ABSTRACT: Pre-existing T cell responses have been correlated with decreased disease severity and positive clinical outcomes during the influenza pandemic of 2009 and the recent COVID-19 pandemic. Therefore, diversifying T cell responses, particularly targeting conserved internal viral proteins such as the influenza virus nucleoprotein (NP), could confer protection against both epidemic and pandemic strains of influenza A virus (IAV). We have designed a mosaic nucleoprotein (MNP) composed of 9-mer epitopes derived from the complete set of 7,422 NP sequences from human, swine, and avian IAVs. We have strong evidence that a vaccine formulation consisting of MNP and a combination adjuvant (Adjuplex + TLR-4 agonist glucopyranosyl lipid A [GLA]) elicits T cell responses that protect against epidemic and pandemic strains of influenza A virus in mice. Avian influenza viruses have decimated poultry in the US and many parts of the world, and most concerningly, these viruses have spilled over to infect multiple mammalian species, including cats and cows. Most notably, the H5N1 avian influenza virus in cows is rapidly spreading to multiple states in the US. Currently, there is serious concern about the possibility of the rapidly spreading avian influenza virus in cows mutating to gain the potential to infect humans, cause severe disease, and efficiently spread by airborne transmission. Therefore, it is critical to develop vaccine strategies to protect humans, and contain avian influenza virus infection and transmission in cows. Significantly, the MNP we generated exhibits close homology to the NP of the H5N1 virus affecting dairy cows in the ongoing outbreak. We hypothesize that the MNP will be immunogenic and that T cell responses elicited by the MNP-based vaccine will provide effective protection against the currently circulating strains of avian influenza virus isolated from cows. The aims of this administrative supplement are: (1) Test the hypothesis that mosaic nucleoprotein (MNP) formulated in the combination adjuvants (Adjuplex+GLA or Adjuplex+CdN) will elicit diverse antibody and T cell responses in circulation and mammary gland of ferrets and cows; (2) Test the hypothesis that mosaic nucleoprotein (MNP) formulated in the combination adjuvants (Adjuplex+GLA or Adjuplex+CdN) will protect ferrets against H5N1 in lungs, upper respiratory tract, and the mammary gland. The proposed studies will have implications in the development of effective vaccines to protect humans against avian influenza, break the transmission cycle in cows and mitigate spillover into humans.