O-Polysaccharide Epitopic Heterogeneity at the Surface ofBrucellaspp. Studied by Enzyme-Linked Immunosorbent Assay and Flow Cytometry

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Abstract
SmoothBrucellastrains are classified into three serotypes, i.e., A+M−, A−M+, and A+M+, according to slide agglutination with A and M monospecific polyclonal sera. The epitopes involved have been located on the O-polysaccharide (O-PS) moiety of the smooth lipopolysaccharide (S-LPS), which represents the most exposed antigenic structure on the surface ofBrucellaspp. By use of monoclonal antibodies (MAbs) a number of epitope specificities on the O-PS have been reported: A, M, and epitopes shared by both A and M dominant strains, which have been named common (C) epitopes. The latter have been further subdivided, according to relative MAb binding in enzyme-linked immunosorbent assays (ELISA) to A- and M-dominantBrucellastrains and to cross-reactingYersinia enterocoliticaO:9, into five epitopic specificities: C (M>A), C (M=A), C/Y (M>A), C/Y (M=A), and C/Y (A>M). In the present study, we studied the occurrence of these epitopes at the surface of representatives of allBrucellaspecies and biovars including the live vaccine strains by analyzing the levels of MAb binding to wholeBrucellacells in ELISA and flow cytometry assays. In ELISA, the level of MAb binding correlated well with the previously defined epitope specificity and the serotype defined by polyclonal sera for eachBrucellaspecies, biovar, or strain. However, MAbs to the C (M=A) and C (M>A) epitopes showed insignificant binding toB. suisbiovar 2 strains and bound at lower titers toB. suisbiovar 3 andB. neotomaethan to the otherBrucellastrains. Some of the flow cytometry results were contradictory to those obtained by ELISA. In fact, it appeared by flow cytometry that all O-PS epitopes, including the A and M epitopes, are shared to different degrees byBrucellaspp. which nevertheless show a high degree of O-PS heterogeneity according to MAb binding intensities. The subdivision of MAb specificities andBrucellaserotypes was therefore less evident by flow cytometry than by ELISA. Whereas in ELISA the MAb specific for the A epitope showed insignificant binding toY. enterocoliticaO:9, this MAb bound strongly toY. enterocoliticaO:9 in flow cytometry. One of the two MAbs specific to the C (M=A) epitope also bound at a low but significant level toB. suisbiovar 2 strains. However, as in ELISA the MAb specific for the C (M>A) epitope did not bind at all toB. suisbiovar 2 strains in flow cytometry. Flow cytometry provided new information regarding specificity of the MAbs and may further explain some aspects of the capacity of passive protection of some MAbs against smoothBrucellainfection in mice. As shown in the present study the occurrence ofBrucellastrains apparently completely devoid of one specific C O-PS epitope (e.g.,B. suisbiovar 2 devoid of the C [M>A] epitope) offers the possibility of obtaining vaccine strains devoid of a diagnostic O-PS epitope, which could further help to resolve the problem of discriminating infected from vaccinated animals that remains a major goal in brucellosis research.