Wed. Aug 10th, 2022

In a recent study posted to the bioRxiv* preprint server, researchers assessed the impact of epistatic interactions on the angiotensin-converting enzyme-2 (ACE2) affinity in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant.

Study: Compensatory epistasis maintains ACE2 affinity in SARS-CoV-2 Omicron BA.1.  Image Credit: Kateryna Kon / Shutterstock
Study: Compensatory epistasis maintains ACE2 affinity in SARS-CoV-2 Omicron BA.1. Image Credit: Kateryna Kon / Shutterstock


Various studies have investigated the effects of mutations in the SARS-CoV-2 Omicron variant on ACE2 affinity as well as antibody binding. These studies focus on the impact of single mutations on only particular genetic backgrounds. However, extensive research is required to understand the interactions between combinations of mutations found in Omicron and their impact on immune evasion and ACE2 affinity.

About the study

In the present study, researchers mapped the epistatic interactions incident between mutations present in the receptor-binding domain of the SARS-CoV-2 Omicron BA.1 sublineage compared to those in the Wuhan strain.

The team employed a combinatorial assembly approach to develop a plasmid library comprising all probable combinations of the 15 mutations observed in the BA.1 RBD. This library included all the evolutionary intermediates possible between the BA.1 and the Wuhan RBDs. The team used a method based on sequencing and high-throughput flow cytometry called Tite-Seq to estimate the binding affinities (KD, app) of all the possible viral RBD variants to the human ACE2. Furthermore, a standard biochemical model of epistasis was fit for the study data.


The study results showed that all the 32,768 SARS-CoV-2 RBD intermediates possible between the Wuhan strain and the Omicron BA.1 variant had a detectable affinity toward ACE2 with a KD, app between 0.1 μM and 0.1 nM. The BA.1 RND had a three-fold increase in binding affinity compared to that of the Wuhan strain.

However, almost 60% of the intermediate RBD sequences displayed weaker ACE2-binding affinity than that observed for the Wuhan strain. This was because most of the BA.1 mutations had either a neutral or a deleterious effect on the affinity of ACE2 in most of the genetic backgrounds. This was especially true for mutations such as G446S, K417N, Q493R, Y505H, and G496S, among which four were involved in the immune evasion from several classes of monoclonal antibodies.

While many of the BA.1 mutations decrease ACE2 affinity, the interactions observed between these mutations result in the increase of BA.1 affinity to ACE2. These mutations were found to be more deleterious for ACE2 affinity in the presence of a few other mutations; however, these mutations prove to be either neutral or beneficial when several other mutations are present.

The team also observed that although most of the 15 RBD mutations decrease affinity to ACE2 in the Wuhan strain background, these mutations tend to be less deleterious and more advantageous in the Omicron background. This showed that the Omicron BA.1 RBD displayed a more robust ACE2 affinity despite comprising mutations that individually decrease ACE2 affinity due to the deleterious effects of the mutations mitigated by epistatic interactions between the mutations.

The team found that the linear effects of the individual mutations were associated with the contact surface area of ​​ACE2 to the corresponding residue. The higher-order coefficients inferred from the biochemical method showed strong compensatory interactions that mitigated the affinity-reducing effects of the individual mutations. The extent of these interactions was similar to that observed for the linear effects, while the epistatic interactions noted were highly positive. This indicated that mutations that reduced ACE2 affinity had become less deleterious with respect to backgrounds with other compensatory mutations.

The epistatic interactions also eliminated the strong deleterious impact of the mutations involved in antibody escape. The team also found that the G446S, K417N, Q493R, Y505H, and G496S mutations had a negative linear effect on viral affinity to ACE2; however, this effect was found to be very rare across the SARS-CoV-2 phylogeny. This suggested that maintaining ACE2 affinity is probably an important characteristic of viral fitness; hence, these mutations were most likely selected against.

The team also found that mutations having negative effects on ACE2 affinity mitigated by epistatic interactions with N501Y were enriched among all SARS-CoV-2 strains that also have N501Y. This further suggested that at least a few pairwise epistatic interactions were present among other viral backgrounds.


To summarize, the study findings showed that the evolution of antibody escape in the SARS-CoV-2 Omicron BA.1 sublineage was possible without reducing ACE2 binding due to the compensatory epistatic interactions occurring with other mutations. The study showed that high-order patterns of epistasis were essential for viral evolution involving adaptive events such as immune evasion. The researchers believe that the generation of specific combinatorial landscapes may help understand the general patterns of epistasis responsible for viral evolution.

* Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice / health-related behavior, or treated as established information.


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