Browsing Theses and Dissertations School of Pharmacy by Author "Gregg, Kelsey"
Novel Lipid A Structures for Adjuvant Discovery and DevelopmentGregg, Kelsey; Ernst, Robert K.; 0000-0002-1483-735X (2017)There is an urgent need to develop effective immunizations against infectious diseases that continue to be a major cause of morbidity and mortality worldwide. Modern subunit vaccines require immunopotentiators or adjuvants to strengthen the protective immune response. Adjuvants in human licensed vaccines include aluminum hydroxide, a TH2-biased adjuvant, and monophosphoryl lipid A (MPLA), a TH1-biased, non-toxic Toll-like receptor 4 (TLR4) agonist. MPLA is chemically derived from Salmonella minnesota R595 lipopolysaccharide and displays reduced endotoxicity. Phosphorylated hexacyl disaccharide (PHAD) is a similar, but synthetically produced, monophosphoryl lipid A adjuvant. We hypothesized that functionally diverse lipid A adjuvant structures could be rationally designed by reprogramming the lipid A biosynthetic pathway by adding or removing lipid A modifying enzymes in a process called bacterial enzymatic combinatorial chemistry (BECC). BECC lipid A molecules were engineered in an avirulent Yersinia pestis strain, purified, and screened in immortalized cell lines for TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) responses. BECC-derived structures exhibit dose-dependent TLR4-driven NF-?B activation with both human and murine TLR4 signaling complexes. Structures that displayed reduced NF-?B activity were screened to identify BECC molecules that induced cytokine secretion patterns similar to PHAD in murine splenocytes and human primary blood mononuclear cells (PBMCs), and induced similar activation markers on human monocyte-derived dendritic cells (DCs). Selected BECC molecules were tested in vivo for their ability to adjuvant the recombinant fusion protein, rF1-V, in a murine subunit vaccination model against Y. pestis, the causative agent of plague. BECC-adjuvanted vaccines elicited strong rF1-V-specific antibody titers in C57BL/6J mice with increased titers of the TH1-associated immunoglobulin, IgG2c. The BECC adjuvant groups' titers were similar to or higher than the aluminum hydroxide and PHAD control adjuvant groups. The BECC-adjuvanted and control adjuvant vaccines were protective against a lethal, intraperitoneal Y. pestis CO92- challenge, and were more protective than the unadjuvanted rF1-V vaccine control. These data demonstrate that BECC can be used to generate functionally diverse TLR4 ligands with potential for use as TH1-biasing vaccine adjuvants.