2010 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.
The hypothesis that the intermolecular linkages between outer membrane proteins are required for induction of protective immunity has been tested. These experiments indicate that the intermolecular linkages between these proteins enhance antibody production but not protective immunity. Thus, epitopes shared by spatially linked molecules are not required to induce protective immunity. Consequently, these findings simplify the approach required to identify protective epitopes. These findings will be written up and submitted for review in the next 6 months.
Msp2 of A. marginale is a hypervariable protein that allows the pathogen to evade the host immune system and establish persistent infection. Consequently, much research has been focused on Msp2 as a potential vaccine component. The hypothesis that the immune response induced by immunization altered the anti-Msp2 antibody repertoire as compared to that induced during infection, shifting the immune response toward conserved and thus broadly protective epitopes was tested. Among immunized animals, the anti-Msp2 antibody response to either the conserved or hypervariable regions of Msp2 did not correlate with protective immunity. Thus, conserved outer membrane proteins, other than Msp2, are deemed responsible for the complete clearance of A. marginale following challenge. Future efforts in vaccine development will focus in the identification of broadly protective, conserved epitopes. Additionally, strains are being collected both regionally and internationally, and in some cases (Australian strain) are being sequenced. The ADODR participated in weekly onsite meetings with WSU collaborators.