2009 Annual Report 1a.Objectives (from AD-416) Develop effective vaccines for anaplasmosis using an integrated genomic and proteomic approach to identify the essential immunogens within the immunoprotective Anaplasma marginale surface protein complex. This research will test whether intermolecular linkages between outer membrane proteins are required for induction of strong memory/effector CD4+ T lymphocyte, high titer IgG2 responses, and protective immunity. The identified immunogens will be tested for conservation among multiple A. marginale strains from endemic regions. 1b.Approach (from AD-416) The structural requirements for induction of memory/effector CD4+ T lymphocytes, high titer IgG2, and protective immunity will be tested using the surface protein immunogen with either intact intermolecular linkages (cross-linked) or disrupted linkages (cross-linked then reduced). Mapping the B and T cell immunogenicity of protein partners within the cross-linked complex will define the minimal contributions required for protection. This is essential as the minimal immunogen will be more amenable to both standardization and development of low-cost vaccine. To ensure that the epitopes required for immunity are broadly represented among currently transmitted strains (rather than only “historical” strains isolated between 1950 and 1995), both outbreak and endemic strains will be isolated, genotyped, and the sequences of the relevant immunogenic proteins determined by targeted sequencing of their encoded genes. The strains will be isolated from endemic regions in United States and from regions in Mexico that export cattle to the U.S. The relevance of strain-specific polymorphisms to protective immunity and vaccine efficacy will be determined using CD4+ T lymphocytes and IgG2 antibody from immunized animals and immunologic differences confirmed by vaccine trials with heterologous strain challenge. 3.Progress Report The immunogens which will be used to test the hypothesis that intermolecular linkages between outer membrane proteins are required for induction of protective immunity have been developed and purified. All animals have been immunized. Currently, the antibody response to the two immunogens is being measured. Subsequently, the animals will be challenged with Anaplasma marginale and the level of protective immunity induced by the different immunogens will be compared. The ADODR participated in weekly onsite meetings with WSU collaborators.