SUBUNIT PEPTIDE-ENCAPSULATING NANOPARTICLE HIV VACCINE TARGETING CYTOTOXIC T LYMPHOCYTE ACTIVATION VIA PROTAC-ENHANCED CROSS PRESENTATION
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Development of an effective prophylactic vaccine against HIV remains an ongoing challenge. Protein subunit vaccines have garnered interest recently, due to desirable safety and stability profiles in comparison to whole-pathogen vaccines. However, immunization with subunit vaccines has a proclivity to elicit humoral, rather than cell-mediated, immune responses. For cytotoxic T lymphocyte (CTL) stimulation, antigenic material in the subunit vaccine must 1) be endocytosed by antigen presenting cells, 2) escape from the endosome, 3) undergo degradation by the ubiquitin proteasome pathway, and 4) be loaded onto MHC I for cross-presentation (CP) to CTLs. In this thesis, a novel vaccine is introduced that specifically targets antigens to the proteolysis pathway for amplified CP and augments endosomal escape in a nanoparticulate drug delivery platform. Inspired by proteolysis-targeting chimera (PROTAC) technologies which have been employed for in situ destruction of cancer-related proteins, this project explores the incorporation of a proteolysis targeting moiety (ProTM) in vaccination. We hypothesize that by conjugating an E3 ubiquitin ligase ligand to synthetic long peptide (SLP) antigens, SLPs can be targeted for proteasomal processing and CP, ultimately resulting in improved CTL activation. Organic synthesis and click-chemistry methods were utilized to covalently modify SLPs with a ProTM. Poly(lactic-co-glycolic acid) nanoparticles that co-encapsulated the modified SLPs and the endosomal escape-promoting molecule, chloroquine, were formulated via single and double emulsion solvent evaporation. Size and loading efficiencies of nanoparticles were characterized using dynamic light scattering and absorbance. In vitro experiments demonstrated uptake of the ~200 nm particles by dendritic cells; quantitative colocalization analysis of fluorescently labeled SLPs and lysosomes was performed to delineate endosomal escape. With this strategy, statistically significant differences were not observed between chloroquine-containing and lacking nanoparticles. mRuby-labeled Galectin-8 cell lines were engineered for future experimental elucidation of endosomal escape. In vivo experiments tested the vaccine in a prime-boost vaccination schedule, showing the highest relative antigen-specific CTL activation in the group receiving the complete formulation. The ProTM-SLP subunit vaccine technology described here can feasibly be delivered in a biodegradable, polymeric nanoparticle formulation and shows strong potential to improve cellular immune responses that are clinically relevant for vaccination against HIV and other pathogens.