| Résumé: | Virus-like particles (VLPs) are assemblies that resemble viruses in their size, shape, and multivalent display of protein subunits, but lack the genetic material to infect cells. VLP-displayed antigens are more immunogenic than in their uncoupled form. SpyCatcher003 is a protein engineered from a fibronectin-binding protein of Streptococcus pyogenes that rapidly forms an irreversible isopeptide bond with its peptide partner, SpyTag. This thesis presents SpyCatcher003-mi3 VLPs, which I generated by fusing SpyCatcher003 to the mi3 scaffold, a modified version of the computationally-designed i301 nanocage. I demonstrate that SpyCatcher003-mi3 can be solubly expressed in Escherichia coli, can be purified via ammonium sulfate precipitation to high yields, and remains stable at temperatures between 25 °C and 95 °C. This VLP can accommodate antigens of diverse sizes and symmetries. Assembly on SpyCatcher003-mi3 elicits high levels of neutralizing antibodies against influenza haemagglutinin and neuraminidase, as well as the receptor-binding domain (RBD) of SARS-CoV-2 and select betacoronavirus variants. I demonstrated that SpyCatcher003-mi3 can be expressed solubly in a mammalian cell-line. Then I engineered N-linked glycosylation sequons on flexible hydrophilic loops on the VLP with the goal of shielding antibody binding to the mi3 scaffold and SpyCatcher003, to focus antibody responses on the displayed antigen. I found no significant change in the level of antibody titers that bound specifically to the mi3 scaffold and the SpyCatcher003:SpyTag003 moiety, relative to that of the displayed antigen (SARS-CoV-2 RBD). Fusion of SpyCatcher003-mi3 with the albumin-binding sequence, ABD035, was also insufficient to change the relative induction of antibodies specific to SARS-CoV-2 RBD, the SpyCatcher003:SpyTag003 moiety and the mi3 scaffold. Further analysis and combination of blocking components will be required to maximize glycan and albumin-dependent antibody shielding. Nonetheless, the high stability, simple purification, and easy functionalization of the existing SpyCatcher003-mi3 platform can potentially accelerate the vaccine development process against many infectious diseases.
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