Highly Conserved Regions of the Immunodominant Major Surface Protein 2 of the Genogroup II Ehrlichial Pathogen Anaplasma marginale Are Rich in Naturally Derived CD4+ T Lymphocyte Epitopes that Elicit Strong Recall Responses

Abstract

Genogroup II ehrlichia, including the agent of human granulocytic ehrlichiosis, Ehrlichia phagocytophila, and the bovine pathogen Anaplasma marginale, express a markedly immunodominant outer membrane protein designated major surface protein 2 (MSP2). MSP2 is encoded by a multigene family, resulting in the expression of variant B cell epitopes. MSP2 variants are sequentially expressed in the repeated cycles of rickettsemia that characterize persistent A. marginale infection and control of each rickettsemic cycle is associated with development of a variant-specific IgG response. Importantly, these persistent rickettsemic cycles are controlled at levels 100-1000 times lower than those responsible for clinical disease during acute infection. Control of rickettsemia during persistence could result from an anamnestic Th lymphocyte response to conserved regions of MSP2 that enhances the primary Ab response against newly emergent variants. Comparison of MSP2 variants reveals conserved N and C termini flanking the central, surface-exposed hypervariable region that represents the variant B lymphocyte epitopes. We demonstrate MSP2-specific CD4+ T lymphocyte recognition of epitopes common to several strains of A. marginale and the related pathogen A. ovis. Furthermore, T lymphocyte lines from three individuals identified six to nine overlapping peptides representing a minimum of four to seven dominant or subdominant epitopes in these conserved N and C termini. Immunodominant peptides induced high levels of IFN-γ, a cytokine associated with protection against ehrlichia and needed for rapid generation of variant-specific IgG2. The presented data support the potential importance of a strong Th lymphocyte response to invariant MSP2 epitopes in controlling rickettsemia during persistent infection to subclinical levels.

Production of IFN-γ by CD4 T Cells Is Essential for Resolving Ehrlichia Infection

“…We demonstrated that CD4 T cells from IOE-infected mice proliferated in response to IOE Ags and secreted type I cytokines that were essential for host defense. The conclusion that CD4 T cells play an essential role during IOE infection is supported by data from other studies that have demonstrated a requirement for CD4 T cells during ehrlichial infection (12,14,31,32) as well as other intracellular bacterial infections (1,(33)(34)(35)(36). One likely role of CD4 T cells during IOE infection is to induce, via inflammatory cytokines, production of reactive nitrogen and/or oxygen species that may be responsible for the killing of intracellular ehrlichiae within macrophages (11).…”

CD4 + T Lymphocytes from Anaplasma marginale Major Surface Protein 2 (MSP2) Vaccinees Recognize Naturally Processed Epitopes Conserved in MSP3

“…The striking immunodominance of A. marginale MSP2 has been attributed to both protein abundance and the presence of multiple CD4 ϩ T-cell epitopes in the hypervariable region and flanking conserved regions (1,(6)(7)(8). The data presented here provide a third mechanism for immunodominance: processing of MSP2 T-cell epitopes from native MSP2 and MSP3 with presentation to CD4 ϩ T cells.…”

“…In the N terminus of MSP2, amino acids (aa) 46 to 69 are 70.8% identical to those of positions 51 to 74 in MSP3, and aa 116 to 144 in MSP2 are 82.7% identical to those in positions 128 to 156 of MSP3 (14). Previous studies had determined that aa 101 to 142 of MSP2 contained at least two Th-cell epitopes recognized by MSP2 vaccinees (animals 59 and 61 [7]), enabling us to test whether CD4 ϩ T cells from these cattle would respond to the corresponding sequence in MSP3. To this end, peptides P6 and P7, previously shown to stimulate CD4 ϩ T-cell lines from MSP2-immunized animals 61 and 59, respectively (7), and peptides representing the homologous sequence in MSP3 (Table 1) were tested with short-term T-cell lines from…”

“…The importance of MSP2 antigenic variation in vivo as an immune escape mechanism was indicated by the finding that newly emerging variants in persistently infected cattle stimulated a primary variant-specific antibody response (11). Furthermore, MSP2 contains CD4 ϩ T-cell epitopes both in the conserved regions flanking the central hypervariable region and in the hypervariable region itself (6,7). CD4 ϩ T-helper (Th)-cell epitopes conserved among MSP2 variants may allow for rapid anamnestic Th-cell responses and efficient IgG production against sequentially emerging variant organisms, controlling the bacteremia to subclinical levels (7).…”

“…1; Table 1). MSP2 was eluted from sodium dodecyl sulfate-polyacrylamide gels as described previously (7,20), and MSP3 was immunoaffinity purified with MSP3-specific monoclonal antibody AmG75C2 as described previously (13). A control CD4 ϩ T-cell clone, 61.4F11 specific for MSP2 peptide P8 (7) was also used (Fig.…”