In this respect, we hypothesize that immune responses to dormant spores at the mucosal surface may inhibit spore uptake across the mucosa and may also target the susceptible emergent vegetative cell, thus preventing bacterial proliferation or enhancing bacterial clearance. a renewed interest in anthrax vaccines and early disease diagnostics.[6] Anthrax vaccine adsorbed (AVA; BioThrax?, Emergent BioSolutions Inc.) is currently the only licensed anthrax vaccine in the US.[7, 8] The principal immunogen of AVA is anthrax toxin protective antigen (PA). Antibody responses against PA Dihydroethidium target and block the toxemia that is a necessary prerequisite of vegetative cell growth and bacteremia. Vaccines comprising additional specific antigens have been proposed as improvements to PA-only formulations as they have potential to target inclusively the toxemia and the vegetative cell or infectious spore.[9C11] Recently described polysaccharides and glycoproteins of offer exciting new targets for these vaccine formulations and also for the development of improved diagnostics for has been characterized,[12] chemically synthesized, [13C18] and immunologically evaluated. The latter studies demonstrated that this oligosaccharide is exposed to the immune system[14] and has an ability to elicit relevant antibodies.[13] Recently, we reported the structure of a unique polysaccharide released from the vegetative cell wall of and synthetic compounds 1 and 2. As part of a project to determine antigenic determinates of the polysaccharide of and to establish it as a diagnostic or vaccine candidate, we report here the chemical synthesis and immunological properties of trisaccharides 1 and 2 (Scheme 1). These compounds, which are derived from polysaccharide, contain a 5-aminopentyl spacer for selective conjugation to carrier proteins required for enzyme linked immunosorbent assays (ELISA). It has been found that sera of rabbits exposed to live and irradiated-killed spores of Sterne 34F2 or immunized with polysaccharide conjugated to KLH recognize the isolated polysaccharide and the synthetic compounds 1 and 2. The data provide a proof-of-concept step in the Rabbit polyclonal to AGER development of vegetative and spore-specific reagents for detection and targeting of nonprotein structures of were prepared for immunizing rabbits and to examine anti-sera for anti-polysaccharide antibodies, respectively. To this end, the polysaccharide was treated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP)[35] to form reactive cyanyl esters, which were condensed with free amines Dihydroethidium of BSA and KLH to give, after rearrangement of isourea-type intermediate, carbamate-linked polysaccharides. The KLH- and BSA-polysaccharide conjugate solutions were purified using centrifugal filter devices (Micron YM 30,000 Da) and then lyophilized. Saccharide loadings of 0.3 mg/mg BSA and 0.96 mg/mg KLH were determined by bicinchoninic acid (BCA; BSA-conjugate) and Bradfords (KLH-conjugate) protein assay and quantitative carbohydrate analysis by HPAEC-PAD. In addition, maltoheptaose was conjugated to BSA using CDAP to obtain a control conjugate to examine for the possible presence of anti-linker antibodies.[36] Rabbits were inoculated intramuscularly four times at bi-weekly intervals with live- or irradiated spores (3 106 total spores),[14] or polysaccharide-KLH conjugate followed by the collection of terminal bleeds fourteen days after the last immunization. ELISA Dihydroethidium was used to examine the pre- and post-immune sera for polysaccharide recognition. Thus, microtiter plates were coated with the polysaccharide-BSA conjugate and serial dilutions of sera added. An anti-rabbit IgG antibody labeled with horseradish peroxidase was employed as a secondary Dihydroethidium antibody for detection purposes. High titers of anti-polysaccharide IgG antibodies had been elicited by the polysaccharide-KLH conjugate (Physique 1A and Table 1). Furthermore, inoculation with live and irradiated spores resulted in the production of IgG antibodies that can recognize the polysaccharide. Antisera obtained from immunizations with polysaccharide-KLH conjugate showed recognition of maltoheptaose linked to BSA albeit at much lower titers than when polysaccharide linked to BSA was used as ELISA coating. This finding indicates that some anti-linker antibodies had been elicited.[36] As expected, antisera from rabbits immunized with live and irradiated spores showed no reactivity towards the maltoheptaose conjugate (Determine 1B). Open in a separate window Physique 1 Immunoreactivity of polysaccharide and trisaccharides 1, and 2 to antisera elicited by Sterne live spores, irradiated-killed spores, and polysaccharide-KLH conjugate. Microtiter plates were coated with polysaccharide-BSA (A), maltoheptaose-BSA (B), 1-BSA (C), and 2-BSA (D) conjugates (0.15 g mL?1 carbohydrate). Serial dilutions of.
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