The L452R mutation a key player in the evolution of SARS-CoV-2

In a recent study published in the bioRxiv* preprint server, researchers analyzed the characteristics of Omicron severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) subvariants BA.4, BA.5 and BA.2.12.1.

Since its appearance at the end of 2019, SARS-CoV-2 has evolved considerably, with increased transmissibility and resistance to immunity. The SARS-CoV-2 Omicron (BA.1) variant, first reported in November 2021, was quickly designated as a variant of concern (VOC) by the World Health Organization (WHO) due to the alarming number of mutations. The BA.1 variant quickly replaced the predominant variants and wreaked havoc with an unprecedented increase in infections, including vaccine breakthrough cases and reinfections.

Additionally, the increasing frequency of new SARS-CoV-2 Omicron subvariants, such as BA.2.12.1, BA.4, and BA.5, further intensifies concerns about evasion-induced immunity. vaccination or infection. The BA.2.12.1 variant, a descendant of Omicron BA.2, is spreading in the United States (US), while the BA.4 and BA.5 variants have become dominant in South Africa. The BA.4 and BA.5 (hereafter BA.4/5) variants share identical spike (S) proteins, although they have unique mutations compared to the other subvariants.

Study: Differential evasion of Delta and Omicron immunity and enhanced fusogenicity of SARS-CoV-2 subvariants Omicron BA.4/5 and BA.2.12.1. Image credit: Juan Gaertner

About the study

In the present study, researchers determined neutralizing antibody (nAb) titers against SARS-CoV-2 Omicron subvariants BA.4/5 and BA.2.12.1 in sera from vaccinated or convalescents (TS). In addition, they assessed the infectivity, processing, and S-protein fusion properties of these Omicron sublines. Finally, the infectivity of Omicron subvariants was tested using pseudotyped lentivirus particles to infect CaLu-3 or HEK292T-ACE2 cells.

The authors noted that the infectivity of Omicron BA.1 was slightly higher than the D614G variant but about six times higher than the Delta variant in 293T-ACE2 cells, but the infectivity of BA.1 in cells CaLu-3 was 7.5 times and 5.6 times lower compared to D614G and Delta variants, respectively. The infectivity of BA.2 was comparable to that of BA.1, while other subvariants showed modest increases in infectivity. A 2.5 and 3.8 fold increase was observed for the BA.4/5 and BA.2.12.1 subvariants compared to D614G.

All Omicron subvariants showed lower infectivity in CaLu-3 cells than the D614G or Delta variant. nAb titers against Omicron subvariants were assessed using a pseudotyped lentivirus neutralization assay. Fifteen healthcare workers vaccinated with two doses of BNT162b2 or mRNA-1273 vaccine were evaluated for nAbs in their sera. The team noted comparable resistance to neutralization by Omicron subvariants with a 20-fold lower nAb title compared to the D614G.

The BA.4/5 and BA.2.12.1 subvariants exhibit stronger immune leakage than BA.1 and BA.2. (A) Pseudotyped virus infectivity in HEK293T cells stably expressing ACE2 (HEK293T-ACE2). (B) Pseudotyped lentivirus infectivity in CaLu-3 cells derived from human lung epithelium. Bars at (A) and (B) represent means ± standard deviation, and significance is determined by one-way repeated measures ANOVA with Bonferroni’s multiple test correction. The results of at least 3 independent experiments are averaged and displayed. (VS) Sera from 15 healthcare workers collected 3–4 weeks after use of the second dose of mRNA vaccine to neutralize the pseudotyped virus, and the resulting geometric means of 50% neutralization titers (NT₅₀) are displayed at the top of the graph with the percentage of individuals with NT₅₀ values ​​above the detection limit (NT₅₀ = 80; dotted line). (D) A heat map showing the patient/vaccinated NT₅₀ values ​​against each variant for 2-dose HCW sera. (E) Sera from 15 HCWs after homologous mRNA booster vaccination were assessed for nAb titers. Bars at (VS) and (E) represent the geometric mean ± 95% confidence interval, and significance relative to D614G is determined by one-way repeated measures ANOVA with Bonferroni’s multiple test correction. (F) A heat map showing the patient/vaccinated NT₅₀ values ​​against each variant for 3-dose HCW sera. Patient/vaccinated numbers are identified as “P” for Pfizer/BioNTech BNT162b2 vaccinated/boosted HCW, “M” for Moderna mRNA-1273 vaccinated/boosted HCW. Throughout, p-values ​​are represented by **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

Notably, the BA.2 variant with the L452Q substitution exhibited the highest resistance with a 36-fold lower nAb titer than the D614G strain. Sera from boosted individuals (third dose recipients) showed higher and improved nAb titers. Next, sera from infected individuals admitted to intensive care units (ICU) during the Delta wave were examined. Sera from these patients showed high nAb titers against the Delta variant, although lower nAb titers were observed for the BA.1 and BA.2 variants.

Interestingly, the BA.2.12.1 and BA.4/5 variants showed less escape from neutralization by convalescent sera and had higher nAb titers. Individuals infected with the BA.1 variant during the Omicron wave exhibited less potent neutralization of emerging Omicron subvariants, particularly the BA.4/5 variant.

Nevertheless, a booster dose of BNT162b2 or mRNA-1273 vaccine significantly improved nAb titers against all variants tested. Researchers studied membrane fusion by S proteins of different SARS-CoV-2 variants. The BA.1 variant showed 4.7 and 11.3 times lower fusogenicity than the D614G and Delta variants. The BA.4/5 and BA.2.12.1 variants exhibited an increased propensity for fusion than the BA.1 or BA.2 variants, although much lower than for the Delta variant.

The expression of protein S on the surface of virus-producing cells was analyzed by flow cytometry. Expression of BA.1 or BA.2 S protein was slightly higher than D614G or the Delta variant. Emerging Omicron subvariants had similar surface expression. Finally, the team assessed protein S processing in lysates of virus-producing cells and purified virus particles.

Previously, the authors reported a lower propensity of the BA.1 S protein for furin cleavage, indicated by the lower S1/S ratio. Processing of BA.1 and BA.2 S proteins was comparable, with slightly improved processing of BA.2.12.2 and BA.4/5 S proteins with an increase of approximately 1.4 and 1.2 times compared to to the D614GS protein. Similar results were observed for the purified virus particles, and this was assumed due to the L452Q/R substitution.

conclusion

The authors analyzed the immune characteristics induced by vaccines or natural infection against the SARS-CoV-2 Omicron BA.2.12.1 and BA.4/5 subvariants, including their protein S fusogenicity characteristics and of furin cleavage. There was no evidence that two doses of either vaccine were sufficient to neutralize Omicron variants of SARS-CoV-2.

Although booster doses amplified neutralizing titers, they were less potent against emerging Omicron subvariants. The researchers revealed that substitutions at protein S residue 452 could be critical and lead to resistance to neutralization. Taken together, these findings underscore the need for continued monitoring of new variants as well as a detailed understanding of protein S biology and its influence on SARS-CoV-2 pathogenicity and immunity.

*Important Notice

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