In a recent study posted to the bioRxiv* preprint server, researchers investigated the impact of amino acid (AA) substitutions of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron on spike (S) protein function, processing, and neutralization susceptibility.

Study: Determinants of Spike Infectivity, Processing and Neutralization in SARS-CoV-2 Omicron subvariants BA.1 and BA.2. Image Credit: Naeblys/Shutterstock
Study: Determinants of Spike Infectivity, Processing and Neutralization in SARS-CoV-2 Omicron subvariants BA.1 and BA.2. Image Credit: Naeblys/Shutterstock


SARS-CoV-2 Omicron is the fifth variant of concern (VOC), which emerged in November 2021 and has rapidly outcompeted the previously predominant Delta VOC. It contains many mutations, particularly in the S protein, and exhibits high transmissibility and resistance to sera from convalescent or vaccinated individuals.

Studies have reported that the Omicron variant evolved independently from a chronically immunocompromised person, poorly surveilled human population, or spilled from an unknown non-human species.

The S protein is the main target of humoral immune responses, and all SARS-CoV-2 vaccines are based on the S protein. Thereby, AA changes in the S protein’s N-terminal (NTD) and receptor-binding (RBD) domains increase the resistance to neutralization by antibodies. However, the impact of these mutations on the Omicron variant relative to the ancestral Wuhan Hu-1 strain remain unclear.

The study and findings

In the current study, researchers determined the functional impact of AA substitutions that distinguish the Omicron BA.1 and BA.2 variants from the early SARS-CoV-2 Wuhan isolate. The 43 non-synonymous substitutions in the S protein of BA.1 and BA.2 variants were introduced individually into the Wuhan Hu-1 strain by site-directed mutagenesis. Vesicular stomatitis virus (VSV) pseudo particles (pp) were coated with ancestral and mutant S proteins. The 20 common AA changes of BA.1 and BA.2 variants had no significant impact on VSVpp infectivity. The D614G substitution enhanced infectivity while S375F impaired it. The S371L change of BA.1 or S371F of BA.2 variant strongly impaired infectivity.

Automated quantitation assays of VSVpp infection of Caco-2 cells revealed that mutant S proteins had similar but varying infection kinetics that frequently reduced efficiency. Some common changes such as N440K and D614G, and BA.1-specific D69-70, D211, L981F, 214EPE insertion enhanced the infection efficiency.

Overall, the individual AA changes in the serine residues (S371L/F, S373P, and S375F) located in the small loop region and its adjacent BA.2-specific substitution (T376A) severely impaired infectivity. The researchers observed that the S371L, S373F, S375F, and T376A substitutions decreased the S protein processing efficiency. The S375F and T376A S protein mutants were hardly processed. In sum, the substitutions T19I, D24-26, T376A, S375F, and Q954H reduced the infectivity of VSVpp by affecting the S protein processing.

Next, the impact of Omicron mutations on the angiotensin-converting enzyme 2 (ACE2) binding was investigated using an in vitro S:ACE2 binding assay. The S371F, S373P, D614G, N856K, and L981F changes (introduced) in the Hu-1 S protein had little effect on the S:ACE2 binding. The N501Y substitution enhanced the binding of S protein to ACE2, consistent with previous reports.

However, S:ACE2 binding was reduced by the presence of individual AA changes of S375F or T376A and triple mutation (S371F/L-S373P-S375F) in the Hu-1 S protein. Cell-cell fusion assays revealed the formation of large syncytia by co-expressing ACE2 and ancestral Hu-1 S protein or individual S373P, N501Y, D614G, N856K, and L981F mutants. The syncytia formation was promoted by the pan-VOC D614G substitution and the Omicron-specific L981F substitution, but abrogated by the presence of S375F, T376A, or the triple mutation.

Subsequently, the neutralization sensitivity of Hu-1, Delta, BA.1, and BA.2 S proteins by sera from five BNT162b2-vaccinated individuals was evaluated. The team observed that sera collected two weeks post-second dose had substantially lower neutralization of BA.1 and BA.2 S proteins than Hu-1 or Delta S proteins. The common and Omicron-lineage-specific AA changes had reduced neutralization sensitivity.

Moreover, AA substitutions found in the NTD of BA.1 or BA.2 S protein decreased the neutralization. Notably, the D142-144 in the BA.1 S protein and G142D change in the BA.2 variant had a nine-fold reduction in neutralization. Overall, 27 of the 43 AA changes enhanced the resistance to antibody-mediated neutralization by more than two folds.

The research team noted that imdevimab, a therapeutic monoclonal antibody (mAb), failed to inhibit the BA.1 variant; contrastingly, the BA.2 variant was still susceptible to imdevimab. Both BA.1 and BA.2 were completely resistant to neutralization by bamlanivimab. Another mAb, casivirimab, neutralized BA.2 but had no appreciable neutralization against BA.1.


The authors of the current study systematically evaluated the functional impact of AA substitutions of SARS-CoV-2 Omicron BA.1 and BA.2 variants. Several individual and shared mutations of BA.1 and BA.2 VOCs strongly impaired S protein’s infectivity, processing, and neutralization.

The S375F substitution had the most striking effect, completely disrupting S protein function and processing. Many AA changes in the S NTD or RBD reduced neutralization by sera from vaccinated subjects as well as by therapeutic antibodies. More research is needed to comprehensively understand the consequences of AA changes in the SARS-CoV-2 Omicron on its infectivity and pathogenesis.

*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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