Wednesday, June 20, 2007 - 10:30 AM
Cottonwoods (Boise Centre on the Grove)

Immunoproteomic Analysis of the Protective Outer Membrane Fraction of the Rickettsial Pathogen Anaplasma marginale

Wendy C. Brown1, Job E. Lopez1, Paul A. Beare2, and Robert A. Heinzen2. (1) Washington State University, Pullman, WA, (2) Rocky Mountain Laboratories, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT

Rickettsial pathogens in the genera Anaplasma and Ehrlichia cause acute infection in immunologically na´ve hosts and are major causes of tick-borne disease in animals and humans. Immunization with Anaplasma marginale purified outer membranes (OM) induces complete protection against anaplasmosis in 75% of cattle, whereas immunization with the immunodominant major surface proteins such as MSP2 has provided little or no protection. The completed genome sequence of A. marginale facilitated the identification of subdominant and less abundant immunogenic proteins in the OM fraction, using two approaches. First, 2-D electrophoresis and immunoblotting of OM with immune sera identified numerous antigenic protein spots. Analysis of individual excised proteins by LC-MS/MS identified 21 novel antigens including three type IV secretion system (TFSS) proteins, conjugal transfer protein, VirB9, and VirB10. These were expressed and shown to stimulate IgG2, and CD4+ T cell proliferation and IFN-γ production, responses associated with protective immunity in outer membrane vaccinates. A second approach involved in vitro transcription and translation (IVTT) of ORFs encoding proteins predicted to be membrane localized or to have a signal peptide. PCR products of selected ORFs engineered to express antibody-binding sequence tags were amplified and expressed using IVTT. As proof of principal, VirB9 and additional outer membrane proteins known to stimulate T cell responses were expressed by IVTT and affinity purified by binding to anti-tag antibody coupled to protein-G bound beads, and the beads were added to APC and used to stimulate immune CD4+ T cell proliferation. This novel technology can be used to rapidly screen a large number of proteins from a given pathogen for recognition by both antibody and T lymphocytes if the genome sequence is available.