Design, Biophysical Characterization, and Serum Epitope Mapping of SARS-CoV-2 RBD Vaccine Immunogens

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been circulating in the human population for over four years. This has resulted in more than 770 million infections and ~7 million deaths to date. Multiple waves of infections were caused by the emergence of multiple different and more divergent variants of concern (VOCs), and their emergence was largely driven by escape from host immunity generated through natural infections and vaccination programs. The Receptor Binding Domain (RBM) of the Spike surface glycoprotein is the primary target for neutralising antibodies, which makes it an ideal vaccine candidate. The work done in this thesis represents a comprehensive and systematic study of vaccine development focused on the RBD, and includes biophysical characterization, epitope mapping, immune refocusing, and investigating adjuvant role in vaccine stability. Initially, we report the biophysical characterization of a previously reported mammalian cell-expressed RBD derivative, mRBD1-3.2, which has higher thermal stability and greatly enhanced immunogenicity relative to the wild-type mRBD. We have investigated the folding pathway of both WT and stabilized RBD and found that the chemical denaturation of RBD proceeds through a stable equilibrium intermediate. Further characterization of the folding of the stabilized RBD revealed higher stability represented by higher Cm, faster refolding, slower unfolding, and enhanced resistance to proteolytic cleavage relative to WT. Collectively, these data suggest that the enhanced immunogenicity results from reduced conformational fluctuations that likely enhance in vivo half-life as well as reduce the exposure of irrelevant non-neutralizing epitopes to the immune system. Next, we developed a rapid and efficient method for epitope mapping, employing a barcoded charged scanning mutagenesis library of the SARS-CoV-2 RBD displayed on the yeast surface, and screened using flow cytometry coupled to deep sequencing. Once validated by mapping the known RBD interacting residues with the ACE2 receptor, the approach was further used to map epitopes targeted in polyclonal sera of BALB/c and transgenic mice immunized with different SARS-CoV-2 immunogens. The epitopes targeted by antibodies in the sera of Omicron RBD immunized BALB/c mice differ from those targeted by the sera of K18-hACE2 mice, immunized with the same antigen. This approach was used to map the epitopes targeted by the polyclonal sera obtained from a set of individuals vaccinated with two doses of the ChAdOx1 nCoV-19 viral vector vaccine and who had subsequently undergone breakthrough infections. Antibodies targeting either class 1 epitopes or the rare cryptic class 5 epitope were observed in the three human sera subjected to detailed epitope mapping studies. The class 5 epitope residues identified in this study are highly conserved across all SARS-CoV-2 variants, consistent with the observed neutralizing potency of the selected serum samples against heavily mutated Omicron BA.1, BA.5, and the recent XBB.1.5 variant. Our findings illustrate how a combination of vaccinated and breakthrough infections in a highly exposed population can elicit diverse antibody classes that confer potent neutralizing activity with broad cross-reactivity. Given the previous findings and aiming to focus the immune response towards eliciting class 4 antibodies, we designed two constructs by truncating the RBM region at different points to minimize the perturbation to the protein conformation. The designed constructs were expressed successfully in mammalian cells in good yields and folded properly, as indicated by their biophysical characterization. The sera from mice pre-immunized with full length RBD and boosted with the RBM truncated derivatives, could effectively neutralize all the tested VOCs and sarbecoviruses belonging to different clades. The new design strategy, coupled with the long gap between immunizations, has proven to be moderately efficacious in enhancing elicitation of class 4 neutralizing antibodies and potentially broadening the cross-protection against a panel of sarbecoviruses from different clades. Finally, given the important role of adjuvants in enhancing the breadth, durability, and magnitude of the immune response, we investigated the stability of SARS-CoV-2 RBD and different proteins formulated with a class B CpG adjuvant. CpG is a widely used adjuvant, including in a recently approved COVID-19 vaccine. This work demonstrated that RBD and several other proteins are less stable and more sensitive to proteolysis in the presence of CpG than without it, and these effects are enhanced with prolonged incubation, which can potentially impact immunogenicity. This is a factor to be considered carefully when using this adjuvant. In conclusion, the work presented in this thesis provides insights into the different aspects that need to be considered in vaccine design, including vaccine stability, the knowledge of relevant epitopes targeted by the immune response, and the choice of adjuvant.