Joey Sheff

HDX-MS and SARS-CoV-2 spike protein: From epitope mapping to informing vaccine design

Joey Sheff1, M.A. Rossotti1, J. Tanha1, J.F. Kelly1, Y. Durocher1, M. Stuible1, J. Schrag1

1. Human Health Therapeutics Research Centre, Life Sciences Division, National Research Council Canada, Ottawa, ON, Canada

Introduction: Declared a pandemic in March 2020 by the WHO, COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a severe global health and economic burden. The toll on public health has been exacerbated with the emergence of SARS-CoV-2 variants of concern (VoCs). Key to SARS-CoV-2 infection is its surface-displayed spike glycoprotein, a homotrimer where each protomer consists of S1 and S2 domains. S1 is further delineated by an N-terminal domain (NTD), a receptor-binding domain (RBD) and subdomains SD1 and SD2. The spike protein remains the antigen of choice for SARS-CoV-2 vaccines and biotherapeutics. We describe how hydrogen-exchange mass spectrometry (HDX-MS) platform was used to characterize behaviour of therapies emerging from NRC laboratories invoking the spike trimer.

Methods: A versatile bottom-up HDX-MS workflow, enabled by online electrochemical reduction and careful protease selection, was developed to probe the conformational behaviour of the spike protein. This enabled the analysis of an array of spike protein samples, including truncated RBD (76% sequence coverage), S1 (71%) and S2 (72%) domains, as well as the full length spike homotrimer ectodomain (75%). All data was collected using an HDx-3 Pal coupled to a UPLC-MS (Waters Synapt G2-Si) system. Deuteration was assigned with MS Studio, and in all cases significant changes were assigned based on a pooled two-state student T-Test performed for each state and timepoint (ΔD > 3 x SD, 1-p value > 0.98).

Preliminary data: First, the conformational behaviour of pre-fusion stabilized soluble spike constructs produced in CHO cells to support the development of COVID-19 vaccines and treatments as well as diagnostic and serological assays was probed. Thorough biophysical characterization identified a mixture of trimer conformations (trimer 1 and trimer 2) existing in solution and highlighted that this behaviour is driven by VoC-specific mutations, formulation buffer pH and temperature, and the choice of trimerization domain. HDX-MS was used to compare, where it was determined that trimer 2 populations are significantly stabilized relative to a trimer 1 reference state. Notably, regions where HDX stabilization is observed for trimer 2 preparations are concentrated in the S2 domain which hosts a significant portion of the inter-protomer interface buried within the core of the trimer. Trimer 1 showed no HDX differences relative a monomeric protomer, suggesting that trimer 1 is composed of loosely associated individual protomers, with minimal inter-protomer contact, while trimer 2 is a more stable, compact conformation. Further, we identified antibodies that preferentially bind trimer 2. These results have implications for the development of cross-protective spike-based vaccine antigens and epitope accessibility informed by conformational data. Next, we investigated the epitopes of a collection of 24 single-domain antibodies raised against the spike protein. These have broad domain specificity, epitopic and mechanistic diversity, cross-reactivity across many VoCs, and high in vitro neutralization potencies. After coarse epitope binning was assigned by surface plasmon resonance and sandwich ELISA, the HDX-MS workflow was deployed to probe the conformational nature of the epitope bins at a peptide-level resolution. Unique binding modes emerged between bins, allowing for an improved understanding of the underlying binding and neutralization mechanisms. This granularity assists in understanding and predicting neutralization potency as novel variants emerge, and will guide the development of various therapeutic cocktails and multi-paratopic formats.

Novel aspect: We study demonstrate the unique versatility of HDX-MS in informing the development of therapies targeting the Covid-19 spike protein.

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