In a recent study posted to the bioRxiv* preprint server, researchers demonstrated a viral amplification procedure to isolate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants.
Additionally, they examined their sensitivity to a panel of six therapeutic monoclonal antibodies (mAbs) and sera from vaccinated individuals.
Omicron BA.2, BA.4, and BA.5 lineages have given rise to several new subvariants, including BA.2.75.2, and BA.4.6. and BQ.1.1. Successive sub-lineages of Omicron have infected nearly 80% of the world population in less than a year. Due to the increased transmissibility and immune evasion potential of Omicron subvariants, vaccines offer inadequate protection against them, which, in turn, has increased the incidence of breakthrough infections even in triply vaccinated individuals.
The R346T spike (S) mutation found in Omicron sublineages, BA.2-derived BA.2.75.2, BA.4.6, and BQ.1.1, has also been associated with escape from mAbs and vaccine-induced antibodies. The convergent evolution of the SARS-CoV-2 S glycoprotein suggests that the different circulating Omicron subvariants experienced similar selective pressure, likely exerted by preexisting or imprinted immunity. It makes the characterization of these new Omicron-derived viruses crucial.
About the study
Omicron relies more on endocytic proteases and less on transmembrane protease serine, 2 (TMPRSS2) than prior SARS-CoV-2 variants. Hence, its isolates grow less efficiently in Vero E6 and Vero-TMPRSS2+ cells. However, Omicron isolates demonstrated a high sensitivity to ovarian carcinoma-derived IGROV-1 cells, naturally expressing low angiotensin-converting enzyme 2 (ACE2) and TMPRSS2 levels, as assessed by flow cytometry (FC).
Omicron BA.1 was particularly more sensitive to IGROV-1 cells. As with BA.1, numerous foci of infected cells were detected at two days post-infection (p.i.), and supernatants were harvested at days two or three p.i., yielding high titers with the S-Fuse reporter cells. S-Fuse cells form syncytia and become GFP+ upon infection, allowing overnight measurement of viral infectivity and neutralizing antibody (nAb) activity. Sequences of the variants after one passage on IGROV-1 cells identified BA.4.6 and BQ.1.1, indicating that no adaptative mutations were generated during this short culture period.
Syncytia were also observed in BA.2.75.2, and BA.4.6. and BQ.1.1-infected S-Fuse cells. The three variants generated syncytia of similar size that were smaller than those formed by the ancestral D614G strain. It will be worth further examining whether other Omicron subvariants may display different fusogenic potentials in different cell types.
The team collected 72 sera samples from a cohort of 35 healthcare workers in Orleans, France, who received three doses of the BNT162b2 vaccine. Thirty out of these 35 individuals experienced a mild symptomatic breakthrough Omicron infection 60 to 359 days after the third vaccination. To this end, the team analyzed 18 individuals early, i.e., one-month post the third dose and ten individuals at four months post the third dose. They investigated whether vaccine-elicited antibodies neutralized the novel Omicron subvariants, BA.2.75.2, and BA.4.6. and BQ.1.1. They used the D614G ancestral strain belonging to the B.1 lineage and BA.1 and BA.5 as controls. Finally, the team calculated the half-maximal effective dose (ED50) for each combination of serum and virus.
Next, the researchers examined the impact of BA.1/BA.2 breakthrough infections on the cross-neutralizing activity of serum antibodies. They analyzed 18 individuals at three months; they resampled 11 of these 18 individuals eight months after infection to evaluate the evolution of the humoral response. The distinct neutralization profile of BA.2.75.2, BA.4.6. and BQ.1.1 after BA.1/BA.2 reinfections prompted researchers to examine the consequences of a BA.5 breakthrough infection on neutralization. They assessed the sera of 15 individuals nearly a month after the BA.5 infection.
Finally, the researchers assessed the sensitivity of BA.2.75.2 and BA.4.6. and BQ.1.1 to mAbs currently authorized (cilgavimab, tixagevimab, and bebtelovimab) or withdrawn because of Omicron escape (sotrovimab, casirivimab, and imdevimab) using the S-Fuse assay.
Neutralizing titers declined varyingly depending on the Omicron isolate. After three months, the researchers noted a strong augmentation of neutralization against D614G and BA.1, with ED50 above 104. Compared to BA.1, the nAb titers were reduced by about seven-fold against BA.5 and BA.4.6 and 18-fold against BA.2.75.2 and BQ.1.1. Eight months after infection, while nAb titers remained high against D614G and BA.1, the decline was more against BA.5 and BA.4.6 and even more, marked against BA.2.75.2 and BQ.1.1. Therefore, post-vaccination breakthrough infection by BA.1/BA.2 led to an increase in Omicron-specific nAb titers, with disparities between variants. The anti-BA.1 response was higher than against BA.5 and BA.4.6, whereas BA.2.75.2 and BQ.1.1 were less sensitive to neutralization.
ED50 attained values of 3x104 against D614G, similar to what the researchers observed for BA.1/BA.2 breakthrough infections. The neutralization of BA.5 and BA.5-derived variants BQ.1.1 was high but lower for the BA.2-derived BA.2.75.2 strain. Notably, the neutralization activity against BA.1 was less potent after a BA.5 infection than after a BA.1/BA.2 infection. Conversely, a BA.1/BA.2 breakthrough infection favored neutralizing BA.1, and BA.2 derived strains relative to the BA.5 lineage.
ED50 were high for D614G but decreased by eight- and 15-fold for BA.1 and BA.5, respectively, after a month of boosting, confirming the antibody escape properties of these previous sublineages. For BA.4.6. and BQ.1.1, the ED50 was low but within the range as observed with the parental BA.5 strain. BA.2.75.2 neutralization titers were 11-fold lower than BA.1. The researchers noted a similar trend four months after the third vaccination, indicating that vaccinees' sera either poorly neutralized or could not neutralize BA.2.75.2, BA.4.6. and BQ.1.1 subvariants.
All mAbs used as pre- or post-exposure prophylaxis (PrEP) belong to the four anti-RBD antibody classes defined by their binding site. Prophylaxis based on ronapreve and evusheld cocktails provided ~80% protection against symptomatic infection, while post-therapy with sotrovimab prevented COVID-19-related hospitalization or death with 85% efficacy. Cilgavimab and tixagevimab, even in combination, and casirivimab, lost all neutralization activity against the three Omicron variants. Imdevinab inhibited BA.4.6, with half maximal inhibitory concentration (IC50) of 220 ng/ml but was inactive against BA.2.75.2 and BQ.1.1. Bebtelovimab was efficient against BA.4.6 and BA.2.75.2 but did not neutralize BQ.1.1. Sotrovimab was weakly active against BA.2.75.2, BA.4.6. and BQ.1.1, with IC50s ranging from 2,874 to 19,391 ng/ml, representing a 45-to-300-fold increase compared to D641G.
Cocktail mAbs, ronapreve (casirivimab and imdevimab), and evusheld (cilgavimab and tixagevimab) lost antiviral efficacy against BA.2.75.2 and BQ.1.1, whereas sotrovimab remained weakly active. BQ.1.1 was also resistant to Bebtelovimab. The evolutionary trajectory of novel Omicron subvariants facilitated their spread in immunized populations and raised concerns about the efficacy of most currently available mAbs. Together, these results demonstrated that the prevalent BA.2.75.2 and BQ.1.1 strains are resistant or weakly sensitive to currently approved mAbs.
The IGROV-1 cells recapitulated the permissibility of primary human nasal or alveolar cells to Omicron subvariants. They allowed rapid infectivity assessments using samples from infected individuals and one-passage amplification of Omicron subvariants. Future work will help understand viral entry pathways and replication in IGROV-1 cells and their underlying cellular mechanisms. Combining viral isolation in IGROV-1 cells with the S-Fuse neutralization assay provided a rapid method to evaluate the properties of novel yet-to-emerge SARS-CoV-2 variants of concern (VOCs).
Furthermore, the study demonstrated that the currently approved or withdrawn coronavirus disease 2019 (COVID-19) therapeutic mAbs lost most of their neutralization potential against these Omicron subvariants. Only sotrovimab retained a relatively low neutralization activity against all strains, with IC50 ranging from three to more than nine µg/ml. It also displayed non-neutralizing antiviral activities, including antibody-dependent cellular cytotoxicity (ADCC). The current findings could help address the debate on the need to reassess the World Health Organization (WHO) therapeutics and COVID-19 guidelines on mAbs.
The higher neutralization titers against D614G highlighted the role of immune imprinting in anamnestic responses. However, the neutralizing response after BA.1/BA.2 or BA.5 breakthrough infection in vaccinated individuals was dichotomous. Breakthrough infections in triply vaccinated individuals stimulated cross-neutralizing responses with distinct efficacies depending on the infecting Omicron variant. The evolution trajectory of the novel Omicron subvariants likely reflects their continuous circulation in immunized populations. In summary, the study findings showed that a few convergent mutations in the BA.2 or BA.5 S led to resistance to most of the clinically used mAbs and strongly impaired the efficacy of vaccine-elicited antibodies.
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