DEVELOPMENT AND OPTIMIZATION OF A P47-BASED PLASMODIUM VACCINE TO BLOCK MALARIA TRANSMISSION

Malaria is an infectious disease caused by Plasmodium parasites that are transmitted to hosts by infected Anopheles mosquitoes. Over the last two decades, the widespread deployment of effective interventions, such as drugs and insecticides, has resulted in significant reductions of malaria cases. However, without an effective vaccine, the recent emergence of drug-resistant parasites and insecticide-resistant mosquitoes are threats to this progress, motivating the need for newer tools to control and ultimately eliminate malaria. Recently, reducing Plasmodium transmission from humans to mosquitoes has become an actively pursued approach to eradicate malaria. One unique strategy to achieve this goal is through transmission-blocking vaccines (TBVs). TBVs generate antibodies in immunized individuals that are transferred to mosquitoes during a blood meal to block the Plasmodium life cycle. Recently, our laboratory discovered that the P. falciparum surface protein P47 (Pfs47) allows parasites to evade mosquito immune system. This makes Pfs47 critical for the parasite’s survival, and a valuable target for a TBV. The work in this dissertation reveals the potential of P47 as a TBV target in two models of malaria. In the first aim, the development, optimization, and efficacy of a P47 vaccine were investigated using Pfs47 as an antigen. Recombinant Pfs47 protein was expressed in Escherichia coli, and vaccine immunogenicity was assessed in mice. Antibodies targeting a key region of Pfs47 reduced Plasmodium density in mosquito. This result supports TBV as an effective approach to control the spread of malaria. Since delivering vaccines using traditional injection is challenging in developing countries, new technologies that improve vaccine accessibility are also needed. Thus, Pfs47 vaccine was loaded into microneedles, dissolvable micron-scale structures, and tested for function. In the second aim, the efficacy of a P47 vaccine was evaluated in a challenge model of malaria using the Plasmodium berghei mouse malaria antigen Pbs47. The key region in Pbs47 where antibody binding confers protection was mapped. This in vivo system provides preclinical evidence that a vaccine targeting Pfs47 could be effective in humans. Together, this thesis presents P47 as a new malaria vaccine target and introduces MNs as an effective platform to deliver vaccines in resource-poor settings

Ethanol Induces Enhanced Vascularization Bioactivity of Endothelial Cell-Derived Extracellular Vesicles via Regulation of MicroRNAs and Long Non-Coding RNAs

Abstract Extracellular vesicles (EVs), such as exosomes, have been identified as regulators of vascular remodeling and have promise as therapeutics for vascularization applications. Towards development of EVs as therapeutics, it has been demonstrated that physiological stimuli of angiogenic phenotypes in EV-producing cells can enhance the potency of EVs for vascularization. The goal of this study was to assess whether ethanol, which induces angiogenic phenotypes in endothelial cells, could be employed to enhance endothelial-derived EV vascularization bioactivity. The results indicate that ethanol conditioning of endothelial cells increases the ability of endothelial EVs to induce a pro-vascularization response. This response is due in part to increased CD34 expression in recipient endothelial cells that may result from downregulation of microRNA-106b in EVs isolated from ethanol-conditioned producer endothelial cells. Further, ethanol-induced upregulation of long non-coding RNAs (lncRNAs) HOTAIR and MALAT1 in endothelial EVs was observed to play a significant role in mediating pro-angiogenic effects of these vesicles. Overall, these studies validate ethanol conditioning as a method to enhance the bioactivity of endothelial EVs via regulation of EV-associated microRNAs (miRNAs) and, especially, lncRNAs. Further, the results suggest that alcohol consumption may activate endothelial EVs towards a pro-vascularization phenotype, which could have implications for alcohol-induced tumor angiogenesis