Identifying Escape Mutants on the Sars-Cov-2 Spike Receptor Binding Domain Using Yeast Screening

The increased prevalence of SARS-CoV-2 variants like Delta (B.1.617) demonstrates the need for understanding the impact of novel mutants on the human antibody response, with impacts both for development of vaccine boosters and effective monoclonal antibody therapies. The SARS-CoV-2 Spike (S) receptor binding domain (RBD) is primarily responsible for infecting human cells by binding to human angiotensin-converting enzyme 2 (ACE2). Blocking this binding is a main correlate of protection, and so vaccines are developed primarily by the ability to elicit high amounts of antibodies which block this binding, thus neutralizing infectivity of the virus. However, current circulating variants have several RBD mutations which minimize neutralization by many of these antibodies. This work presents a complete experimental pipeline for identifying potential antibody escape mutations. We tested multiple antibodies, including CC6.29, CC12.1 and CC12.3, with yeast screening and deep mutational scanning. A competitive binding experiment was used to test the antibody’s ability to bind in the presence of ACE2 after a mutation is introduced on the S RBD. Following the experimental procedures, custom software was developed to read and filter through output data from deep sequencing. The software also performs a statistical analysis to identify potential escape mutants. A Microsoft Excel per-position heatmap is generated, highlighting each position in the library that was identified as an escape mutation. With this approach, certain escape mutant positions repeatedly appeared for multiple antibodies: K417, D420, Y421 and Q493. Many of the identified positions were located on the perimeter of the ACE2 binding site on the S RBD. A primary limitation of these experiments is that the antibody tested must bind competitively with ACE2 on the S RBD. If the antibody binds to another region of the S RBD, then this method for identifying escape mutations will be insufficient. Even so, many antibodies currently being studied directly compete with ACE2 on the S RBD and these methods provide a relatively quick and reliable approach for determining escape mutations. Having the ability to test for escape mutants given an antibody will be imperative for better understanding this virus and to the future development of monoclonal antibody treatments for SARS-CoV-2.