Neutralization of Omicron sublineages and Deltacron SARS-CoV-2 by three doses of BNT162b2 vaccine or BA.1 infection

ABSTRACT Distinct SARS-CoV-2 Omicron sublineages have evolved showing increased fitness and immune evasion than the original Omicron variant BA.1. Here, we report the neutralization activity of sera from BNT162b2 vaccinated individuals or unimmunized Omicron BA.1-infected individuals against Omicron sublineages and “Deltacron” variant (XD). BNT162b2 post-dose 3 immune sera neutralized USA-WA1/2020, Omicron BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA.4/5-, and XD-spike SARS-CoV-2s with geometric mean titres (GMTs) of 1335, 393, 298, 315, 216, 103, and 301, respectively; thus, BA.4/5 SARS-CoV-2 spike variant showed the highest propensity to evade vaccine neutralization compared to the original Omicron variants BA.1. BA.1-convalescent sera neutralized USA-WA1/2020, BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA.4/5-, and Deltacron-spike SARS-CoV-2s with GMTs of 15, 430, 110, 109, 102, 25, and 284, respectively. The unique mutation F486V in the BA.4/5 spike contributes to the increased evasion of antibody neutralization by sublineage BA.4/5. The low neutralization titres of vaccinated sera or convalescent sera from BA.1 infected individuals against the emerging and rapidly spreading Omicron BA.4/5 variants provide important results for consideration in the selection of an updated vaccine in the current Omicron wave. Trial registration: ClinicalTrials.gov; identifier: NCT04368728.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has emerged as the fifth variant of concern (VOC) after Alpha, Beta, Gamma, and Delta. Omicron exhibited the greatest evasion from vaccine-and infection-elicited neutralizing antibodies among all the VOCs [1,2]. Since the emergence of Omicron in late November 2021, distinct sublineages have evolved around the world: BA.1 was responsible for the initial surge and has now been replaced by BA.2 and BA.2.12.1 in the USA (https://covid.cdc.gov/covid-data-tracker/#varia nt-proportions); BA.4 and BA.5 have become prevalent in South Africa and, more recently, are becoming prevalent in some areas in Europe and are increasing in the USA; BA.3 currently remains at low frequency. The co-circulation of Omicron and Delta has also led to a hybrid variant "Deltacron" [3]. Since these newly emerged Omicron sublineages and Deltacron have distinct mutations in the spike glycoproteins ( Figure S1 (A)), it is important to examine their susceptibility to vaccine-or infection-elicited antibody neutralization.
Using the recombinant SARS-CoV-2s, we determined the 50% fluorescent focus-reduction neutralization titres (FFRNT 50 ) for two panels of human sera. The first serum panel (n = 22) was collected from individuals at one-month post-dose 3 (PD3) of BNT162b2 vaccine. We chose the PD3 sera because (i) many people have already received 3 doses of BNT162b2 and (ii) two doses of BNT162b2 did not elicit robust neutralization against Omicron BA.1 [2]. The second serum panel (n = 20) was collected from unvaccinated COVID-19 patients who had been infected by BA.1 during the initial Omicron emergence in the USA [4]. The genotype of the infecting virus was verified for each patient by Sanger sequencing of nasal swabs. Tables S1 and S2 summarize the data from the PD3 BNT162b2-vaccinated-and BA.1-infected individuals, respectively.
The current study has confirmed and expanded our previous results on Omicron sublineage neutralization [1,2,4]. Our results have potential implications for the strategy of updating the current COVID-19 vaccines in which, even within the Omicron lineages, substantial differences exist in the potential of vaccinated or BA.1-infected individuals to neutralize Omicron sublineages. The data suggest that Omicron BA.4 and BA.5 sublineages are adapted to evade neutralization in both prototype vaccinated and Omicron BA.1infected individuals. The antigenic distinctions of spike glycoproteins among Omicron sublineages, Deltacron XD, and USA-WA1/2020 as well as prevalence of Omicron sublineages must be carefully considered when deciding to update the vaccine to new variants.
Our study has some limitations. This study lacks analyses of T cells and non-neutralizing antibodies that can mediate Fc-mediated effector functions. Both of these immune arms, in conjunction with neutralizing antibodies, protect patients from severe disease. After vaccination or infection, the majority of the T cell epitopes are preserved in Omicron spikes [7]. Caution should also be taken when comparing the differences in variant neutralization between BNT162b2-vaccinated (PD3 sera) and BA.1-infected individuals. This is because (i) a third dose booster vaccination increases both the magnitude and breadth of neutralization [8], which might explain the observation that the neutralizing GMTs of BA.1 infected convalescent sera against heterologous variants or sublineages were reduced more than those from BNT162b2 PD3 sera. (ii) BA.1-infected sera used in this study (collected on days 8-62 after a positive RT-PCR test) were heterogeneous, with some sera collected at an acute plasma blast stage and other sera at a convalescent IgG dominant phase; and (iii) the relatively small sample size. Regardless, our results consistently showed the most significant reduction in neutralization against BA.4/5 spike, underscoring the importance of closely monitoring the prevalence of sublineages BA.4 and BA.5 globally and taking the data into account when making decisions on updating current COVID-19 vaccines.

Human sera
Two human serum panels were used in the study. The first panel of serum samples was collected from BNT162b2 vaccines participating in the phase 1 portion of the ongoing phase 1/2/3 clinical trial (Clinical-Trials.gov identifier: NCT04368728). The protocol and informed consent were approved by institutional review boards for each of the investigational centres 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 previously [1,9,10]. BNT162b2-vaccinated sera (n = 22) were collected one month after dose 3 and used for neutralization test. Table S1 summarizes the patient information which was previously reported [8,11].
The second serum panel (n = 20) was collected from COVID-19 patients who were not vaccinated but infected by BA.1 sublineage during the initial Omicron emergence at the University of Texas Medical Branch (UTMB) [4]. The research protocol regarding the use of human sera was reviewed and approved by the UTMB Institutional Review Board (IRB number 20-0070). The de-identified human sera were heat-inactivated at 56°C for 30 min before the neutralization test. The genotype of infecting virus was verified by Sanger sequencing. Table S2 summarizes the serum information which was recently reported [4].

Recombinant Omicron sublineage-or Deltacron XD-spike mNG SARS-CoV-2s
Recombinant Omicron sublineage BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA.4/5-, and Deltacron XD-spike mNG SARS-CoV-2s were constructed by engineering the complete spike gene from the indicated variants into an infectious cDNA clone of mNG USA-WA1/ 2020 [12] (Figure S1(A)). All amino acid substitutions, deletions, and insertions in the variant-spike glycoproteins were introduced into the infectious cDNA clone of mNG USA-WA1/2020 using PCR-based mutagenesis as previously described [13]. The BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA4/5-, and XD-spike sequences were based on GISAID EPI_ISL_6640916, EPI_ISL_6795834.2, EPI_ISL_12115772, EPI_ISL_7 605591, EPI_ISL_11542270 (BA.4 spike is identical to BA.5 spike EPI_ISL_11542604), and EPI_ISL_1 0819657, respectively. Figure S1(A) depicts the spike mutations from different Omicron sublineages and Deltacron XD variant. The full-length cDNA of viral genome bearing the variant spike was assembled via in vitro ligation and used as a template for in vitro transcription. The full-length viral RNA was then electroporated into Vero E6-TMPRSS2 cells. On day 3-4 post electroporation, the original P0 virus was harvested from the electroporated cells and propagated for another round on Vero E6 cells to produce the P1 virus. The infectious titre of the P1 virus was quantified by fluorescent focus assay on Vero E6 cells ( Figure S1(B)) and sequenced for the complete spike gene to ensure no undesired mutations. The P1 virus was used for the neutralization test. The protocols for the mutagenesis of mNG SARS-CoV-2 and virus production were reported previously [14].

Fluorescent focus reduction neutralization test (FFRNT)
FFRNT was performed to measure the neutralization titres of sera against USA-WA1/2020, BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA4/5-, and XD-spike mNG SARS-CoV-2s. The FFRNT protocol was reported previously [15]. Vero E6 cells were seeded onto 96-well plates with 2.5 × 10 4 cells per well (Greiner Bio-one™) and incubated overnight. On the next day, each serum was two-fold serially diluted in culture medium and mixed with 100-150 FFUs of mNG SARS-CoV-2. The final serum dilution ranged from 1:20 to 1:20,480. After incubation at 37°C for 1 h, the serum-virus mixtures were loaded onto the preseeded Vero E6 cell monolayer in 96-well plates. After 1 h infection, the inoculum was removed and 100 μl of overlay medium containing 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 Cytation™ 7 (BioTek) armed with 2.5× FL Zeiss objective with a wide-field of view and processed using the software settings (GFP [469,525] threshold 4000, object selection size 50-1000 µm). The fluorescent mNG foci were counted in each well and normalized to the non-serum-treated controls to calculate the relative infectivities. The FFRNT 50 value was defined as the minimal serum dilution to suppress >50% of fluorescent foci. The neutralization titre of each serum was determined in duplicate assays, and the geometric mean was taken. Tables S1 and S2 summarize the FFRNT 50 results.