Saccharides cross-reactive with Bacillus anthracis spore glycoprotein as an anthrax vaccine component

Bacillus anthracis is a spore-forming bacterium that causes anthrax in humans and in other mammals. The glycoprotein BclA (Bacillus collagen-like protein of anthracis) is a major constituent of the exosporium, the outermost surface of B. anthracis spores. The glycosyl part of BclA is an oligosaccharide composed of 2-O-methyl-4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-d-glucose, referred to as anthrose, and three rhamnose residues. A structure similar to anthrose, 4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-d-glucose is found in the side chain of the capsular polysaccharide (CPS) of Shewanella spp. MR-4. Under certain growth conditions the bacteria produce a variant CPS lacking one methyl group on the hydroxybutyrate, 4-(3-hydroxybutanamido)-4,6-dideoxy-d-glucose. Contrary to anthrose, neither of the Shewanella CPSs is 2-O methylated. Here, we report that both Shewanella CPS variants react with anti-B. anthracis spore sera. We also found that these antisera reacted with flagellae of Pseudomonas syringae, reported to be glycosylated with a similar terminal saccharide, 4-(3-hydroxybutanamido)-4,6-dideoxy-2-O-methyl-d-glucose. Sera produced by immunization with Shewanella or P. syringae cells bound to B. anthracis spores but not to Bacillus cereus spores in a fluorescent microscopy assay. These experiments show that methylation of the anthrose at the O-2 of the sugar ring and at the C-3 of 3-hydroxybutyrate are not essential for induction of cross-reactive antibodies. We report the preparation, characterization, and antibody responses to protein conjugates of the two variants of Shewanella CPS. Both conjugates induced antibodies that bound to both Shewanella CPS variants by ELISA and to B. anthracis spores, as detected by fluorescent microscopy. We propose the use of Shewanella CPS conjugates as a component of an anthrax vaccine.