Vaccinogenic and Therapeutic Applications of Burkholderia Polysaccharides
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Authors
Orne, Caitlyn Emily
Issue Date
2022
Type
Dissertation
Language
Keywords
Immunology , Melioidosis , Therapeutic , Vaccinology
Alternative Title
Abstract
Burkholderia pseudomallei the causative agent of melioidosis, is a Gram-negative, facultative-intracellular bacterium that can cause disease in humans and animals. The diagnosis and treatment of melioidosis can be difficult, and at present no licensed vaccines currently exist. Previous studies in our laboratory have revealed that melioidosis subunit vaccine candidates consisting of a B. pseudomallei capsular polysaccharide (CPS)-based glycoconjugate, CPS-CRM197, co-formulated with recombinant B. pseudomallei hemolysin co-regulated protein 1 (Hcp1) or TssM provide high-level protection against acute inhalational challenges of mice with B. pseudomallei. Extending upon these findings in Chapters 2 and 3, we set out to investigate the potential use of Hcp1 and TssM as novel carrier proteins for developing next-generation polysaccharide-based glycoconjugates to immunize against melioidosis. Additionally, we sought to evaluate the potential of FliC and an active-site mutant of the AhpC protein, AhpCC57G, as novel carrier proteins. To facilitate these studies, recombinant Hcp1, TssM, and FliC engineered to possess a single C-terminal cysteine residue (designated Hcp1-Cys, TssM-Cys, and Hcp1-Cys) and the active-site mutant protein AhpCC57G were expressed in Escherichia coli and purified using tandem nickel-cobalt affinity chromatography. Once purified, the cysteine-modified proteins and AhpCC57G were labeled at their C-terminal cysteines using various biotin compounds to confirm the proteins could be modified at the cysteine residues. Following this, Hcp1-Cys and TssM-Cys were modified with heterofunctional linkers to enable site-specific, single-point attachment of CPS or O-polysaccharide (OPS) to the proteins. The glycoconjugates generated in this study were characterized using SDS-PAGE and Western immunoblotting. To assess the immunogenic potential of the glycoconjugates, C57BL/6 mice were immunized with the various glycoconjugates produced in these studies, following which humoral and cellular responses were assessed by ELISA and ELISpot assays, respectively. Results of these studies indicated that the glycoconjugates stimulated the production of high-titer antigen-specific IgG responses. Furthermore, we found that robust interferon (IFN)-γ secreting T-cell responses were raised against the carrier proteins, TssM-Cys and Hcp1-Cys. Finally, we found that the most immunogenic glycoconjugate generated during these studies, OPS-Hcp1, was capable of conferring significant protection in an aerosol challenge with B. pseudomallei K96243. Collectively, our findings suggest that the production of glycoconjugates using site-specific, single-point attachment of Burkholderia-expressed polysaccharides to novel carrier proteins may represent a promising approach for developing a safe and effective melioidosis vaccine comprised exclusively of Burkholderia-specific antigens. In Chapter 4, we sought to develop a process for the isolation, production, and characterization of monoclonal antibodies (mAbs) against CPS for the development of an immunotherapeutic for pre- and post-exposure prophylaxis of melioidosis. B. pseudomallei expresses a variety of structurally conserved protective antigens that are being pursued by our lab as potential vaccine candidates and therapeutic targets. In the present study, we explored the use of 10x Genomics technology to enable the rapid identification and production of CPS-specific mAbs. To facilitate these studies, purified CPS was chemically activated and covalently linked to the carrier protein CRM197 to form the immunogenic glycoconjugate, CPS-CRM197. Mice were then immunized with the glycoconjugate material following which regional lymph nodes were harvested. To isolate B cells from the lymphoid tissues, single-cell suspensions were prepared and depleted of non-target cells using magnetic separation. The enriched B cells were then depleted of naïve IgM+/IgD+ cells resulting in a population of activated B cells that were subjected to 10x single-cell RNA sequencing (scRNAseq). Paired heavy- and light-chain sequences associated with high-frequency clonotypes were synthesized and cloned into expression vectors enabling the production of recombinant mAbs by CHO cells. Using this approach, eight mAbs were purified and characterized by ELISA. The results of these analyses demonstrated that six of the eight recombinant mAbs reacted strongly with purified CPS. The most reactive mAb identified, LN2-3, was further evaluated for potential as an immunotherapeutic. The LN2-3 mAb was successfully subclass-switched to recombinant murine mAbs with different constant regions as well as human chimeric antibodies (ximAbs) for further evaluation and characterization using Western immunoblotting and opsonophagocytosis (OPC) assays. The recombinant murine LN2-3 mAb constructs LN2-3 IgG1 and LN2-3 IgG2c assessed in these studies were capable of conferring protection against an aerosol challenge after passive immunization that was greatly augmented with the administration of co-trimoxazole. Collectively, our findings demonstrate that scRNAseq of activated B cells from mouse lymph nodes represents a promising strategy for the rapid identification and production of antigen-specific mAbs with the potential to serve as an adjunct therapy for the treatment of melioidosis.