Neutralization of Omicron BA.1, BA.2, and BA.3 SARS-CoV-2 by 3 doses of BNT162b2 vaccine

Ethical statement

All virus work was performed in a biosafety level 3 (BSL-3) laboratory with redundant fans in the biosafety cabinets at The University of Texas Medical Branch at Galveston. All personnel wore powered air-purifying respirators (Breathe Easy, 3M) with Tyvek suits, aprons, booties, and double gloves.


Vero E6 (ATCC® CRL-1586) was purchased from the American Type Culture Collection (ATCC, Bethesda, MD), and maintained in a high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; HyClone Laboratories, South Logan, UT) and 1% penicillin/streptomycin at 37 °C with 5% CO2. All culture media and antibiotics were purchased from ThermoFisher Scientific (Waltham, MA). The cell line was tested negative for mycoplasma.

Human serum

Serum samples were collected from BNT162b2 vaccinees participating in the phase 1 portion of the ongoing phase 1/2/3 clinical trial ( identifier: NCT04368728). The protocol and informed consent were approved by institutional review boards for each of the investigational centers participating in the study. The study was conducted in compliance with all International Council for Harmonisation Good Clinical Practice guidelines and the ethical principles of the Declaration of Helsinki. The primary outcomes for phase 1 were reported previously15,16. BNT162b2-vaccinated serum (n = 22) was collected on the day of the third dose of BNT162b2, which was administered at 7.9–8.8 months after the second dose; and sera collected 1 month after the third dose were used in the present study for neutralization testing. Table S1 summarizes the patient information (e.g., age and gender) and sample collection time points. The information for the serum panels were reported previously17,18.

Construction and characterization of recombinant Omicron sublineage spike mNG SARS-CoV-2s

Recombinant Omicron BA.1-, BA.2-, and BA.3-spike mNG SARS-CoV-2s were constructed by engineering the complete spike gene from Omicron sublineages into an infectious cDNA clone of mNG USA-WA1/202019 (Fig. S1a). All spike mutations, deletions, and insertions were introduced into the infectious cDNA clone of mNG USA-WA1/2020 using PCR-based mutagenesis as previously described20. The BA.1, BA.2, and BA.3 spike sequences were based on GISAID EPI_ISL_6640916, EPI_ISL_6795834.2, and EPI_ISL_7605591, respectively. The full-length cDNA of viral genome containing the complete Omicron spike was assembled via in vitro ligation. The resulting full-length cDNA was used as a template for in vitro transcription of genome-length viral RNA. The in vitro transcribed viral RNA was electroporated into Vero E6 cells. On day 3 post electroporation, the original viral stock (P0) was harvested from the electroporated cells. The P0 virus was amplified for another round on Vero E6 cells to produce the P1 stock for neutralization testing. The infectious titer of the P1 virus was quantified by fluorescent focus assay on Vero E6 cells (Fig. S1b). We sequenced the complete spike gene of the P1 virus to ensure no undesired mutations. Only the P1 viruses were used for the neutralization test. The protocols for the mutagenesis of mNG SARS-CoV-2 and virus production were reported previously21. To determine the specific infectivity, we quantified the P1 virus stocks for their fluorescent focus units (FFU) and genomic RNA contents by fluorescent focus assay on Vero E6 cells and RT-qPCR, respectively. The methods for fluorescent focus assay and RT-qPCR were reported previously4,22. The specific infectivity of each virus was measured by the genomic RNA-to-FFU ratios (genome/FFU).

Fluorescent focus reduction neutralization test

Neutralization titers of human sera were measured by FFRNT using the USA-WA1/2020, BA.1-, BA.2-, and BA.3-spike mNG SARS-CoV-2s. The details of the FFRNT protocol were reported previously4. Briefly, 2.5 × 104 Vero E6 cells per well were seeded in 96-well plates (Greiner Bio-one™). The cells were incubated overnight. On the next day, each serum was 2-fold serially diluted in the culture medium with the first dilution of 1:20 (final dilution range of 1:20 to 1:20,480). The diluted serum was incubated with 100-150 FFUs of mNG SARS-CoV-2 at 37 °C for 1 h, after which the serum-virus mixtures were loaded onto the pre-seeded Vero E6 cell monolayer in 96-well plates. After 1 h infection, the inoculum was removed and 100 μl of overlay medium (supplemented with 0.8% methylcellulose) was added to each well. After incubating the plates at 37 °C for 16 h, raw images of mNG foci were acquired using CytationTM 7 (BioTek) armed with 2.5× FL Zeiss objective with a wide-field of view and processed using the Gene 5 software settings (GFP [469,525] threshold 4000, object selection size 50–1000 µm). The foci in each well were counted and normalized to the non-serum-treated controls to calculate the relative infectivities. The FFRNT50 value was defined as the minimal serum dilution that suppressed >50% of fluorescent foci. The neutralization titer of each serum was determined in duplicate assays, and the geometric mean was taken. All attempts at replication were successful. Table S1 summarizes the FFRNT50 results. Data were initially plotted in GraphPad Prism 9 software and assembled in Adobe Illustrator.


The nonparametric Wilcoxon matched-pairs signed-rank test was used to analyze the statistical significance in Fig. 1.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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