stem domain near to the FP, which could generate poly IgG antibodies highly cross reactive to different subtype flu A viruses and both lineage flu B viruses. A neuraminidase (NA) epitope (NA2e) near to the enzymatic active site was also identified to be universally conserved in all flu A and B viruses. NA2e specific monoclonal antibody was used to quantitate the NA contents in different flu vaccine lots. NA immunity is considered an independent protective correlate in addition to HA. Influenza A virus M2 protein extracellular domain (M2e), which is highly conserved among flu A viruses, has been shown to be a promising target for developing universal flu A vaccines. However, M2e immunity alone would not provide sufficiently high efficacy of cross protection. No vaccines incorporating universally conserved multi epitopes including M2e, FP, stem domains and NA2e have been developed. Virus-like particle (VLP) platform has been demonstrated to be a promising delivery vehicle for poor immunogenic but cross protective epitopes. Preliminary data showed that NA2e monoclonal antibody was able to provide protection after passive inoculation. We hypothesize that new VLP vaccine constructs inducing antibodies to universal FP-stem domain epitopes and NA2e epitopes in addition to M2e will enhance the efficacy of cross protection against different strains of flu A and B viruses. In the aim 1, we will determine whether new recombinant VLP vaccine constructs containing FP-stem, NA2e (or NA), and M2e epitopes as a standalone vaccine or in combination with HA-based vaccine will enhance the cross protection against influenza A viruses. In the aim 2, we will determine whether vaccines inducing antibodies to FP-stem and NA2e epitopes as a standalone vaccine or when supplemented in inactivated virus vaccines will enhance cross protection against influenza B viruses. This proof-of-concept of two-year project on developing cross protective vaccines inducing immunity universally conserved new epitopes in both flu A and B viruses would provide supporting data warranting further advanced preclinical studies.