ACE2-fused nanobody targeting a cryptic RBD epitope broadly neutralizes SARS-like viruses

Area of Science:

• Virology
• Immunology
• Biotechnology

Background:

• SARS-CoV-2 and SARS-CoV exhibit high mutation rates, leading to antibody ineffectiveness and cross-species transmission risks.
• Developing broad-spectrum countermeasures is crucial for pandemic preparedness against emerging SARS-like viruses.

Purpose of the Study:

• To generate cross-reactive nanobodies with broad-spectrum neutralizing capacity against SARS-like viruses.
• To engineer potent therapeutic agents for current and future SARS-like viral threats.

Main Methods:

• Employed an alternating immunization strategy using SARS-CoV and SARS-CoV-2 Receptor Binding Domains (RBDs) in alpacas.
• Identified and characterized cross-binding nanobodies, including aSR29, which targets a conserved, cryptic epitope on the RBD.
• Engineered a bispecific fusion protein (aSR29-ACE2-Fc) combining nanobody binding with ACE2 receptor blockade.

Main Results:

• Four cross-reactive nanobodies (aSR29, aSR196, aSR347, aSR348) demonstrated high affinity for SARS1/SARS2 RBDs and Omicron subvariants.
• Nanobody aSR29 potently neutralized SARS1, SARS2, and multiple Variants of Concern (VOCs), including XBB.1.5 and BQ.1.1, by targeting a conserved inner RBD epitope.
• The engineered aSR29-ACE2-Fc fusion protein exhibited a 33-fold increase in neutralization potency against SARS-CoV-2 and enhanced resistance to immune escape.

Conclusions:

• An efficient strategy for generating broad-spectrum nanobodies against SARS-like viruses was validated.
• Engineered VHH-ACE2 fusion proteins represent promising broad-spectrum therapeutics for combating current and emerging SARS-like viral infections.