Novel mutations that are capable of reaching high frequency in SARS-CoV-2 genomes are possible due to the intrinsic error rate of the SARS-CoV-2 RNA replication process. The rise of these variants may affect vaccine and therapeutic development as they spread throughout the population.

Furthermore, to prevent the derailing of promising vaccines or antibody-based prophylactics or treatments, it is critical to understand how and whether SARS-CoV-2 may evolve to evade antibody-dependent immunity.

The researchers examined the immunodominant SARS-CoV-2 RBM, a portion of the receptor-binding domain (RBD) that mediates viral entry and is a major target of neutralizing antibodies in the body. They found it to be a highly variable region of the spike protein in circulating viruses, accounting for the top 10% of entropy (rate of mutation). Generally, the results suggest that the RBM is able to accommodate amino acid changes without disrupting human angiotensin-converting enzyme 2 (ACE2) binding.

Effect of RBM mutations in SARS-CoV-2

More specifically, the team sought to define the clinical and epidemiological effect, molecular features, and immune response to the RBM mutation N439K -- cytosine-to-adenine transversion in the third codon position, resulting in an amino acid change from asparagine to lysine. The variant was first identified in March 2020 in Scotland, and as of January 2021, has two lineages. This mutation has been observed in over 30 countries and is the second most commonly observed RBD mutation worldwide, and the sixth most common spike mutation.

An equivalent position to N439K in the SARS-CoV RBM forms a salt bridge with ACE2 with a positively charged amino acid, according to the team. Therefore, they hypothesized that the N439K SARS-CoV-2 variant may form a similar salt bridge at the RBD-ACE2 interface. They determined the crystal x-ray structure of the N439K RBD complex with ACE2 at 2.8 å resolution and found that the salt bridge does form. Thus, the N439K mutation may add strength to the interaction at the binding interface.

They next evaluated the effect of the mutation on viral fitness by examining clinical data and outcomes associated with the virus carrying the N439K mutation, as well as in vitro data. The researchers found that the mutation is associated with a similar clinical spectrum of disease and slightly higher viral loads in vivo compared to viruses with the wild-type N439 residue.

"A significant finding from this paper is the extent of variability found in the immunodominant RBM on the spike protein," said senior author Gyorgy Snell, PhD, senior director of structural biology at Vir Biotechnology, in a statement.

Resistance to monoclonal antibodies

Lastly, the researchers tested whether the N439K mutation promotes evasion of antibody-mediated immunity. To do this, they evaluated the effectiveness of monoclonal antibodies and polyclonal immune serum from 442 recovered individuals, including six donors who were infected by the SARS-CoV-2 N439K variant, to recognize the N439K RBD. Of those, 6.8% of the tested sera showed a greater than two-fold reduction in binding to N439K RBD as compared to controls.

To further understand the effect of the mutation on monoclonal antibody binding, the scientists screened a panel of 140 monoclonal antibodies isolated from individuals who recovered from SARS-CoV-2 infection, which are a representative sample of the RBD-targeting antibodies generated after infection, as well as therapeutic antibodies that are in clinical stage development or already approved for emergency use authorization (REGN10933, REGN10987, LY-CoV555, and S309).

Overall, 16.7% of the monoclonal antibodies demonstrated a greater than two-fold reduction of RBD-binding in response to the N439K mutation. RBD-binding competition experiments with ACE2 and three structurally distinct epitopes on the RBD revealed that monoclonal antibodies with sensitivity to N439K were enriched for one of the epitopes (S2H14/site I) and showed no ACE2 blockade. This is consistent with the positioning of the N439K mutation at the edge of the RBM.

Importantly, the N439K mutation allowed pseudoviruses to resist neutralization by a monoclonal antibody that has been approved by the U.S. Food and Drug Administration (FDA) for emergency use as part of a two-antibody cocktail (LY-CoV555). Resistance to one antibody in a cocktail could reduce the overall efficacy of the treatment as a monotherapy, according to the authors.

To minimize the effect of monoclonal antibody escape mutations of SARS-CoV-2, the authors suggested that researchers must develop monoclonal antibodies with epitopes that are highly resistant to viral escape, such as conserved epitopes outside of the RBM, or else screen patients for the presence of resistant variants prior to drug administration.

An additional challenge for studying SARS-CoV-2, according to Snell, is the limited amount of sequencing. With more than 90 million cases of reported COVID-19, only about 350,000 virus variants have been sequenced.

"That's only 0.4% -- just the tip of the iceberg," Snell explained. "This underscores the need for broad surveillance, a detailed understanding of the molecular mechanisms of the mutations, and for the development of therapies with a high barrier to resistance against variants circulating today and those that will emerge in the future."

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