Deeya Patel

SARS-CoV-2, the virus that has led to COVID-19, requires powerful, yet affordable therapeutics due to its ability to evolve into different strains. XBB.1.5 and BA.1 are the specific Omicron subvariants of interest. The virus-surface spike protein of SARS-CoV-2 facilitates its entry into the host cell. Nanobodies (single-chain antibodies) have been demonstrated to be easy to produce, cost-efficient, and effective at binding the receptor binding domain (RBD) of the spike protein, thus inhibiting its binding to the ACE2 receptor for entry into the host. However, as new mutated strains arise, the need to produce new nanobodies with the ability to bind and neutralize the new strains becomes imperative. Previously identified by the Li Lab is Nanosota-3A, a nanobody demonstrated to successfully bind and neutralize the prototypic spike and BA.1 spike. However, unable to bind or neutralize XBB.1.5 spike, Nanosota-3A was affinity-maturated and subjected to random mutations of three residues that were found to be different between XBB.1.5 and BA.1/prototypic spike proteins. This created a new nanobody, Nanosota-3B. ELISA analysis was performed to detect binding ability between Nanosota-3B and XBB.1.5 spike ectodomain. Further, to evaluate the neutralizing strength of Nanosota-3B, pseudovirus entry assays were performed. Pymol was used to observe the detailed structure at the interface between Nanosota-3A and the prototypic spike and Nanosota-3B and XBB.1.5. Comparisons between Nanosota-3A and Nanosota-3B binding sites revealed that the prototypic spike protein contains phenylalanine at position 490, whereas XBB.1.5 contains serine. Furthermore, compared to Nanosota-3A, Nanosota-3B differs by two mutations: V50F and Q58S. The results indicated that Nanosota-3B was able to successfully bind and neutralize XBB.1.5. These results support structural guided affinity maturation as an effective tool for adapting nanobodies to new strains of SARS-CoV-2.