Mapping Antigenic Sites of an Immunodominant Surface Lipoprotein of Mycoplasma agalactiae , AvgC, with the Use of Synthetic Peptides

Abstract

As a first step toward the design of an epitope vaccine to prevent contagious agalactia, the strongly immunogenic 55-kDa protein of Mycoplasma agalactiae was studied and found to correspond to the AvgC protein encoded by the avgC gene. The avg genes of M. agalactiae, which encode four variable surface lipoproteins, display a significant homology to the vsp (variable membrane surface lipoproteins) genes of the bovine pathogen Mycoplasma bovis at their promoter region as well as their N-terminus-encoding regions. Some members of the Vsp family are known to be involved in cytoadhesion to host cells. In order to localize immunogenic peptides in the AvgC antigen, the protein sequence was submitted to epitope prediction analysis, and five sets of overlapping peptides, corresponding to five selected regions, were synthesized by Spot synthesis. Reactive peptides were selected by immunobinding assay with sera from infected sheep. The three most immunogenic epitopes were shown to be surface exposed by immunoprecipitation assays, and one of these was specifically recognized by all tested sera. Our study indicates that selected epitopes of the AvgC lipoprotein may be used to develop a peptide-based vaccine which is effective against M. agalactiae infection.

Development of a Sensitive and Specific Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigens for Rapid Detection of Antibodies against Mycoplasma agalactiae in Sheep

“…Many studies have now reported that P30, P48, P55, and P80 are the immunodominant surface proteins of M. agalactiae (3,14,16,22). The immunoreactivities and suitability of these proteins as diagnostic antigens have been demonstrated.…”

“…This might suggest that P55 shares antigenic similarity with P26. In a previous study, Santona et al (16) showed by N-terminal sequencing that P55 was identical to the AvgC protein encoded by the avgC gene (5). The M. agalactiae variable gene (avg) system is a cluster of four genes that encode a family of surface lipoproteins characterized by high-frequency phase and size variations (5).…”

“…In a previous work, we identified and characterized the surface membrane proteins involved in the immunological response of sheep (21). Among the proteins we characterized at the molecular level, some are stable proteins, such as P80, P48, and P30 (3,14,22), while others belong to a family of variable surface lipoproteins which undergo high-frequency phase and size variation (6,16); one of them (P40), however, plays an important role in attachment to the host cell (4). These proteins are expressed with remarkable variability among M. agalactiae strains and isolates (17,19).…”

An Introduction to B-Cell Epitope Mapping and In Silico Epitope Prediction

“…The dot blot requires the purified molecule to be spotted on the membrane and is mainly used for qualitative detection [43]. The peptides can be synthesized on pins (PEPSCAN®), on a cellulose membrane support (SPOT method®), or on peptide microarrays [44, 45]. Such techniques simplify the handling of large numbers of peptides and eliminate the need for identification of positive peptides by sequencing or by mass spectrometry.…”

Genetic immunization with the immunodominant antigen P48 of Mycoplasma agalactiae stimulates a mixed adaptive immune response in BALBc mice

“…Membrane surface proteins can also play a basic role in pathogenesis of mycoplasmas especially by acting as cytoadhesins in the attachment of mycoplasmas to host cells (Zhang et al, 1995;Santona et al, 2002). Evasion of immune responses through surface antigen variability and modulation of immunity may render vaccination ineffective.…”