Omicron infection enhances Delta antibody immunity in vaccinated persons

The extent to which Omicron infection1–9, with or without previous vaccination, elicits protection against the previously dominant Delta (B.1.617.2) variant is unclear. Here we measured the neutralization capacity against variants of severe acute respiratory syndrome coronavirus 2 in 39 individuals in South Africa infected with the Omicron sublineage BA.1 starting at a median of 6 (interquartile range 3–9) days post symptom onset and continuing until last follow-up sample available, a median of 23 (interquartile range 19–27) days post symptoms to allow BA.1-elicited neutralizing immunity time to develop. Fifteen participants were vaccinated with Pfizer's BNT162b2 or Johnson & Johnson's Ad26.CoV2.S and had BA.1 breakthrough infections, and 24 were unvaccinated. BA.1 neutralization increased from a geometric mean 50% focus reduction neutralization test titre of 42 at enrolment to 575 at the last follow-up time point (13.6-fold) in vaccinated participants and from 46 to 272 (6.0-fold) in unvaccinated participants. Delta virus neutralization also increased, from 192 to 1,091 (5.7-fold) in vaccinated participants and from 28 to 91 (3.0-fold) in unvaccinated participants. At the last time point, unvaccinated individuals infected with BA.1 had low absolute levels of neutralization for the non-BA.1 viruses and 2.2-fold lower BA.1 neutralization, 12.0-fold lower Delta neutralization, 9.6-fold lower Beta variant neutralization, 17.9-fold lower ancestral virus neutralization and 4.8-fold lower Omicron sublineage BA.2 neutralization relative to vaccinated individuals infected with BA.1. These results indicate that hybrid immunity formed by vaccination and Omicron BA.1 infection should be protective against Delta and other variants. By contrast, infection with Omicron BA.1 alone offers limited cross-protection despite moderate enhancement.

as BA.2 and other variants is still unclear. We isolated live Omicron BA.1, Omicron BA.2, ancestral, Beta and Delta viruses and neutralized viruses with plasma from participants enrolled and longitudinally sampled during the Omicron BA.1 infection wave in South Africa, with all participants having a confirmed diagnosis of SARS-CoV-2 by quantitative PCR. To quantify neutralization, we used a live virus neutralization assay and calculated the focus reduction neutralization test (FRNT 50 ) titre, the inverse of the plasma dilution required for 50% neutralization, as measured by the reduction in the number of infection foci. We enrolled 41 participants who reported symptoms from late November 2021 to January 2022. We successfully sequenced the infecting virus in 26 participants, and all sequences corresponded to Omicron BA.1 (Extended Data Table 1). Two participants had advanced human immunodeficiency virus (HIV) disease on the basis of a low CD4 count (<200 cells μl −1 throughout the study) and unsuppressed HIV infection, and we excluded these participants because of our previous data showing an atypical response to SARS-CoV-2 in advanced HIV disease 15 . Extended Data Table 2 summarizes the characteristics of the remaining 39 participants.
Of the 39 participants, 27 were admitted to hospital because of coronavirus disease 2019 symptoms. Seven required supplemental oxygen and one died. Fifteen participants were vaccinated and had a breakthrough Omicron BA.1 infection. The median time post vaccination was 139 days (interquartile range (IQR) 120-178), a time interval that would predict considerable waning of the vaccine-elicited immune response 16 , which may have contributed to the breakthrough infections. Eight participants were vaccinated with two doses of Pfizer's BNT162b2 and seven were vaccinated with Johnson & Johnson's Ad26. CoV2.S (six with a single dose and one with two doses; Extended Data Table 1). The length of hospital stay was shorter in the vaccinated (3.5 days) relative to unvaccinated (8 days; Extended Data Table 2) participants. Three participants self-reported having a previous SARS-CoV-2 infection (Extended Data Table 1).
Participants were sampled at enrolment at a median of 6 days (IQR 3-9 days) after symptom onset, and again at weekly follow-up visits that were attended as practicable because of the Christmas holidays in South Africa. The last follow-up visit was a median of 23 days (IQR 19-27 days) post-symptom onset (Extended Data Table 1). Examining neutralization at all available time points per study participant showed that neutralization of the Omicron BA.1 variant increased substantially in most participants from enrolment to the time of the last follow-up (Extended Data Fig. 1), consistent with developing a neutralizing antibody response to Omicron BA.1 infection. We therefore analysed neutralization at enrolment (baseline for the study) and the last follow-up visit to quantify the increase in neutralization capacity after Omicron infection.
We observed that Omicron BA.1 neutralization increased in vaccinated individuals from a low geometric mean titre (GMT) FRNT 50 of 42 at the enrolment visit to 575 at the last follow-up visit about 2 to 3 weeks later, a 13.6-fold change (95% CI confidence interval (CI) 3.7-50.2; Fig. 1a Fig. 2). The trend for the other variants and the ancestral virus was that neutralization was higher relative to Omicron BA.1 in vaccinated participants but lower relative to Omicron BA.1 in unvaccinated participants, although the differences were mostly not significant (Fig. 2a). As a result of the relatively moderate fold change, higher participant numbers would probably be required to make the trends statistically significant.
The comparison of the other variants to Omicron BA.1 within the vaccinated or unvaccinated group does not indicate the differences in neutralization capacity elicited by Omicron BA.1 between the vaccinated and unvaccinated participants. We therefore compared neutralization of each variant between the vaccinated and unvaccinated groups at the last time point directly (Fig. 2b). The smallest difference between vaccinated and unvaccinated participants was in neutralization of Omicron BA.1, the infecting variant, with the vaccinated participants showing 2.2-fold higher neutralization. For the other variants, neutralization was higher in vaccinated participants by a factor of 4.8-fold for Omicron BA.2, 9.6-fold for Beta, 12.0-fold for Delta and 17.9-fold for ancestral (Fig. 2b). All differences were significant, and the 95% CIs for the GMT FRNT 50 of vaccinated and unvaccinated participants did not overlap for BA.2, Beta, Delta or ancestral virus neutralization (Fig. 2b). For the unvaccinated participants, the absolute neutralization capacity against the BA.2, Beta, Delta and ancestral viruses was low 18 , with GMT FRNT 50 being about or below FRNT 50 = 100 (Fig. 2b).

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We also tested neutralization of Omicron BA.1 by Delta-variant-elicited immunity. We collected 18 plasma samples from 14 participants (including pre-vaccination and post-vaccination samples from 4 participants) previously infected in the Delta variant wave in South Africa, 8 of whom were vaccinated either before or after infection (Extended Data Table 3). Confirming previously reported results 19 , we observed similar escape of Omicron BA.1 from Delta-elicited immunity across all samples tested, manifested as a 22.5-fold decrease (95% CI 14.4-35.0) in Omicron BA.1 neutralization compared to Delta virus neutralization (Fig. 3).
The large fold drop in Delta-infection-elicited neutralization capacity against Omicron BA.1 contrasts with the moderate and nonsignificant fold drops, or even fold increases, in neutralization of other variants relative to Omicron BA.1 in individuals infected with Omicron BA.1. However, in unvaccinated individuals, even though fold drops in neutralization were moderate and nonsignificant, the absolute levels of neutralization of the other variants, and of Omicron BA.1 itself, were low and on a similar scale to the cross-neutralization capacity against Omicron in Delta-infection-elicited immunity. This is consistent with other recently reported results 20 , and possibly indicates that Omicron is poorly immunogenic. In agreement with recent reports 21 Our results may be supportive of a scenario in which hybrid immunity formed by Omicron infection combined with vaccination protects as well or better against reinfection with variants such as Delta relative to reinfection with Omicron itself. By contrast, unvaccinated participants infected with Omicron BA.1 only, have low neutralization capacity against the Omicron BA.2, Beta, Delta and ancestral viruses.
Limitations of this study include heterogeneity in participant immune history, including two vaccination types and one boost. On the basis of the high seroprevalence observed in South Africa 25,26 , some participants may also have had unreported previous infection. However, including two vaccine types did not mask the differences between vaccinated and unvaccinated participants, and the low levels of neutralization in unvaccinated participants against the ancestral, Beta and Delta viruses (the dominant strains in the preceding South African infection waves) support the notion that these participants were either not previously infected, or that immunity has waned completely. Participants were also mostly hospitalized, which may not be typical of Omicron infection 13,27 . Increased disease severity has been shown to lead to higher anti-SARS-CoV-2 antibody titres 28 . This should help in the detection of the neutralization response, but whether it would affect the trend we observed is unclear. Omicron infection is unlikely benign to the extent that hospitalization is an outlier outcome: in the USA, the number of individuals with coronavirus disease 2019 who died in the Omicron wave was similar to the number who died in the Delta

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Reagent availability
Virus isolates and the cell line are available from the corresponding author. A Biosafety Level 3 facility is required for laboratories receiving live SARS-CoV-2.
Whole-genome sequencing, genome assembly and phylogenetic analysis RNA was extracted on an automated Chemagic 360 instrument, using the CMG-1049 kit (Perkin Elmer). The RNA was stored at −80 °C before use. Libraries for whole-genome sequencing were prepared using either the Oxford Nanopore Midnight protocol with Rapid Barcoding or the Illumina COVIDseq Assay. For the Illumina COVIDseq assay, the libraries were prepared according to the manufacturer's protocol. Briefly, amplicons were tagmented, followed by indexing using the Nextera UD Indexes Set A. Sequencing libraries were pooled, normalized to 4 nM and denatured with 0.2 N sodium acetate. An 8 pM sample library was spiked with 1% PhiX (PhiX Control v3 adaptor-ligated library used as a control). We sequenced libraries on a 500-cycle v2 MiSeq Reagent Kit on the Illumina MiSeq instrument (Illumina). On the Illumina NextSeq 550 instrument, sequencing was performed using the Illumina COVIDSeq protocol (Illumina), an amplicon-based next-generation sequencing approach. The first-strand synthesis was carried using random hexamer primers from Illumina, and the synthesized cDNA underwent two separate multiplex PCR reactions. The pooled PCR-amplified products were processed for tagmentation and adaptor ligation using IDT for Illumina Nextera UD Indexes. Further enrichment and cleanup was performed as per protocols provided by the manufacturer (Illumina). Pooled samples were quantified using a Qubit 3.0 or 4.0 fluorometer (Invitrogen) through the Qubit dsDNA High Sensitivity Assay according to the manufacturer's instructions. The fragment sizes were analysed using TapeStation 4200 (Invitrogen). The pooled libraries were further normalized to 4 nM concentration and 25 μl of each normalized pool containing unique index adaptor sets was combined in a new tube. The final library pool was denatured and neutralized with 0.2 N sodium hydroxide and 200 mM Tris-HCl (pH 7), respectively. A 1.5 pM sample library was spiked with 2% PhiX. Libraries were loaded onto a 300-cycle NextSeq 500/550 HighOutput Kit v2 and run on the Illumina NextSeq 550 instrument (Illumina). For Oxford Nanopore sequencing, the Midnight primer kit was used: cDNA synthesis was performed on the extracted RNA using LunaScript RT mastermix (New England BioLabs) followed by gene-specific multiplex PCR using the Midnight Primer pools that produce 1,200-base-pair amplicons that overlap to cover the 30-kb SARS-CoV-2 genome. Amplicons from each pool were pooled and used neat for barcoding with the Oxford Nanopore Rapid Barcoding kit as per the manufacturer's protocol. Barcoded samples were pooled and bead purified. After the bead cleanup, the library was loaded on a prepared R9.4.1 flow cell. A GridION X5 or MinION sequencing run was initiated using MinKNOW software with the base-call setting switched off. We assembled paired-end and nanopore.fastq reads using Genome Detective 1.132 (https:// www.genomedetective.com), which was updated for the accurate assembly and variant calling of tiled primer amplicon Illumina or Oxford Nanopore reads, and the Coronavirus Typing Tool. For Illumina assembly, the GATK HaploTypeCaller -min-pruning 0 argument was added to increase mutation calling sensitivity near sequencing gaps. For Nanopore, low-coverage regions with poor alignment quality (<85% variant homogeneity) near sequencing/amplicon ends were masked to be robust against primer drop-out experienced in the spike gene, and the sensitivity for detecting short inserts using a region-local global alignment of reads was increased. In addition, we also used the wf_artic (ARTIC SARS-CoV-2) pipeline as built using the nextflow workflow framework. In some instances, mutations were confirmed visually with .bam files using Geneious software V2020.1.2 (Biomatters). The reference genome used throughout the assembly process was NC_045512.2 (numbering equivalent to MN908947.3). For lineage classification, we used the widespread dynamic lineage classification method from the Phylogenetic Assignment of Named Global Outbreak Lineages software suite (https://github.com/hCoV-2019/pangolin).

Cells
Vero E6 cells (originally ATCC CRL-1586, obtained from Cellonex in South Africa) were propagated in complete growth medium consisting of Dulbecco's modified Eagle medium with 10% fetal bovine serum (Hyclone) containing 10 mM HEPES, 1 mM sodium pyruvate, 2 mM l-glutamine and 0.1 mM nonessential amino acids (Sigma-Aldrich). Vero E6 cells were passaged every 3-4 days. The H1299-E3 cell line (H1299 originally from ATCC as CRL-5803) was propagated in growth medium consisting of complete Roswell Park Memorial Institute 1640 medium with 10% fetal bovine serum containing 10 mM HEPES, 1 mM sodium pyruvate, 2 mM l-glutamine and 0.1 mM nonessential amino acids. Cells were passaged every second day. The H1299-E3 (H1299-ACE2, clone E3) cell line was derived from H1299 as described in our previous work 1,17 . Cell lines were not authenticated. Cell lines tested negative for mycoplasma contamination.

Virus expansion
All work with live virus was performed in Biosafety Level 3 containment using protocols for SARS-CoV-2 approved by the Africa Health Research Institute Biosafety Committee. ACE2-expressing H1299-E3 cells were seeded at 4.5 × 10 5 cells in a 6-well plate well and incubated for 18-20 h. After one Dulbecco's phosphate-buffered saline (PBS) wash, the subconfluent 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 (completion of passage 1 (P1)), cells were trypsinized, centrifuged at 300 RCF for 3 min and resuspended in 4 ml growth medium. All cells from the P1 infection were added to Vero E6 cells that had been seeded at 2 × 10 5 cells ml −1 , 20 ml total, 18-20 h earlier in a T75 flask for cell-to-cell infection. Foci were stained with a rabbit anti-spike monoclonal antibody (BS-R2B12, GenScript A02058) at 0.5 μg ml −1 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, and then washed with wash buffer containing 0.05% Tween-20 in PBS. Secondary goat anti-rabbit antibody conjugated to horseradish peroxidase (Abcam ab205718) was added at 1 μg ml −1 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 ImmunoSpot Ultra-V S6-02-6140 Analyzer ELISPOT instrument with BioSpot Professional built-in image analysis (Cellular Technology Limited).

Statistics and fitting
Statistical methods were not used to predetermine sample size, and blinding and randomization were not used. All statistics and fitting were performed using custom code in MATLAB v.2019b. Neutralization data were fitted to: D Tx = 1/1 + ( /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 ID 50 is the plasma dilution giving 50% neutralization. FRNT 50 = 1/ID 50 . Values of FRNT 50 < 1 are set to 1 (undiluted), the lowest measurable value. We note that the most concentrated plasma dilution was 1:25 and therefore FRNT 50 < 25 data were extrapolated. To calculate CIs, FRNT 50 or fold change in FRNT 50 per participant was log transformed and the arithmetic mean plus 2 s.d. and the arithmetic mean minus 2 s.d. were calculated for the log-transformed values. These were exponentiated to obtain the upper and lower 95% CIs on the geometric mean FRNT 50 or the fold change in FRNT 50 geometric means.

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