FUSEDESIGN · Model-guided design of a stabilized pre-fusion class III viral fusogen, rabies virus glycoprotein

Model-guided design of a stabilized pre-fusion class III viral fusogen, rabies virus glycoprotein

Multiple human pathogens are enveloped viruses that require specific membrane proteins (also known as fusogens) to perform the first stage of viral infection, namely fusion of viral and host membranes. Often such fusogens invoke strong immune responses in infected hosts and induce the production of neutralizing antibodies, thus making them excellent candidates for vaccine antigens. Before viral and host membrane fusion takes place, fusogenic proteins exist in high-energy activated pre-fusion form, which changes to the low-energy post-fusion form after membrane fusion is carried out. Pre-fusion protein state differs from post-fusion by its three-dimensional (3D) structure, moreover, each of such protein forms determines the production of overlapping but distinct antibody repertoire in hosts. Differences in antibody responses dictated by antigen structure might influence the efficacy and longevity of immune response upon vaccination. Thus, knowing the detailed 3D structure of the potential vaccine antigen might guide the rational design of the best vaccine candidate. However, in the past, it has been proven difficult to study pre-fusion forms of fusogens from clinically important human viruses due to the protein complexity and instability in physiologically relevant pre-fusion form. One such clinically relevant human pathogen is the rabies virus (RV). Up to now, the structure of its fusogen rabies glycoprotein (RVG) was unknown, which made a rational improvement of the existing rabies vaccine difficult. The overall objective of this project was to design a better RVG antigen for a novel improved vaccine candidate and to achieve a better understanding of the host immune protection against RV. To meet such objectives, RVG was stabilized in the physiologically relevant trimeric pre-fusion form through a large screening of protein point mutants. Further on, a 3D structure of such stabilized antigen was elucidated in complex with a licensed monoclonal therapeutic antibody using a cryo-electron microscopy approach. Obtained structural information provided the much-needed pre-fusion structure of RVG as well as contributed towards a better understanding of the neutralizing action of the therapeutic antibody.

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