Anaplasma marginale major surface protein 2 CD4+-T-cell epitopes are evenly distributed in conserved and hypervariable regions (HVR), whereas linear B-cell epitopes are predominantly located in the HVR

Abstract

Organisms in the genus Anaplasma express an immunodominant major surface protein 2 (MSP2), composed of a central hypervariable region (HVR) flanked by highly conserved regions. Throughout Anaplasma marginale infection, recombination results in the sequential appearance of novel MSP2 variants and subsequent control of rickettsemia by the immune response, leading to persistent infection. To determine whether immune evasion and selection for variant organisms is associated with a predominant response against HVR epitopes, T-cell and linear B-cell epitopes were localized by measuring peripheral blood gamma interferon-secreting cells, proliferation, and antibody binding to 27 overlapping peptides spanning MSP2 in 16 cattle. Similar numbers of MSP2-specific CD4(+) T-cell epitopes eliciting responses of similar magnitude were found in conserved and hypervariable regions. T-cell epitope clusters recognized by the majority of animals were identified in the HVR (amino acids [aa] 171 to 229) and conserved regions (aa 101 to 170 and 272 to 361). In contrast, linear B-cell epitopes were concentrated in the HVR, residing within hydrophilic sequences. The pattern of recognition of epitope clusters by T cells and of HVR epitopes by B cells is consistent with the influence of protein structure on epitope recognition.

FIG. 1.

Hydropathic profile of full-length A. marginale MSP2. The central hypervariable region, aa 190 to 272, which represents the MSP2 A variant from the Florida strain (6), is predominantly hydrophilic and identified by shading. Amino acid position is indicated on the x axis, and the hydropathy index, representing the average value over a window of 19 amino acids, is indicated on the y axis. The overlapping peptides are represented under the hydropathic profile as black bars in groups of five to illustrate the relative position in the whole protein. The numbering of the amino acids corresponds to the msp2 11.2 genomic DNA clone (24).

FIG. 2.

Identification of T-lymphocyte subsets that secrete IFN-γ detected by ELISPOT assay. PBMC from MSP2-alum-CpG ODN-immunized animals no. 87 (panel A) and no. 78 (panel B), untreated and after depletion of either CD4⁺ cells or CD8⁺ cells and γδ T cells, were tested by an IFN-γ ELISPOT assay. The results are presented as the mean number of spot-forming cells (SFC) plus 1 standard deviation per 10⁶ PBMC cultured with 10 μg of antigen per ml after subtracting the mean number of SFC in cells cultured with medium.