Polysaccharide Vaccines for Enteric Pathogens: The Next Generation Multivalent Diarrhea Vaccine
Diarrheal disease due to enteric pathogens is a global health problem and three major contributors are enterotoxigenic Escherichia coli (ETEC), Shigella flexneri and Campylobacter jejuni. The effects of some gastric infections also go beyond the intestinal tract, such as C. jejuni and its link to Guillain-Barré Syndrome, and the autism associated pathogen, Clostridium bolteae. The first section of this thesis is an extension of my MSc research, in which I discovered the capsule polysaccharide of the autism associated bacteria, C. bolteae. Here, I show the conjugation of the C. bolteae polysaccharide to a carrier protein in preparation for upcoming immunological testing. More significantly, I also divulge the chemical synthesis of the C. bolteae capsule polysaccharide non-reducing end terminus with an aminopentyl linker at the reducing end: α-D-Manp-(1→4)-b-D-Rhap-(1→O(CH2)5NH2. The capsule polysaccharide of a key representative strain of C. jejuni serotype complex HS:5 was fully characterized in this work. C. jejuni HS:5 capsule polysaccharide was determined to be highly complex composed of 3,6-dideoxy-α-D-ribo-heptopyranose, glucitol, α-D-glycero-D-manno-heptopyranose, MeOPN, and diester phosphate:. Another interesting structural finding came from the analysis of an isogenic mutant of C. jejuni serotype HS:23/36 (PG3718), in which one of the two genes responsible for the addition of O-methyl-phosphoramidate (MeOPN) to the capsule polysaccharide was deactivated. In this case, the MeOPN was not connected to position 2 or 6 of galactose as in the wild-type strain, but to the 4 position of the said galactose. This isogenic strain was found to be more resistant to complement killing than the wild-type strain. Two other classes of polysaccharide conjugate vaccines are also described in this work; those composed of (i) C. jejuni polysaccharides (from serotypes HS:4AB and HS:53) and diphtheria toxoid (CRM197), and (ii) S. flexneri polysaccharide and the ETEC protein CfaEB. These polysaccharide conjugate vaccines were made using a scheme developed in the Monteiro laboratory, where the polysaccharides were first stoichiometric oxidized at primary hydroxyls with 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and bleach and then conjugated to the immunostimulatory protein. These polysaccharide conjugate vaccines were shown to be highly immunogenic (high IgG levels) in a mouse model and represent a next generation multivalent diarrhea vaccine.