Characterisation of a unique epitope at the non-reducing end of the Brucella O-polysaccharide

Abstract

Brucellosis is a global zoonosis caused by the Gram negative bacteria Brucella. In animals the disease causes reproductive complications and in humans the disease causes debilitating illness. There is no vaccine for humans, wildlife or swine and the licensed vaccines for ruminants are protective but have disadvantages. The vaccines are live attenuated strains that can cause abortions in livestock. Humans can become infected by these vaccine strains. In animals the most protective vaccines contain O-polysaccharides (OPS) which induce antibodies that cannot be differentiated, by serodiagnostic tests, from antibodies raised during Brucella infection. The O-polysaccharide (OPS) of Brucella species is an unbranched homopolymer of 4,6-dideoxy-4-formamido-α-D-mannopyranosyl residues (D-Rha4NFo). These residues are linked either by α1-2 or α1-3 glycosidic linkages. It is the sequence of these linkages that define the different epitopes of Brucella OPS. There are currently three described epitopes of the Brucella OPS that co-exist within the same OPS molecule; the A, M and C/Y epitopes. There is also a fourth epitope, the C epitope, which has not yet been formally agreed upon. The aim of this project was to determine whether the terminal D-Rha4NFo residues at the non-reducing end of the Brucella OPS form a previously undescribed series of tip epitope structures, defined by the necessity of inclusion of the terminal D-Rha4NFo to enable antibody binding. The putative tip epitope was investigated by comparing antibody binding to whole cells, lipopolysaccharides (LPS), modified (so as to remove the tip) OPS and synthetic oligosaccharide-BSA antigens. The oligosaccharide-BSA antigens represent different epitopes and permit discrimination of different antibody populations. Results showed that terminal residues at the non-reducing end of the OPS bind antibodies and these antibodies are a separate anti-tip antibody population, different from the antibodies induced by the internal linear epitopes of the OPS. Antibody binding studies showed that the in-tact terminal D-Rha4NFo is required for antibody binding to the tip epitope. Mice immunised with modified OPS-glycoconjugates, with linear epitopes and no tip epitope, raised anti-linear antibodies but did not raise anti-tip antibodies. These findings may show a pathway towards the generation of an OPS-glycoconjugate vaccine for brucellosis that permits differentiation of infected from vaccinated animals (DIVA) when paired with oligosaccharide-BSA diagnostic antigens containing the tip and M epitope. This is important for supporting livestock vaccination programmes to control brucellosis in order to reduce its impact upon human and animal health.