HexaPro (Mahidol University)

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has accumulated more than 700 million infection cases and 6.9 million deaths. New variants have affected antibody interaction with the surface spike protein. We defined the domain specificities and measured hexa-proline stabilized spike protein (HexaPro) binding kinetics of a large panel of antibodies sourced by the Coronavirus Immunotherapeutics Consortium. Epitope binning analysis of antibodies competing for HexaPro binding separated the fine specificities of the majority of antibodies to four regions: top, outer, mesa/valley, or cryptic site of receptor binding domain (RBD). Most of the top-RBD-specific antibodies showed >3-fold loss of binding and authentic-virus neutralization activity for the B.1.351 variant. Remarkably, among RBD mesa/valley-specific or cryptic-site-specific antibodies, 55% showed >3-fold stronger affinities, and at least 60% maintained neutralization activity for the B.1.351 variant. These data also highlighted the diversity of SARS-CoV-2-specific antibodies that retain high spike affinities and antiviral functions across variants.

Multiple SARS-CoV-2 variants of concern have emerged and caused a significant number of infections and deaths worldwide. These variants of concern contain mutations that might significantly affect antigen-targeting by antibodies. It is therefore important to further understand how antibody binding and neutralization are affected by the mutations in SARS-CoV-2 variants. We highlighted how antibody epitope specificity can influence antibody binding to SARS-CoV-2 spike protein variants and neutralization of SARS-CoV-2 variants. We showed that weakened spike binding and neutralization of Beta (B.1.351) and Omicron (BA.1) variants compared to wildtype are not universal among the panel of antibodies and identified antibodies of a specific binding footprint exhibiting consistent enhancement of spike binding and retained neutralization to Beta variant. These data and analysis can inform how antigen-targeting by antibodies might evolve during a pandemic and prepare for potential future sarbecovirus outbreaks.

The coronavirus disease 2019 (COVID-19) pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. We characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting higher expression than its parental construct (by a factor of 10) as well as the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. A cryo-electron microscopy structure of HexaPro at a resolution of 3.2 angstroms confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).