Expression of major surface protein 2 antigenic variants during acute Anaplasma marginale Rickettsemia

Antigenic variants of Anaplasma marginale major surface protein 2 (MSP-2), a target of protective immune responses, have been detected by use of copy-specific monoclonal antibodies reactive with some, but not all, organisms during acute rickettsemia. The presence of polymorphic msp-2 genes was confirmed by cloning and sequencing two gene copies, 11.2 and DF5, each of which encodes a full-length MSP-2 with a unique amino acid sequence. Transcription of msp-2 genes during acute rickettsemia was analyzed by use of cDNA cloning of hybrid-selected msp-2 mRNA. Sequencing of cDNA clones, designated AR1 to AR14, indicated that DF5 msp-2 was transcribed during acute rickettsemia. Two classes of variant msp-2 genes were also transcribed during acute rickettsemia. The first class of variant transcripts, typified by clones AR3, AR4, AR7, and AR14, each encoded a single or small number of amino acid substitutions relative to DF5. The second type, AR5, encoded a large region of amino acid polymorphism, including additions, deletions, and substitutions, as compared to DF5. Specific antibody directed against the AR5 polymorphic region bound a unique MSP-2 expressed on A. marginale that was not recognized by antibody generated against DF5. Similarly, anti-AR5 peptide antibody reacted with a different MSP-2 that was not bound by anti-DF5 antibody. This expression confirmed that variant msp-2 transcripts encode structurally distinct MSP-2 molecules which bear unique B-cell epitopes. These results support the hypothesis that the large msp-2 gene family, which constitutes a minimum of 1% of the genome, encodes antigenic variants critical to evasion of a protective immune response directed against surface MSP-2 epitopes.