Immunogenicity and linked recognition of Anaplasma marginale type IV secretion system proteins

Like several other bacterial pathogens, Anaplasma marginale has an outer membrane (OM) that induces protection from infection and disease. However proteins that confer protective immunity and whether the protection requires linked T-cell and immunoglobulin G epitopes and/or interacting proteins are not known. Our goal was to target the conserved type IV secretion system (T4SS) to identify immunogenic membrane proteins that are interacting and linked recognition candidates. Linked recognition is a process by which B cells are optimally activated by helper T cells responding to the same or physically associated antigen. The T4SS is a membrane complex within many bacterial pathogens secreting virulence factors and promoting host cell invasion and intracellular survival. A. marginale T4SS proteins VirB2, VirB4-1, VirB4-2, VirB6-1, VirB7, VirB8-2, VirB9-1, VirB9-2, VirB10, VirB11 and VirD4 were screened for their ability to induce IgG and stimulate CD4 + T cells from OM vaccinated cattle. VirB9-1, VirB9-2, and VirB10 induced the strongest IgG and T cell responses in the majority of cattle, although animals with major histocompatibility complex class II (MHC class II) DRB3 RFLP types 3/16, 8/23, and 16/27 lacked T-cell responses to VirB9-1, VirB9-1 and VirB9-2, or VirB9-2 and VirB10, respectively. For these animals-specific IgG production may result from T cell help provided by responses to an interacting protein partner(s). Interacting partners were determined by far western blotting and confirmed by immunoprecipitation assays, and revealed, for the first time, specific interactions of VirB9-1 with VirB9-2 and VirB10. Since, MHC class II molecules influence antigen-specific CD4+ T-lymphocyte responses, specific bovine leukocyte antigens required for presentation of peptides from VirB9-1, VirB9-2, and VirB10 were also determined. Overlapping peptides spanning each protein were tested in T cell assays with autologous antigen presenting cells (APC) and artificial APC expressing combinations of bovine DR and DQ molecules. Twenty immunostimulatory peptides were identified, of which, four DRA/DRB3 presented fifteen peptides, four DQA/DQB presented seven peptides, and three functional mixed isotype (DQA/DRB3) were identified. The immunogenicity, interactions, and broad MHC class II presentation of VirB9-1, VirB9-2, and VirB10 justify their testing as a linked multi-epitope vaccine against A. marginale