Neutralizing nanobodies against SARS-CoV-2 recognizing highly conserved epitopes at the Spike's S2 subunit

Area of Science:

• Virology
• Immunology
• Structural Biology

Background:

• The SARS-CoV-2 Spike protein's S2 subunit, specifically heptad repeat regions 1 and 2 (HR1 and HR2), is crucial for viral membrane fusion.
• HR1-mimicking proteins (CoVS-HR1) have shown potential in blocking viral fusion and are recognized by convalescent COVID-19 antibodies.

Purpose of the Study:

• To generate and characterize nanobodies (NBs) targeting the HR1 region of the SARS-CoV-2 Spike protein.
• To investigate the neutralizing potential and binding characteristics of these NBs.

Main Methods:

• Generation of nanobodies (VHHs) against HR1-mimetic proteins.
• Affinity and epitope mapping using biophysical techniques (e.g., hydrogen-deuterium exchange mass spectrometry).
• Structural analysis via crystallography of a ternary complex.
• Assessment of neutralizing activity in cell infection assays.

Main Results:

• Selected NBs exhibited high affinity for HR1 and competed with HR2.
• NBs targeting the C-terminal half of HR1 showed mild neutralizing activity.
• NBs targeting the N-terminal half had lower affinities and no detectable neutralizing activity.
• Structural studies confirmed NB binding and competition with HR2.
• Cooperative binding observed between NBs targeting different HR1 halves, but without synergistic inhibition.

Conclusions:

• Neutralizing epitopes exist within the SARS-CoV-2 Spike S2 HR1 region.
• HR1-mimetic proteins are valuable tools for developing targeted nanobodies.
• Further development of NBs targeting specific epitopes may lead to enhanced viral fusion inhibitors.