Intelligence Disclosure on Deploying SpyTag/SpyCatcher for Nanoparticle Assembly

Immunization with antigens presented multivalently on virus-like particles elicits potent immune responses. The SpyTag/SpyCatcher system, which allows spontaneous covalent bond formation between two proteins, provides a simple route to functionalize virus-like particles for multivalent presentation. Termed SpyVLPs, these reactive nanoparticles allow modular plug-and-display of different antigens. SpyCatcher003-mi3 is a resilient SpyVLP that can be produced at high yield, allows efficient coupling, and can be applied to vaccines against a diverse range of pathogens, as well as against cancer and allergy. Here, we describe the procedure for the expression and purification of SpyCatcher003-mi3. We outline considerations related to antigen design, multivalent display, nanoparticle validation, and immunogen preparation that are critical for effective implementation of SpyCatcher003-mi3 and other SpyVLPs. The methods discussed are applicable beyond vaccines, extending to therapeutic targeting, diagnostics, and catalysis.