A lipoteichoic acid has recently been shown to be conserved in the majority of strains from Clostridium difficile and as such is being considered as a possible vaccine antigen. In this study we examine the candidacy of the conserved lipoteichoic acid by demonstrating that it is possible to elicit antibodies against C. difficile strains following immunisation of rabbits and mice with glycoconjugates elaborating the conserved lipoteichoic acid antigen. The present study describes a conjugation strategy that utilises an amino functionality, present at approximately 33 % substitution of the N-acetyl-glucosamine residues within the LTA polymer repeating unit, as the attachment point for conjugation. A maleimide-thiol linker strategy with the maleimide linker on the carboxyl residues of the carrier protein and the thiol linker on the carbohydrate was employed. Immunisation derived antisera from rabbits and mice, recognised all strains of C. difficile vegetative cells examined, despite an immune response to the linkers also being observed. These sera recognised live cells in an immunofluorescence assay and were also able to recognise the spore form of the bacterium. This study has illustrated that the LTA polymer is a highly conserved surface polymer of C. difficile that is easily accessible to the immune system and as such merits consideration as a vaccine antigen to combat C. difficile infection.

The C chemokine lymphotactin has been characterized as a T cell chemoattractant both in vitro and in vivo. To determine whether lymphotactin expression within tumors could influence tumor growth, we transfected an expression vector for lymphotactin into SP2/0 myeloma cells and tested their ability to form tumors in BALB/c and nude mice. Transfection did not alter cell growth in vitro. Whereas SP2/0 cells gave rise to a 100% tumor incidence, lymphotactin-expressing SP2/0-Lptn tumors invariably regressed in BALB/c mice and became infiltrated with CD4+ and CD8+ T cells and neutrophils. Regression of the SP2/0-Lptn tumors was associated with a type 1 cytokine response and dependent on both CD4+ and CD8+ T cells, but not NK cells. Both SP2/0 and SP2/0-Lptn tumors grew in nude mice, but growth of the latter tumors was retarded and associated with heavy neutrophil responses; this retardation of SP2/0-Lptn tumor growth was reversed by neutrophil depletion of the mice. Our data also indicate that mouse neutrophils express the lymphotactin receptor XCR1 and that lymphotactin specifically chemoattracts these cells in vitro. Thus, lymphotactin has natural adjuvant activities that may augment antitumor responses via effects on both T cells and neutrophils and thereby could be important in gene transfer immunotherapies for some cancers.

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Dental caries remains a major health issue and the Gram-positive bacterium Streptococcus mutans is considered as the major pathogen causing caries. More recently, S. mutans has been recognised as a cause of endocarditis, ulcerative colitis and fatty acid liver disease along with the likelihood of increased cerebral hemorrhage following a stroke if S. mutans is present systemically. We initiated this study to examine the vaccine candidacy of the serotype specific polysaccharides elaborated by S. mutans. We have confirmed the carbohydrate structures for the serotype specific rhamnan containing polysaccharides from serotypes c, f and k. We have prepared glycoconjugate vaccines using the rhamnan containing polymers from serotypes f and k and immunised mice and rabbits. We consistently obtained a robust immune response to the glycoconjugates with cross-reactivity consistent with the structural similarities of the polymers from the different serotypes. We developed an opsonophagocytic assay which illustrated the ability of the post-immune sera to facilitate opsonophagocytic killing of the homologous and heterologous serotypes at titers consistent with the structural homologies. We conclude that glycoconjugates of the rhamnan polymers of S. mutans are a potential vaccine candidate to target dental caries and other sequelae following the escape of S. mutans from the oral cavity.

Pseudomonas aeruginosa was added to the World Health Organization’s priority pathogen list for research and development of new antibiotics in 2017. Alongside the development of new antibiotics to fight antimicrobial-resistant P. aeruginosa, vaccines would be an appealing addition to the toolbox health professionals have against this bacteria, which causes life-threatening respiratory infections. Recently, the structure of a novel immunogenic terminal carbohydrate moiety on the cell surface of P. aeruginosa was elucidated, consisting of a 3-O-methyl (1→4)-α-d-rhamnan penta­saccharide. As isolating this oligo­saccharide from P. aeruginosa in sufficient amounts for producing a conjugate vaccine is challenging, herein we describe the synthesis of 3-O-methyl d-rhamnose oligo­saccharide. We also report the conjugation of the synthetic penta­saccharide to human serum albumin and its resulting immunogenicity.

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Pseudomonas aeruginosa produces a variety of cell surface glycans. Previous studies identified a common polysaccharide (PS) antigen often termed A-band PS that was composed of a neutral D-rhamnan trisaccharide repeating unit as a relatively conserved cell surface carbohydrate. However, nuclear magnetic resonance (NMR) spectra and chemical analysis of A-PS preparations showed the presence of several additional components. Here, we report the characterization of the carbohydrate component responsible for these signals. The carbohydrate antigen consists of an immunogenic methylated rhamnan oligosaccharide at the nonreducing end of the A-band PS. Initial studies performed with the isolated antigen permitted the production of conjugates that were used to immunize mice and rabbits and generate monoclonal and polyclonal antibodies. The polyclonal antibodies were able to recognize the majority of P. aeruginosa strains in our collection, and three monoclonal antibodies were generated, one of which was able to recognize and facilitate opsonophagocytic killing of a majority of P. aeruginosa strains. This monoclonal antibody was able to recognize all P. aeruginosa strains in our collection that includes clinical and serotype strains. Synthetic oligosaccharides (mono-to pentasaccharides) representing the terminal 3-Omethyl D-rhamnan were prepared, and the trisaccharide was identified as the antigenic determinant required to effectively mimic the natural antigen recognized by the broadly cross-reactive monoclonal antibody. These data suggest that there is considerable promise in this antigen as a vaccine or therapeutic target.

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