2010 Annual Report 1a.Objectives (from AD-416) To improve Foot-and-Mouth Disease Virus (FMDV) vaccine potency and duration of immunity, ARS, PIADC and the University of Vermont (UV) will study the cellular immune response to infection and the ability to refine the killed virus vaccine for FMDV or the recombinant empty capsid vaccine. The objective of this agreement is to analyze T cell responses to FMDV infection in swine and cattle. 1b.Approach (from AD-416) ARS, PIADC in cooperation with the University of Copenhagen is developing state of the art technology for analysis of FMDV vaccine performance in cattle. The product of this collaboration, MHC tetramers for tracking T cell responses to vaccination, will be used to analyze genetically defined cattle. The University of Vermont collaborators maintain a genetically homogenous herd of Holstein cattle that make it possible to use the tetramer technology to analyze the response to vaccination with FMDV new vaccines. ARS, PIADC will collaborate with UV faculty to analyze responses to infection of these cattle with FMDV and mastitis. 3.Progress Report The major histocompatibility complex (MHC) class I and class II genes are critical in the T-cell mediated immune response to intracellular pathogens including viruses (class I MHC) and the adaptive immune response mediated through antigen presenting cells (class II MHC). The bovine class I and class II gene structure and sequence diversity are poorly understood, representing an obstacle in our understanding of antigen specific responses to pathogens and vaccine candidates. The goal of this project is to determine allele diversity and frequency of the MHC class I genes in a herd of pure-bred registered Holstein dairy cattle (University of Vermont Research Herd). The planned experiments involve tissue typing the MHC of Holstein dairy cattle and developing tools to study the T cell response to Foot- and- Mouth Disease (FMD) virus. The first objective is to quantify the extent of MHC diversity; we will establish the potential for use of tetramer technology to study the immune response to FMD virus and a FMD vaccine candidate currently under development by USDA-ARS. The second objective is to select representative MHC types and evaluate the Holstein immune response to an adenovirus vectored FMD vaccine using tetramer technology. In FY 2010 we accomplished the following: 1. Whole blood samples have been collected from approximately 50 Holstein cattle. Aliquots have been cataloged in frozen storage. Genomic DNA and mRNA has been extracted from samples and cDNA synthesized from mRNA. DNA samples have been cataloged in frozen storage building a library of samples for the Holstein herd. 2. PCR methods for MHC class I genes have been adapted and the class I sequence type has been determined for 20 Holstein cattle. All 20 cattle express at least one of two alleles of Class I MHC proteins, demonstrating the homogeneity of class I genes in this population. 3. A graduate student has been recruited to begin working on the project (start date, August 15, 2010). In FY 2011 we will continue objective 1, identifying the MHC class 1 genotype of at least 100 cattle in the Holstein herd. When the dominant gene variants are identified, the protein products will be used to synthesize MHC class I tetramers for flow cytometry experiments. This collaborative research project was monitored through email and telephone exchange as well as by site visits to ARS, PIADC and the University of Vermont.