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Syntheses of Carbohydrate Antigens Expressed by Gastric-intestinal Bacteria and Conjugates Thereof

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Title: Syntheses of Carbohydrate Antigens Expressed by Gastric-intestinal Bacteria and Conjugates Thereof
Author: Jiao, Yuening
Department: Department of Chemistry
Program: Chemistry
Advisor: Monteiro, Mario
Abstract: Diarrhea is one of the most common and debilitating ailments in the world and gastro-intestinal microorganisms are responsible for a majority of cases. This thesis describes the creation of synthetic carbohydrate conjugate vaccines against two key diarrheal bacterial pathogens, Clostridium difficile (antibiotic-associated diarrhea) and Campylobacter jejuni (food-borne traveller’s diarrhea). The synthesis of a conjugate containing two C. difficile antigens, the cell surface PS-I pentasaccharide repeating block, α-L-Rhap-(1→3)-β-D-Glcp-(1→4)-[α-L-Rhap- (1→3)]-α-D-Glcp-(1→2)-α-D-Glcp-(1→O(CH2)5NH2, and a subunit peptide of exotoxin B (ToxB), the 248–262 fragment of the N-terminal NGESFNLYEQELVER, was achieved. Immunodetection studies using the aforementioned synthetic PS-I revealed that sera of healthy horses contained anti-PS-I IgG antibodies. This conjugate represents the first dual antigen C. difficile vaccine that has the potential to control colonization and disease by targeting a surface antigen and an exotoxin simultaneously. In the past, we discovered that C. jejuni surface capsule polysaccharides (CPSs) are decorated with O-methyl-phosphoramidates (MeOPN) that serve as key structural markers responsible for C. jejuni serotype classification. Our previous C. jejuni studies have also led to an efficacious CPS conjugate vaccine that is now in phase 1 human clinical trials. The immunization experiments with native C. jejuni CPS-based conjugates also revealed that the MeOPN-glycan linkages were highly immunogenic. Thus, we envisaged that conjugates rich in synthetic MeOPN-CPS epitopes could perhaps make more efficacious anti-C. jejuni conjugate vaccines. To this end, the main body of this thesis teaches the chemical syntheses of MeOPN-sugar antigens exposed by serotypes HS:23/36 and HS:1. The synthetic MeOPN-monosaccharide units were shown to react with C. jejuni whole-cell antisera, which indicated the presence of antibodies specific for these MeOPN-sugar linkages, especially against MeOPN→6-Gal. Interestingly, whole-cell sera raised by C. jejuni with CPSs with MeOPN at primary positions (C-6 of hexoses or C-7 of heptoses) were also seen to react with the synthetic MeOPN→6-Gal. Based on the observed heighten immunogenicity of MeOPN→6-Gal, a MeOPN→6-Gal-CRM197 conjugate was synthesized, using an aminopentyl linker at C-1, and shown to be capable of raising antibodies against C. jejuni HS:23/36 cell surface with accompanying bactericidal activity. In order to increase the display of the low molecular weight MeOPN→6-Gal in a conjugate format, an approach involving the attachment of multiple MeOPN→6-Gal units to activated starch was also explored. The syntheses of C. jejuni CPS-associated MeOPN glycan antigens described in this thesis represent a significant advancement in the development of a next generation C. jejuni synthetic vaccine that in time may replace the native CPS-based vaccine.
Date: 2017-03

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