Abstract This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 23-045. The proposed supplement project aims to validate the immunogenicity of a total 10 public tumor- specific neoantigen (pTSNA) candidates identified by our parent R01CA252713 project, entitled “Canine MHC- I genotyping and tumor specific neoantigen determination”. These are defined as mutant peptides that are: 1) derived from hotspot mutations in canine and human cancers; and 2) predicted to bind MHC class I (MHC-I) alleles dominant in canine/human populations and/or subpopulations (breeds or ethnic groups). We will collaborate with Drs. Biao He and Dong An, two leading vaccine developers to perform two studies. 1) We will clone the pTSNA candidates in tandem into vaccine vectors parainfluenza virus 5 (PIV5) and PIV5- based self-amplifying virus-like particle (AVLP). PIV5 and AVLP, developed by Drs He and An, are ideal vaccine vectors especially for cancer vaccine development, as they elicit more robust cellular immune responses, compared to other strategies. 2) We will infecting peripheral blood mononuclear cells (PBMCs) isolated from dogs harboring the required MHC-I alleles with rPIV5-pTSNA and rAVLP-pTSNA viruses, identify pTSNAs that are presented by the MHC-I alleles via mass-spectrometry, and identify immunogenic pTSNAs via interferon-g assays. This proposed supplement study will significantly enhance Aim 2c of the parent R01, by accelerating immunogenic pTSNA discovery. Furthermore, the study will yield critical reagents (rPIV5-pTSNA amd rAVLP- pTSNA vaccines) and knowledge for future cancer vaccine development that impacts a large population of both dogs and humans. The study is especially significant, considering that PIV5-based vaccines are shown to be safe and immunogenic for dogs, and PIV5-based vaccines are often administrated intranasally.