Highly pathogenic avian influenza (HPAI) H5N1 virus has been causing severe disease leading to high mortality outbreaks in several avian and mammalian species in recent years. Notably, domestic cats are one of the most affected mammalian species, presenting with a wide range of clinical signs varying from respiratory to neurological disease often with a fatal outcome. The zoonotic nature of these viruses poses a significant burden to public health and infection of cats, a companion animal with close interaction with humans, could lead to spillover of the virus into humans. Importantly, currently there are no vaccines available to protect cats from HPAI H5N1 virus-induced morbidity and mortality. The goal of proposed study is to develop new tools (i.e. vaccines and diagnostics) to combat HPAI H5N1 virus in cats, and thus reduce the burden and risk posed by the virus to both felids and to humans. To achieve this goal, we propose two specific aims: 1) To evaluate the immunogenicity of vectored and recombinant protein HPAI H5N1 virus vaccine candidates in cats; and 2) To develop and validate a companion DIVA diagnostic assay for use in felids. In Aim 1, we will assess the immunogenicity of viral vectored and recombinant protein vaccine candidates expressing the HPAI H5N1 virus hemagglutinin (NP) and nucleoprotein (NP) in domestic cats. Following immunization both antibody and T cell responses to the vaccine candidates will be characterized. Effector antibody responses will be evaluated by virus neutralization assays and hemagglutination inhibition (HI) assays. Additionally, the ability of each individual vectored vaccine platform to induce antibody-dependent cellular cytotoxicity (ADCC) will also be evaluated following immunization. To characterize T cell responses, we will perform flow cytometry-based lymphocyte proliferation and intracellular cytokine staining assays, following in vitro recall stimulation of lymphocytes collected from immunized animals. In Aim 2, we will develop and validate a multiplex Luminex assay based on the HA, NP and neuraminidase (NA) HPAI H5N1 viral proteins. This assay will allow differentiation of antibodies elicited by vaccination or natural infection. We expect to identify a highly immunogenic vaccine platform eliciting both humoral and cellular immune responses in cats and potentially protective against HPAI infections