Cross-neutralization of Omicron BA.1 against BA.2 and BA.3 SARS-CoV-2

Omicron SARS-CoV-2 comprises multiple sublineages with distinct spike mutations. BA.1 drove the initial Omicron wave and has been replaced in many regions by BA.2, while BA.3 circulates at low levels. This epidemiological shift highlights the need to define the extent of serological cross-neutralization among Omicron sublineages to inform protection from reinfection and vaccine strategies. The study’s purpose was to quantify neutralizing activity in sera from BA.1-infected individuals against homologous BA.1 and heterologous BA.2, BA.3, and an early pandemic strain (USA/WA1-2020).

Emerging evidence supports booster vaccination to mitigate Omicron risk: two doses of BNT162b2 elicit weak neutralization against Omicron, whereas a third dose induces robust Omicron-neutralizing activity, though durability beyond four months remains to be clarified. Prior non-Omicron SARS-CoV-2 infection alone does not elicit strong neutralization against Omicron, indicating that previously infected individuals benefit from vaccination. T cell immunity and Fc-mediated functions likely contribute to protection against severe disease; most T cell epitopes remain conserved in Omicron spikes. Real-world effectiveness data show that three BNT162b2 doses confer protection against Omicron disease, with some waning over time but overall efficacy remaining high up to six months. These data provide context for interpreting cross-neutralization findings and guiding booster timing and vaccine design.

Ethics and biosafety: All virus work was conducted in BSL-3 labs at the University of Texas Medical Branch (UTMB) with powered air-purifying respirators and full PPE. The UTMB IRB (IRB number 20-0070) approved use of de-identified leftover human sera; informed consent was not required. Cells: Vero E6 (ATCC CCL-81) cells were maintained in high-glucose DMEM with 10% FBS and 1% antibiotics; cells were mycoplasma-negative. Viruses: Recombinant mNeonGreen (mNG) SARS-CoV-2s were generated by engineering full-length Omicron BA.1, BA.2, or BA.3 spike genes into the USA-WA1/2020 backbone (mNG inserted at ORF7). Spike sequences were from GISAID accessions EPI_ISL_649616 (BA.1), EPI_ISL_675934 (BA.2), and EPI_ISL_705691 (BA.3). Virus stocks were produced from infectious cDNA clones; P1 stocks, sequence-verified to exclude undesired mutations, were used for neutralization assays. Pseudoviruses were characterized by genome RNA-to-infectivity ratios as previously reported. Human sera: De-identified sera from individuals infected with Omicron BA.1 were used. Twenty specimens were tested; demographic and FRNT50 details are summarized in Table 1. Sera were collected 8–62 days after positive RT-PCR, reflecting heterogeneous immune states. Neutralization assay: A fluorescent focus reduction neutralization test (FRNT/FRTNT) using mNG SARS-CoV-2 measured 50% neutralization titers (FRNT50). Two-fold serial dilutions of sera were incubated with virus and used to infect Vero E6 monolayers in 96-well plates. After 1 h adsorption, inoculum was removed and overlay medium (0.5% methylcellulose) was added; plates were incubated at 37 °C for 18 h. Fluorescent foci were imaged (Cytation 7, BioTek) and counted. Infection rates were normalized to controls. The neutralization titer for each virus-serum pair was determined in duplicate, and the geometric mean was used. For analysis, FRNT50 values <20 were treated as 10 to differentiate low titers from no activity. Statistics: Geometric mean titers (GMTs) and 95% confidence intervals (CIs) were calculated. Paired comparisons used two-tailed Wilcoxon matched-pairs signed-rank tests. Data and source files are provided with the paper.

- Sera from BA.1-infected individuals (n=20) neutralized Omicron sublineages and an early strain with the following GMTs: BA.1 = 445, BA.2 = 107, BA.3 = 102, USA/WA1-2020 = 16. - Relative to homologous BA.1, neutralization was reduced by 4.2-fold (BA.2), 4.4-fold (BA.3), and 28.4-fold (USA/WA1-2020). - All paired comparisons of GMTs between BA.1 and BA.2, BA.3, or USA/WA1-2020 were statistically significant (two-tailed Wilcoxon matched-pairs signed-rank test; P < 0.0001). - Individual-level FRNT50 values showed consistent patterns across sera, with some variability; values <20 were set to 10 for analysis. - Findings indicate substantial but incomplete cross-neutralization from BA.1 infection against BA.2 and BA.3, and markedly lower activity against the ancestral USA/WA1-2020 strain.

The study directly addresses whether infection with Omicron BA.1 elicits neutralizing antibodies that cross-react with other Omicron sublineages. The observed 4.2- to 4.4-fold reduction in neutralizing titers against BA.2 and BA.3 relative to BA.1 demonstrates partial cross-protection within Omicron, suggesting individuals recently infected with BA.1 may retain meaningful antibody-mediated protection against BA.2 and BA.3. In contrast, the dramatically lower titers against the early pandemic strain underscore antigenic drift from ancestral SARS-CoV-2. These results integrate with emerging vaccine data indicating that booster doses are required to achieve robust Omicron neutralization and that T cell responses and non-neutralizing antibody functions also contribute to protection from severe disease. Together, the cross-neutralization profile supports expectations of reduced susceptibility to BA.2 following BA.1 infection and informs booster strategies aimed at maximizing breadth and durability of protection.

This work quantifies cross-neutralizing antibody responses in BA.1-infected human sera against Omicron BA.2 and BA.3, revealing a modest 4.2–4.4-fold reduction compared to homologous BA.1 and much lower activity against an ancestral strain. These findings imply that recent BA.1 infection may confer partial protection against BA.2/BA.3 and provide immunological context for vaccine booster strategies. Future research should include larger cohorts, standardized timepoints post-infection or vaccination, and longitudinal sampling to define durability of cross-neutralization and to evaluate cellular immune contributions to protection.

- Small sample size (n=20) of serum specimens. - Heterogeneous sampling times (8–62 days post positive RT-PCR), encompassing different immune phases (e.g., acute plasmablast vs. later convalescent stages). - Variability in immune status across donors may affect generalizability; additional specimens collected at later timepoints are needed to substantiate observations. - FRNT50 values below 20 treated as 10 may reduce resolution at low titers.