Protection against Experimental Melioidosis with a Synthetic manno-Heptopyranose Hexasaccharide Glycoconjugate.

Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei and is associated with high morbidity and mortality rates in endemic areas. Antibiotic treatment is protracted and not always successful; even with appropriate therapy, up to 40% of individuals presenting with melioidosis in Thailand succumb to infection. In these circumstances, an effective vaccine has the potential to have a dramatic impact on both the scale and the severity of disease. Currently, no vaccines are licensed for human use. A leading vaccine candidate is the capsular polysaccharide consisting of a homopolymer of unbranched 1→3 linked 2-O-acetyl-6-deoxy-ß-d-manno-heptopyranose. Here, we present the chemical synthesis of this challenging antigen using a novel modular disaccharide assembly approach. The resulting hexasaccharide was coupled to the nontoxic Hc domain of tetanus toxin as a carrier protein to promote recruitment of T-cell help and provide a scaffold for antigen display. Mice immunized with the glycoconjugate developed IgM and IgG responses capable of recognizing native capsule, and were protected against infection with over 120 × LD50 of B. pseudomallei strain K96243. This is the first report of the chemical synthesis of an immunologically relevant and protective hexasaccharide fragment of the capsular polysaccharide of B. pseudomallei and serves as the rational starting point for the development of an effective licensed vaccine for this emerging infectious disease.

Characterization of the Burkholderia pseudomallei K96243 capsular polysaccharide I coding region.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to regions of Southeast Asia and Northern Australia. Both humans and a range of other animal species are susceptible to melioidosis, and the production of a group 3 polysaccharide capsule in B. pseudomallei is essential for virulence. B. pseudomallei capsular polysaccharide (CPS) I comprises unbranched manno-heptopyranose residues and is encoded by a 34.5-kb locus on chromosome 1. Despite the importance of this locus, the role of all of the genes within this region is unclear. We inactivated 18 of these genes and analyzed their phenotype using Western blotting and immunofluorescence staining. Furthermore, by combining this approach with bioinformatic analysis, we were able to develop a model for CPS I biosynthesis and export. We report that inactivating gmhA, wcbJ, and wcbN in B. pseudomallei K96243 retains the immunogenic integrity of the polysaccharide despite causing attenuation in the BALB/c murine infection model. Mice immunized with the B. pseudomallei K96243 mutants lacking a functional copy of either gmhA or wcbJ were afforded significant levels of protection against a wild-type B. pseudomallei K96243 challenge.