Periodic Reporting for period 1 - FUSEDESIGN (Model-guided design of a stabilized pre-fusion class III viral fusogen, rabies virus glycoprotein)
Período documentado: 2019-07-01 hasta 2021-06-30
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.
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.
To rationally guide the mutational stabilization of RVG antigen, two approaches have been employed. Initially, an RVG homology model was generated using Rosetta CM software. Additionally, certain mutations were chosen manually based on the literature survey and sequence alignment with the closest structurally related fusogen. Such a combined approach helped to rationally choose 75 single-point mutations. RVG genes carrying one of each identified mutation were synthesized, and proteins were assessed for protein expression and pre-fusion stability in a physiological membrane-bound state using a fluorescence-activated cell sorting technique. The best-performing mutant RVG carried a single histidine 270 to proline helix-breaking mutation. This protein variant was further complexed with two monoclonal antibodies 1112-1 and 17C7, the latter being a licensed therapeutic antibody for rabies post-exposure prophylaxis. A 3D structure of such a triple complex was elucidated using a cryo-electron microscopy approach, yielding a 2.8 Å resolution structure. In addition, studying RVG-Ab protein-protein interactions by means of surface plasmon resonance allowed us to conclude that antibodies against site III antigenic region (that binds 17C7 as well) exhibit their neutralizing function by physically locking RVG in the pre-fusion state, thus precluding membrane fusion. Stabilized RVG antigen was tested as a vaccine candidate in the context of the chimpanzee adenovirus vaccine platform, however, it did not induce a more potent immune response in mice in this specific delivery format. In the second iteration of RVG optimization, further 25 double RVG mutants were designed by pair-wise combination of best-performing single mutations, the effects of such mutational analysis are currently being investigated.
Due to the outcomes of this project, for the first time in the rabies vaccinology field, it is understood how anti-rabid therapeutic antibody executes their protective function. Additionally, a novel 3D structure can inform further on the sites of protein vulnerability and guide the development of therapeutic antibody cocktails that target such regions.
Despite the fact that the RVG H270P mutant failed to elicit a better antibody response in the chimpanzee adenovirus vaccine platform, such an outcome might be exclusively platform-related and can be proven different in proteinaceous or mRNA-based vaccine formats. Having obtained proof-of-concept results for pre-fusion stabilization of type III fusogen, this project is a stepping stone for further optimization of RVG antigen aiming at improved protein trimeric stability and expression yields.
No website has been developed for the project.
Despite the fact that the RVG H270P mutant failed to elicit a better antibody response in the chimpanzee adenovirus vaccine platform, such an outcome might be exclusively platform-related and can be proven different in proteinaceous or mRNA-based vaccine formats. Having obtained proof-of-concept results for pre-fusion stabilization of type III fusogen, this project is a stepping stone for further optimization of RVG antigen aiming at improved protein trimeric stability and expression yields.
No website has been developed for the project.