| Summary: | <p>Protein nanoparticles have been used as platforms to multimerize and present otherwise weakly immunogenic antigens to the immune system. Virus-like particles (VLPs) are self-assembling protein cages, forming the basis of safe and immunogenic vaccines that have been administered to millions of people. However, the creation of chimeric VLPs that display heterologous antigens through either genetic fusion or chemical coupling continues to be a challenge. To address this challenge, a modular vaccine platform has been previously developed that takes advantage of the spontaneous isopeptide bond between the protein domain SpyCatcher and its peptide partner SpyTag. Fusion of SpyCatcher to the surface-exposed N-terminus of the bacteriophage VLP AP205 afforded a particle that could be covalently coupled to a SpyTag-antigen simply by mixing. In the first part of this thesis I establish a methodology for the directed evolution of SpyCatcher-VLPs. I evolve the AP205 coat protein to attempt to address limitations in solubility and yield of the SpyCatcher-AP205 platform. I then extend the scope of SpyCatcher/SpyTag-mediated vaccines by characterizing a new modular platform based on the computationally designed I3-01 nanoparticle. I3-01 was rationally engineered to reduce aggregation by removing two surface-exposed cysteines. This mutated protein was given the name MI3. Genetic fusion of SpyCatcher to the N-terminus of MI3 yielded a platform that was purified in high yield from <em>E. coli</em>. I show that SpyCatcher-MI3 is robust and tolerates heating, freezing, and lyophilization. Immunization with SpyCatcher-MI3 particles conjugated with the SpyTagged malarial antigen CyRPA yielded high-avidity antibodies against CyRPA. The SpyCatcher-MI3 platform's stability, simple production method, and modular quantitative coupling should facilitate the development of new vaccines.</p>
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