ENGINEERING BACTERIAL GLYCOBIOLOGY FOR THE CREATION OF GLYCOCONJUGATE VACCINES
Stevenson, Taylor Currie
The use of vaccines has led to the effective eradication of several human diseases which were once epidemic such as smallpox and polio. Vaccines have also dramatically decreased the incidence of other diseases including rabies, mumps, and measles. A common theme to these diseases is that their etiological pathogens are viruses. In general, it has proven to be a much more challenging task to develop effective vaccines against bacterial pathogens. The field of bacterial vaccinology has seen success in recent decades with the development of glycoconjugate vaccines. A canonical glycoconjugate vaccine uses polysaccharides isolated from bacterial pathogens and chemically conjugates them to strong protein antigens, thus allowing the immune system to generate a robust and long lasting immune response against a component of the pathogen that is an ideal target for immune system. This approach has been very successful against some bacterial pathogens including Heamophilus influenzae, Streptococcus pneumoniae, and Salmonella enterica. However, the semi-synthetic process for making these glycoconjugates is lengthy, requiring several rounds of purification before and after conjugation chemistry links protein to polysaccharide. This work will discuss alternative wholly biosynthetic processes for the creation of glycoconjugate vaccines. I have done preliminary work engineering the glycobiology of Escherichia coli to display azide-linked sugars on lipid-linked oligosaccharides with the overall goal of performing in vivo N-linked protein glycosylation to create glycoprotein vaccines that can be functionalized through well-characterized bioorthogonal chemistry. I was able to observe the presence of these azide-linked sugars on the outer membrane of E. coli by fluorescence based screening in a flow cytometer. Additionally, I have leveraged the use of recombinant outer membrane vesicles (OMVs) combined with in vivo and in vitro bacterial N-linked glycosylation to formulate new glycoconjugate vaccines against the bacterial pathogen Francisella tularensis. The presence of these glycoproteins in and on the OMVs was verified through Western blotting, proteinase protection assays, and density gradient ultracentrifugation. I have also formulated a non-canonical glycoconjugate vaccine by producing OMVs coated in the common bacterial extra-cellular polysaccharide poly-N-acetylglucosamine (PNAG). I have shown that these PNAG OMVs are able to elicit a strong, class-switched glycan-specific antibody response against PNAG in mice. Mice vaccinated with these PNAG OMVs are partially protected against lethal challenge with Staphylococcus aureus (8/16 survivors in PNAG OMV immunized mice as compared to 3/16 survivors in PBS control mice). I have also shown that sera from mice immunized with PNAG OMVs has bactericidal activity against the attenuated strain F. tularensis LVS, suggesting mice immunized with PNAG OMVs may be protected from a wide range of pathogens. These studies provide several routes for novel formulation of glycoconjugate vaccines against bacterial pathogens.
Chemical engineering; Biomedical engineering; glycobiolology; glycoconjugate; OMV; PNAG; polysaccharide; vaccine
Chang, Yung-Fu; Putnam, David A.
PHD of Biomedical Engineering
Doctor of Philosophy
dissertation or thesis