The Tip of Brucella O-Polysaccharide Is a Potent Epitope in Response to Brucellosis Infection and Enables Short Synthetic Antigens to Be Superior Diagnostic Reagents.

Brucellosis is a global disease and the world's most prevalent zoonosis. All cases in livestock and most cases in humans are caused by members of the genus Brucella that possess a surface O-polysaccharide (OPS) comprised of a rare monosaccharide 4-deoxy-4-formamido-D-mannopyranose assembled with α1,2 and α1,3 linkages. The OPS of the bacterium is the basis for serodiagnostic tests for brucellosis. Bacteria that also contain the same rare monosaccharide can induce antibodies that cross-react in serological tests. In previous work we established that synthetic oligosaccharides, representing elements of the Brucella A and M polysaccharide structures, were excellent antigens to explore the antibody response in the context of infection, immunisation and cross reaction. These studies suggested the existence of antibodies that are specific to the tip of the Brucella OPS. Sera from naturally and experimentally Brucella abortus-infected cattle as well as from cattle experimentally infected with the cross-reactive bacterium Yersinia enterocolitica O:9 and field sera that cross react in conventional serological assays were studied here with an expanded panel of synthetic antigens. The addition of chemical features to synthetic antigens that block antibody binding to the tip of the OPS dramatically reduced their polyclonal antibody binding capability providing conclusive evidence that the OPS tip (non-reducing end) is a potent epitope. Selected short oligosaccharides, including those that were exclusively α1,2 linked, also demonstrated superior specificity when evaluated with cross reactive sera compared to native smooth lipopolysaccharide (sLPS) antigen and capped native OPS. This surprising discovery suggests that the OPS tip epitope, even though common to both Brucella and Y. enterocolitica O:9, has more specific diagnostic properties than the linear portion of the native antigens. This finding opens the way to the development of improved serological tests for brucellosis.

A Three Component Synthetic Vaccine Containing a ß-Mannan T-Cell Peptide Epitope and a ß-Glucan Dendritic Cell Ligand.

Glycoconjugates prepared from the capsular polysaccharide of several pathogenic bacteria and carrier proteins, such as CRM 197 or tetanus toxoid, have been one of the most successful public health measures to be implemented in the last quarter century. A crucial element in the success of conjugate vaccines has been the recruitment of T-cell help and systematic induction of a secondary immune response. The seminal discovery, that degraded polysaccharide fragments with attached peptide are presented to the T-cell receptor of carbohydrate specific T-cells by MHC-II molecules that bind to the peptide component of degraded vaccine, suggests potentially novel designs for conjugate vaccines. A fully synthetic conjugate vaccine was constructed from a 1,2-linked ß-mannose trisaccharide conjugated to a T-cell peptide, previously shown to afford protection against Candida albicans. This combined B- and T-cell epitope was synthesized with a C-terminal azidolysine residue for subsequent conjugation by click chemistry. Four copies of a ß-1,3 linked hexaglucan dendritic cell epitope were conjugated to an asymmetric dendrimer bearing an alkyne terminated tether. Click chemistry of these two components created a conjugate vaccine that induced antibodies to all three epitopes of the fully synthetic construct.

Novel Solutions for Vaccines and Diagnostics To Combat Brucellosis.

Brucellosis is diagnosed by detection of antibodies in the blood of animals and humans that are specific for two carbohydrate antigens, termed A and M, which are present concurrently in a single cell wall O-polysaccharide. Animal brucellosis vaccines contain these antigenic determinants, and consequently infected and vaccinated animals cannot be differentiated as both groups produce A and M specific antibodies. We hypothesized that chemical synthesis of a pure A vaccine would offer unique identification of infected animals by a synthetic M diagnostic antigen that would not react with antibodies generated by this vaccine. Two forms of the A antigen, a hexasaccharide and a heptasaccharide conjugated to tetanus toxoid via reducing and nonreducing terminal sugars, were synthesized and used as lead vaccine candidates. Mouse antibody profiles to these immunogens showed that to avoid reaction with diagnostic M antigen it was essential to maximize the induction of anti-A antibodies that bind internal oligosaccharide sequences and minimize production of antibodies directed toward the terminal nonreducing monosaccharide. This objective was achieved by conjugation of Brucella O-polysaccharide to tetanus toxoid via its periodate oxidized terminal nonreducing monosaccharide, thereby destroying terminal epitopes and focusing the antibody response on internal A epitopes. This establishes the method to resolve the decades-long challenge of how to create effective brucellosis vaccines without compromising diagnosis of infected animals.

Synthetic glycoconjugates characterize the fine specificity of Brucella A and M monoclonal antibodies.

The dominant cell wall antigen of Brucella bacteria is the O-polysaccharide component of the smooth lipopolysaccharide. Infection by various Brucella biovars causes abortions and infertility in a wide range of domestic and wild animals and debilitating disease in humans. Diagnosis relies on the detection of antibodies to the A and M antigens expressed in the O-polysaccharide. This molecule is a homopolymer of the rare monosaccharide, 4-formamido-4,6-dideoxy-d-mannopyranose (Rha4NFo). The A epitope is created by a uniform α1,2 linked internal polymeric sequence capped by a distinct tetrasaccharide sequence defining the M antigen. Unique oligosaccharides only available by chemical synthesis and conjugated via reducing and non-reducing residues to bovine serum albumin have revealed the structural basis of the fine specificity that allows the discrimination of these closely related A and M epitopes. All three M specific monoclonal antibodies (mAbs) are inferred to possess groove type binding sites open at each end, and recognize an α1,3 linked Rha4NFo disaccharide as a part of a trisaccharide epitope, which in two mAbs includes the terminal Rha4NFo residue. The binding site of one of these antibodies is sufficiently large to engage up to six Rha4NFo residues and involves weak recognition of α1,2 linked Rha4NFo residues. The third mAb binds an internal trisaccharide epitope of the M tetrasaccharide. Two A specific mAbs also possess groove type binding sites that accommodate six and four α1,2 linked Rha4NFo residues.