Convergent structural features of respiratory syncytial virus neutralizing antibodies and plasticity of the site V epitope on prefusion F.

Respiratory syncytial virus (RSV) is a global public health burden for which no licensed vaccine exists. To aid vaccine development via increased understanding of the protective antibody response to RSV prefusion glycoprotein F (PreF), we performed structural and functional studies using the human neutralizing antibody (nAb) RSB1. The crystal structure of PreF complexed with RSB1 reveals a conformational, pre-fusion specific site V epitope with a unique cross-protomer binding mechanism. We identify shared structural features between nAbs RSB1 and CR9501, elucidating for the first time how diverse germlines obtained from different subjects can develop convergent molecular mechanisms for recognition of the same PreF site of vulnerability. Importantly, RSB1-like nAbs were induced upon immunization with PreF in naturally-primed cattle. Together, this work reveals new details underlying the immunogenicity of site V and further supports PreF-based vaccine development efforts.

Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein Degradation Pathway Observed in a Stabilized Form of RSV Prefusion F Protein.

Stability is one of the critical attributes of a protein-based therapeutic or vaccine product, which is directly linked to product quality and efficacy. Elucidating protein degradation pathways is required to obtain thorough understanding of the product and ensure degradation products are properly monitored. We observed a unique protein degradation involving nonenzyme catalyzed loss of a complete N-linked glycan under stress condition from an engineered respiratory syncytial virus (RSV) prefusion F protein (RSVPreF3). Investigations involving mass spectrometry, molecular modeling, and mutagenesis revealed that the glycan shedding was site-specific, dependent on structural elements, and required a glycine residue immediately following the site of glycosylation. The glycan loss did not negatively affect the binding between the main immunogenic epitope Site Ø and the neutralizing antibody D25. Further study indicated that the glycan shedding followed a similar but different mechanism than that of conventional deamidation. Since glycosylation is an important attribute for many recombinant therapeutic proteins or vaccine antigens, the finding from this study suggests the need to monitor this new type of degradation, especially when glycosylation has an impact on efficacy or safety.

Structural and computational design of a SARS-CoV-2 spike antigen with improved expression and immunogenicity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern challenge the efficacy of approved vaccines, emphasizing the need for updated spike antigens. Here, we use an evolutionary-based design aimed at boosting protein expression levels of S-2P and improving immunogenic outcomes in mice. Thirty-six prototype antigens were generated in silico and 15 were produced for biochemical analysis. S2D14, which contains 20 computationally designed mutations within the S2 domain and a rationally engineered D614G mutation in the SD2 domain, has an ~11-fold increase in protein yield and retains RBD antigenicity. Cryo-electron microscopy structures reveal a mixture of populations in various RBD conformational states. Vaccination of mice with adjuvanted S2D14 elicited higher cross-neutralizing antibody titers than adjuvanted S-2P against the SARS-CoV-2 Wuhan strain and four variants of concern. S2D14 may be a useful scaffold or tool for the design of future coronavirus vaccines, and the approaches used for the design of S2D14 may be broadly applicable to streamline vaccine discovery.