Conformational Dependence ofAnaplasma marginaleMajor Surface Protein 5 Surface-Exposed B-Cell Epitopes

Abstract

The Anaplasma marginale outer membrane is composed of immunogenic major surface proteins (MSPs) linked both covalently and noncovalently in multimeric complexes (M. C. Vidotto, T. C. McGuire, T. F. McElwain, G. H. Palmer, and D. P. Knowles, Infect. Immun. 62:2940–2946). Consequently, effective induction of antibody against surface-exposed MSP epitopes has been postulated to require maintenance of MSP secondary through quatenary structures. Using MSP5 as a model and the approach of epitope mapping with recombinant expressed full-length and truncated proteins, we demonstrated that the immunodominant surface epitope bound by monoclonal antibody (MAb) ANAF16C1 required disparate amino- and carboxy-terminal regions of MSP5, indicating the conformational dependence of this epitope. The required amino-terminal MSP5 region included the cysteines involved in intramolecular disulfide bonding. The dependence of the immunodominant epitope on disulfide bonding was confirmed by loss of MAb ANAF16C1 binding to MSP5 following disulfide bond reduction and covalent modification of the reduced sulfhydryl groups. The recognition of the MSP5 immunodominant epitope by antibody induced by protective immunization with A. marginale outer membranes was also conformationally dependent, as shown by the loss of epitope binding following serum adsorption with native but not reduced and denaturedA. marginale. Importantly, the antibody response to all immunodominant MSP5 surface epitopes was restricted to conformationally dependent epitopes, since the binding of polyclonal anti-MSP5 antibody to the A. marginale surface could be blocked by adsorption with native but not denatured and reduced MSP5. These results confirm the importance of the secondary and tertiary structures of MSP epitopes as immune system targets and support the testing of immunogens which maintain the required conformation.

Genetic diversity ofAnaplasmaspecies major surface proteins and implications for anaplasmosis serodiagnosis and vaccine development

“…The MSP5 sequence is highly conserved and thus similar among strains of A. marginale, as well as between A. marginale, A. centrale, and A. phagocytophilum (Table 2). The cross-reactivity of the MSP5 test with multiple species of Anaplasma has been confirmed through the identification of common regions defined to be essential for ANAF16C1 reactivity (Munodzana et al, 1998). Thus, the MSP5 cELISA does not differentiate Anaplasma species in regions where co-infection with A. phagocytophylum and A. marginale or A. centrale occurs (de la Hofmann-Lehmann et al, 2004;Lin et al, 2004).…”

Persistence of Anaplasma ovis Infection and Conservation of the msp-2 and msp-3 Multigene Families within the Genus Anaplasma

“…2, lanes 2 and 5) and to that of the 44-kDa MSP-2 homologue recently identified in the closely related HGE agent (35). MAb ANAF16C1, which reacts with a genus-conserved MSP-5 epitope (18,33), was the positive antibody control (Fig. 2, lanes 8 [A. ovis] and 3 [A. marginale]).…”

Expression of a Gene Encoding the Major Antigenic Protein 2 Homolog ofEhrlichia chaffeensisand Potential Application for Serodiagnosis

“…Fixing the membrane-bound protein in 20% methanol did not inhibit reactivity with immune sera but resulted in enhancement of the signal, probably associated with increased amounts of protein being fixed to the nitrocellulose membrane. Similarly, conformationally dependent epitopes were also identified on MSP5 of A. marginale (12). In that study, reduction of disulfide bonds using dithiothreitol, followed by treatment with iodoacetamide to modify sulfhydryl groups, completely abolished monoclonal antibody binding to MSP5 in dot blot assays.…”