SARS-CoV-2 Omicron has extensive but incomplete escape of Pfizer BNT162b2 elicited neutralization and requires ACE2 for infection

Abstract

The emergence of the SARS-CoV-2 Omicron variant, first identified in South Africa, may compromise the ability of vaccine and previous infection (1) elicited immunity to protect against new infection. Here we investigated whether Omicron escapes antibody neutralization elicited by the Pfizer BNT162b2 mRNA vaccine in people who were vaccinated only or vaccinated and previously infected. We also investigated whether the virus still requires binding to the ACE2 receptor to infect cells. We isolated and sequence confirmed live Omicron virus from an infected person in South Africa. We then compared neutralization of this virus relative to an ancestral SARS-CoV-2 strain with the D614G mutation. Neutralization was by blood plasma from South African BNT162b2 vaccinated individuals. We observed that Omicron still required the ACE2 receptor to infect but had extensive escape of Pfizer elicited neutralization. However, 5 out of 6 of the previously infected, Pfizer vaccinated individuals, all of them with high neutralization of D614G virus, showed residual neutralization at levels expected to confer protection from infection and severe disease (2). While vaccine effectiveness against Omicron is still to be determined, these data support the notion that high neutralization capacity elicited by a combination of infection and vaccination, and possibly by boosting, could maintain reasonable effectiveness against Omicron. If neutralization capacity is lower or wanes with time, protection against infection is likely to be low. However, protection against severe disease, requiring lower neutralization levels and involving T cell immunity, would likely be maintained.

The emergence of the Omicron variant (https://www.nicd.ac.za/wp-content/uploads/2021/11/Update-of-SA-sequencing-data-from-GISAID-26-Nov_Final.pdf) of SARS-CoV-2 in November 2021 in South Africa has raised concerns that, based on the large number of mutations in the spike protein and elsewhere on the virus (https://covdb.stanford.edu/page/mutation-viewer/#omicron), this variant will have considerable escape from vaccine elicited immunity. Furthermore, several mutations in the receptor binding domain and S2 are predicted to impact transmissibility and affinity for ACE2.

We previously engineered a human lung cell line clone (H1299-ACE2) which expresses the ACE2 receptor (3). Here we used it to both isolate the virus and test neutralization. The H1299-ACE2 cells are similar to Vero-E6 in the way they form infection foci in a live virus infection with ancestral D614G and Beta variant virus but are considerably more sensitive (Fig S2A–B). Infection of cell-free Omicron of our unmodified Vero-E6 cells was low (Fig S2C) so that we were unable to use these cells with the generated virus stock.

Sequencing of the isolated virus confirmed it was an Omicron strain with the R346K mutation. We first tested the isolated virus on the H1299-ACE2 and H1299 parental cells and observed that Omicron infected the ACE2-expressing cells in a concentration dependent manner but did not infect the parental H1299, indicating that ACE2 is required for Omicron entry (Fig. 1A).

Figure 1: ACE2 dependence and partial neutralization of the Omicron variant by Pfizer BNT162b2 elicited immunity.

(A) Titration of live SARS-CoV-2 Omicron on H1299 parental cells and H1299-ACE2 cells. Plot shows result of titration on H1299-ACE2 cells. (B) Neutralization of the Omicron virus compared to D614G ancestral virus participants vaccinated with BNT162b2 and infected by ancestral SARS-CoV-2 (green) or vaccinated only. 14 samples from 12 participants were tested. Red horizontal line denotes most concentrated plasma tested. Numbers in black above each virus strain are geometric mean titers (GMT) of the reciprocal plasma dilution (FRNT50) causing 50% reduction in the number of infection foci. Number in red denote fold-change in GMT between virus strain on the left and the virus strain on the right of each panel. p=0.0018 as determined by the Wilcoxon rank sum test.

We then tested the ability of plasma from BNT162b2 vaccinated study participants to neutralize Omicron versus ancestral D614G virus in a live virus neutralization assay. We tested 14 plasma samples from 12 participants (Table S1), with 6 having no previous record of SARS-CoV-2 infection nor detectable nucleocapsid antibodies indicative of previous infection (Materials and methods). For two of these participants, we used samples from two timepoints. The remaining 6 participants had a record of previous infection in the first SARS-CoV-2 infection wave in South Africa where infection was with ancestral D614G virus (Table S1). To quantify neutralization in the live virus neutralization assay, we calculated the focus reduction neutralization test (FRNT50) value, which is the inverse of the plasma dilution required for 50% reduction in infection focus number. Geometric mean titer (GMT) FRNT50 was 1321 for D614G. These samples therefore had very strong neutralization of D614G virus, consistent with sampling soon after vaccination. GMT FRNT50 for the same samples was 32 for Omicron, a 41-fold decline (Fig 1B). However, the escape was incomplete. Five of the participants, all previously infected, with the highest neutralization of the D614G virus in the group (FRNT50= 15124, 6603, 38336, 89288 and 9811) showed a drop to lower but still reasonably high levels (FRNT50= 503, 355, 645, 1628, 451), indicating that Omicron escape was incomplete. The single previously infected and vaccinated participant with lower neutralization (FRNT50= 902) showed no detectable neutralization of the Omicron variant (FRNT50= 1).

Vaccination with BNT162b2 has been shown to induce neutralizing antibody titers against the ancestral virus of 2.4-fold of the mean convalescent titer (2, 4). Thus, a 41-fold reduction translates to a neutralization level of Omicron of around 5.7% the mean convalescent titer. This is estimated to correspond to a vaccine efficacy of 22.5% (95% CI: 8.5%−40.7%) against symptomatic infection (2, 5), essentially compromising the ability of the vaccine to protect against infection. However, much lower neutralization levels (~3% of convalescent (2, 5)) are sufficient for protection against severe disease, although this estimate is difficult to validate. The current level of residual neutralization with Omicron is above this minimal level and therefore would be roughly sufficient for protection from severe disease.

Shortly after we released these results, several studies reported results (6) including Pfizer- BioNTech (https://www.businesswire.com/news/home/20211208005542/en/). These results are similar to ours, with large fold-drops in neutralization of Omicron versus ancestral virus, although the fold-drop we observe is at the upper range. This may be due to the R346K mutation present in our isolate. This mutation has been previously identified as an escape mutation (7) and is predicted to give additional moderate escape relative to Omicron without the mutation (https://jbloomlab.github.io/SARS2_RBD_Ab_escape_maps/escape-calc/)

Interestingly, the Pfizer- BioNTech study reports that boosting, which strongly increases neutralization capacity, greatly reduces Omicron escape. Given that neutralization of Omicron was low in vaccination in the absence of previous infection, and that waning of vaccine elicited neutralization, which occurs after about the first month post-vaccination (8), would be expected to further decrease neutralization capacity, a reasonable conclusion from this data may be that vaccination my offer very limited protection against Omicron infection. However, since Omicron escape from neutralization is incomplete, our results support a role for Pfizer-BNT162b2 vaccination with in being effective against severe disease which requires lower neutralization levels. T cell immunity which is unlikely to be strongly decreased with Omicron infection (9, 10). While T cells may not prevent infection, they would also protect against more severe illness and death.

Based on these results, high neutralization capacity of SARS-CoV-2, which can arise with a combination of previous infection and vaccination, would be expected to confer protection against Omicron infection. Boosters may confer a similar or even greater protective effect (https://www.businesswire.com/news/home/20211208005542/en/). Boosters based on variant sequences such as Beta should also be tested to investigate if they can result in better protection. While there may be other explanations for a decreased frequency of severe disease in an emerging variant (11), the incomplete escape of this variant from neutralization may predict that severe disease in the Omicron wave would be less than in previous infection waves where immunity to SARS-CoV-2 at the population level was lower.

Materials and Methods

Ethical statement

Blood samples were obtained from hospitalized adults with PCR-confirmed SARS-CoV-2 infection and/or vaccinated individuals who were enrolled in a prospective cohort study approved by the Biomedical Research Ethics Committee at the University of KwaZulu–Natal (reference BREC/00001275/2020). Use of residual swab sample was approved by the University of the Witwatersrand Human Research Ethics Committee (HREC) (ref. M210752).

Whole-genome sequencing, genome assembly and phylogenetic analysis

cDNA synthesis was performed on the extracted RNA using random primers followed by gene-specific multiplex PCR using the ARTIC V.3 protocol (https://www.protocols.io/view/covid-19-artic-v3-illumina-library-construction-an-bibtkann). In brief, extracted RNA was converted to cDNA using the Superscript IV First Strand synthesis system (Life Technologies) and random hexamer primers. SARS-CoV-2 whole-genome amplification was performed by multiplex PCR using primers designed using Primal Scheme (http://primal.zibraproject.org/) to generate 400-bp amplicons with an overlap of 70 bp that covers the 30 kb SARS-CoV-2 genome. PCR products were cleaned up using AmpureXP purification beads (Beckman Coulter) and quantified using the Qubit dsDNA High Sensitivity assay on the Qubit 4.0 instrument (Life Technologies). We then used the Illumina Nextera Flex DNA Library Prep kit according to the manufacturer’s protocol to prepare indexed paired-end libraries of genomic DNA. Sequencing libraries were normalized to 4 nM, pooled and denatured with 0.2 N sodium acetate. Then, a 12-pM sample library was spiked with 1% PhiX (a PhiX Control v.3 adaptor-ligated library was used as a control). We sequenced libraries on a 500-cycle v.2 MiSeq Reagent Kit on the Illumina MiSeq instrument (Illumina). We assembled paired-end fastq reads using Genome Detective 1.126 (https://www.genomedetective.com) and the Coronavirus Typing Tool. We polished the initial assembly obtained from Genome Detective by aligning mapped reads to the reference sequences and filtering out low-quality mutations using the bcftools 1.7–2 mpileup method. Mutations were confirmed visually with BAM files using Geneious software (Biomatters). P2 stock was sequenced and confirmed Omicron with the following substitutions:E:T9I,M:D3G,M:Q19E,M:A63T,N:P13L,N:R203K,N:G204R,ORF1a:K856R,ORF1a:L2084I,ORF1a:A2710T,ORF1a:T3255I,ORF1a:P3395H,ORF1a:I3758V,ORF1b:P314L,ORF1b:I1566V,ORF9b:P10S,S:A67V,S:T95I,S:Y145D,S:L212I,S:G339D,S:R346K,S:S371L,S:S373P,S:S375F,S:K417N,S:N440K,S:G446S,S:S477N,S:T478K,S:E484A,S:Q493R,S:G496S,S:Q498R,S:N501Y,S:Y505H,S:T547K,S:D614G,S:H655Y,S:N679K,S:P681H,S:N764K,S:D796Y,S:N856K,S:Q954H,S:N969K,S:L981F. Deletions: N:E31-,N:R32-,N:S33-,ORF1a:S2083-,ORF1a:L3674-,ORF1a:S3675-,ORF1a:G3676-,ORF9b:E27-,ORF9b:N28-,ORF9b:A29-,S:H69-,S:V70-,S:G142-,S:V143-,S:Y144-,S:N211-.Sequence was deposited in GISAID, accession: EPI_ISL_7358094.

SARS-CoV-2 nucleocapsid enzyme-linked immunosorbent assay (ELISA)

2 μg/ml nucleocapsid protein (Biotech Africa; Catalogue number: BA25-P was used to coat 96-well, high-binding plates and incubated overnight at 4°C. The plates were incubated in a blocking buffer consisting of 5% skimmed milk powder, 0.05% Tween 20, 1x PBS. Plasma samples were diluted to a 1:100 dilution in a blocking buffer and added to the plates. IgG secondary antibody was diluted to 1:3000 in blocking buffer and added to the plates followed by TMB substrate (Thermo Fisher Scientific). Upon stopping the reaction with 1 M H2SO4, absorbance was measured at a 450 nm wavelength.

Cells

Vero E6 cells (ATCC CRL-1586, obtained from Cellonex in South Africa) were propagated in complete DMEM with 10% fetal bovine serum (Hylone) containing 1% each of HEPES, sodium pyruvate, L-glutamine and nonessential amino acids (Sigma-Aldrich). Vero E6 cells were passaged every 3–4 days. The H1299-E3 cell line for first-passage SARS-CoV-2 expansion, derived as described in (3), was propagated in complete RPMI with 10% fetal bovine serum containing 1% each of HEPES, sodium pyruvate, L-glutamine and nonessential amino acids. H1299 cells were passaged every second day. Cell lines have not been authenticated. The cell lines have been tested for mycoplasma contamination and are mycoplasma negative.

Virus expansion

All work with live virus was performed in Biosafety Level 3 containment using protocols for SARS-CoV-2 approved by the AHRI Biosafety Committee. ACE2-expressing H1299-E3 cells were seeded at 4.5 × 10⁵ cells in a 6 well plate well and incubated for 18–20 h. After one DPBS wash, the sub-confluent cell monolayer was inoculated with 500 μL universal transport medium diluted 1:1 with growth medium filtered through a 0.45-μm filter. Cells were incubated for 1 h. Wells were then filled with 3 mL complete growth medium. After 4 days of infection, cells were trypsinized, centrifuged at 300 rcf for 3 min and resuspended in 4 mL growth medium. Then 1 mL was added to Vero E6 cells that had been seeded at 2 × 10⁵ cells per mL 18–20 h earlier in a T25 flask (approximately 1:8 donor-to-target cell dilution ratio) for cell-to-cell infection. The coculture of ACE2-expressing H1299-E3 and Vero E6 cells was incubated for 1 h and the flask was then filled with 7 mL of complete growth medium and incubated for 4 days. The viral supernatant (P2 stock) was used for experiments.

Live virus neutralization assay

H1299-E3 cells were plated in a 96-well plate (Corning) at 30,000 cells per well 1 day pre-infection. Plasma was separated from EDTA-anticoagulated blood by centrifugation at 500 rcf for 10 min and stored at −80°C. Aliquots of plasma samples were heat-inactivated at 56°C for 30 min and clarified by centrifugation at 10,000 rcf for 5 min. Virus stocks were used at approximately 50–100 focus-forming units per microwell and added to diluted plasma. Antibody–virus mixtures were incubated for 1 h at 37°C, 5% CO₂. Cells were infected with 100 μL of the virus–antibody mixtures for 1 h, then 100 μL of a 1X RPMI 1640 (Sigma-Aldrich, R6504), 1.5% carboxymethylcellulose (Sigma-Aldrich, C4888) overlay was added without removing the inoculum. Cells were fixed 18 h post-infection using 4% PFA (Sigma-Aldrich) for 20 min. Foci were stained with a rabbit anti-spike monoclonal antibody (BS-R2B12, GenScript A02058) at 0.5 μg/mL in a permeabilization buffer containing 0.1% saponin (Sigma-Aldrich), 0.1% BSA (Sigma-Aldrich) and 0.05% Tween-20 (Sigma-Aldrich) in PBS. Plates were incubated with primary antibody overnight at 4°C, then washed with wash buffer containing 0.05% Tween-20 in PBS. Secondary goat anti-rabbit horseradish peroxidase (Abcam ab205718) antibody was added at 1 μg/mL and incubated for 2 h at room temperature with shaking. TrueBlue peroxidase substrate (SeraCare 5510–0030) was then added at 50 μL per well and incubated for 20 min at room temperature. Plates were imaged in an ELISPOT instrument with built-in image analysis (C.T.L).

Statistics and fitting

All statistics and fitting were performed using MATLAB v.2019b. Neutralization data were fit to

$$\text{Tx}=1/1+(\text{D}/\text{ID}50).$$

Here Tx is the number of foci normalized to the number of foci in the absence of plasma on the same plate at dilution D and ID50 is the plasma dilution giving 50% neutralization. FRNT50 = 1/ID50. Values of FRNT50 <1 are set to 1 (undiluted), the lowest measurable value.

Estimating vaccine efficacy from neutralization titers

Previously, the fold reduction in neutralization was shown to correlate and predict vaccine efficacy against symptomatic and severe infection with ancestral SARS-CoV-2 infection (2), and more recently with variants of concern (5). The methods used in these previous studies were used here to estimate the vaccine efficacy against Omicron based on the loss of neutralization observed in this study. Briefly, vaccine efficacy (VE) was estimated based on the (log10) mean neutralization titer as a fold of the mean convalescent titer reported for BNT162b2 in phase 1/2 trials (μ), and the (log10) fold drop in neutralization titer to Omicron (f) using the equation:

$$VE(\mu ,f)={\int }_{-\infty }^{\infty }N(x,\mu -f,\sigma )\frac{1}{1+e^{-k\left(x-x_{50}\right)}}dx.$$

Here, N is the probability density function of a normal distribution with mean μ − f and standard deviation σ, and k and x₅₀ are the parameters of the logistic function relating neutralization to protection. All parameters excluding f were estimated previously as, μ = log₁₀ 2.4, σ = 0.46, k = 3 and x₅₀ = log₁₀ 0.2 (2).