Immunogenicity of Anaplasma marginale Type IV Secretion System Proteins in a Protective Outer Membrane Vaccine

Abstract

Rickettsial pathogens in the genera Anaplasma and Ehrlichia cause acute infection in immunologically naive hosts and are major causes of tick-borne disease in animals and humans. Immunization with purified outer membranes induces protection against acute Anaplasma marginale infection and disease, and a proteomic and genomic approach recently identified 21 proteins within the outer membrane immunogen in addition to the well-characterized major surface proteins MSP1 to MSP5. Among the newly described proteins were the type IV secretion system (TFSS) proteins VirB9, VirB10, and conjugal transfer protein (CTP). In other gram-negative bacteria, TFSS proteins form channels, facilitate secretion of molecules, and are required for intracellular survival. However, TFSS proteins have not been explored as vaccine antigens. In this study we demonstrate that in Anaplasma marginale outer membrane-vaccinated cattle, VirB9, VirB10, and CTP are recognized by serum immunoglobulin G2 (IgG2) and stimulate memory T-lymphocyte proliferation and gamma interferon secretion. VirB9 induced the greatest proliferation in CD4+ T-cell lines, and VirB9-specific CD4+ T-cell clones responded to three A. marginale strains, confirming the VirB9-specific T-cell responses are directed against epitopes in the native protein. The three TFSS proteins are highly conserved with orthologous proteins in Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia canis. Recognition of TFSS antigens by CD4+ T cells and by IgG2 from cattle immunized with the protective outer membrane fraction provides a rationale for including these proteins in development of vaccines against A. marginale and related pathogens.

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

“…Partial sequencing of VirB9, VirB10, and EF-Tu from different Brazilian isolates of A. marginale suggests that they are conserved (unpublished observations). VirB9 and VirB10 were found to be highly conserved with orthologous proteins in Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia canis (Lopez et al 2007). …”

“…In other Gram-negative bacteria, TFSS proteins form channels and facilitate secretion of molecules, and are required for intracellular survival (Lopez et al 2007). In prokaryotes, EF-Tu can form ternary complexes with GTP and aminoacyl-tRNA.…”

“…These data corroborate findings of Lopez et al (2005) which demonstrated that VirB9, VirB10 and EF-Tu are recognized by IgG2 of A. marginale outer membrane-vaccinated cattle. These proteins also stimulate memory T-lymphocyte proliferation and IFN-γ secretion Lopez et al (2007). This is important, as protective immune response might require the interaction of humoral and cellular components.…”

“…In A. marginale outer membrane-vaccinated cattle, VirB9 and VirB10 also stimulate proliferation of memory T-lymphocytes 0 and secretion of Interferon-gamma (IFN-γ) (Lopez et al 2007). Protective immunity against this rickettsia has been hypothesized to require production of IFN-γ and induction of IgG2 against outer membrane protein epitopes (Brown et al 1998).…”

Analysis of membrane protein genes in a Brazilian isolate of Anaplasma marginale

“…It is possible that TFSS expression is regulated and associated with survival in intracellular bacteria . Due to its location on the cell surface, its highly conserved nature and intracellular survival requirements, TFSS proteins in Gram-negative bacteria are consistent targets of immunological investigations (Lopez et al 2007). In the present study, the predicted amino acid sequences of two TFSS proteins, VirB3 and VirB9-1 (Sutten et al 2010) were analyzed.…”