Immune sera from all animals bound significantly to whole MSP2 (Table ?(Table3)

Immune sera from all animals bound significantly to whole MSP2 (Table ?(Table3).3). 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 usually consistent with the influence of NFIL3 protein structure on epitope recognition. Tick-borne pathogens in the family infect and persist in reservoir mammalian hosts, with acute disease occurring after transmission to a na?ve host. Acute infection is usually characterized by high-level rickettsemia resulting in clinical disease, whereas persistent infection is usually typified by recurrent low-level, subclinical rickettsemic peaks (10, 13, 17). Common to the pathogens in the genera and are immunodominant outer membrane proteins (OMP) with defined conserved and variable domains (13, 15, 21, 23, 26, 30). In spp., gene conversion of pseudogenes into a single expression site provides an efficient mechanism to generate the large number of OMP variants seen during persistent contamination (1, 2, 4, 12). Novel MSP2 variants arise in sequential rickettsemic peaks, followed by clearance that is associated with generation of primary immunoglobulin G (IgG) antibody responses against variant-specific B-cell epitopes localized to the central hypervariable region (HVR) (12, 13). More recently, epitopes that elicited high levels of Pyridoxine HCl gamma interferon (IFN-) production by CD4+ T cells were identified in conserved and variable domains of MSP2 (6, 7). The induction and recall of T-cell responses, including production of IFN- for macrophage activation and provision of help for antibody production, in particular IgG2, are proposed to be required for control of emergent variants (7, 25). The importance of CD4+ T cells and IFN- in controlling contamination with other spp. has also been reported (3, 14, 20). Detailed knowledge of the epitope specificity of T- and B-lymphocyte responses to Pyridoxine HCl conserved and variable domains of MSP2 is necessary to understand the selection for variants during contamination. If the majority of IFN–secreting CD4+ T lymphocytes were specific for HVR epitopes, control of emergent variants would require primary T-lymphocyte responses in addition to primary B-lymphocyte responses directed against variable region epitopes. Such predominant recognition of variable epitopes could facilitate immune evasion and persistent infection. Previous studies that identified T-cell epitopes in conserved and hypervariable regions of MSP2 were performed with proliferation assays using T-cell lines that were cultured for several weeks and obtained from only three animals (6, 7). Responses by in vitro propagated cell lines derived from few animals might be biased towards recognition of a few immunodominant epitopes. To more comprehensively test the hypothesis that the majority of T- and B-lymphocyte epitopes reside within the HVR, the present study used peripheral blood mononuclear cells (PBMC), rather than T-cell lines, from 16 outbred cattle immunized with MSP2. The animals expressed 10 different major histocompatibility complex (MHC) class II alleles and 11 different combinations of alleles. Pyridoxine HCl Furthermore, the present study measured the number of peptide-specific IFN–secreting cells by enzyme-linked immunospot (ELISPOT) assay in addition to levels of proliferation and IFN- secretion. The strengths of this approach are that this ELISPOT assay specifically quantifies those IFN–producing T lymphocytes that are important for the control of anaplasmosis and that the use of three T-cell assays may reveal a greater number of epitopes, as exhibited by a study examining epitope recognition by PBMC from humans exposed to malaria (11). To determine B-cell epitopes on MSP2, immune sera from the MSP2-immunized animals were also used to identify peptides by enzyme-linked immunosorbent assay (ELISA). All cattle were previously immunized (29) with gel-purified MSP2 (22, 28). Twenty animals were allocated into five groups with four calves per group. Groups I to IV were immunized six occasions with 50 g of MSP2 adsorbed in 2 mg of alum (29). The cattle received no additional adjuvant (group I) or the following: 1 mg of control non-CpG oligodeoxynucleotide (ODN) R2006 (group II), 1 mg of CpG ODN 2006 (group III), or 10 g of recombinant human interleukin-12 (IL-12) (group IV). Calves in group V served as a control group and received alum plus CpG ODN 2006 but no MSP2. The Kyte and Doolittle method (18) was used.