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Omicron infection enhances neutralizing immunity against the Delta variant

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Khadija Khan1,2#, Farina Karim1,2#, Sandile Cele1,2, James Emmanuel San3, Gila Lustig4, Houriiyah
Tegally3,5, Mallory Bernstein1, Yashica Ganga1, Zesuliwe Jule1, Kajal Reedoy1, Nokuthula Ngcobo1,
Matilda Mazibuko1, Ntombifuthi Mthabela1, Zoey Mhlane1, Nikiwe Mbatha1, Jennifer Giandhari3,
Yajna Ramphal3, Taryn Naidoo1, Nithendra Manickchund6, Nombulelo Magula7, Salim S. Abdool
Karim4,8, Glenda Gray9, Willem Hanekom1,10, Anne von Gottberg11,12, , COMMIT-KZN Team§,
Bernadett I. Gosnell6, Richard J. Lessells3,4, Penny L. Moore4,11,12,13, Tulio de Oliveira3,4,5,14, MahomedYunus S. Moosa6, Alex Sigal1,2,15*

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1

Africa Health Research Institute, Durban, South Africa. 2School of Laboratory Medicine and Medical
Sciences, University of KwaZulu-Natal, Durban, South Africa. 3KwaZulu-Natal Research Innovationand Sequencing Platform, Durban, South Africa. 4Centre for the AIDS Programme of Research inSouth Africa, Durban, South Africa. 5Centre for Epidemic Response and Innovation, School of Data
Science and Computational Thinking, Stellenbosch University, Stellenbosch, South Africa.
6
Department of Infectious Diseases, Nelson R. Mandela School of Clinical Medicine, University of
KwaZulu-Natal, Durban, South Africa. 7Department of Internal Medicine, Nelson R. Mandela Schoolof Medicine. University of Kwa-Zulu Natal. 8Department of Epidemiology, Mailman School of Public
Health, Columbia University, New York, NY, United States. 9South African Medical Research Council,
Cape Town, South Africa. 10Division of Infection and Immunity, University College London, London,
UK. 11National Institute for Communicable Diseases of the National Health Laboratory Service,
Johannesburg, South Africa. 12SAMRC Antibody Immunity Research Unit, School of Pathology, Facultyof Health Sciences, University of the Witwatersrand, Johannesburg, South Africa. 13Institute of
Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa.
14
Department of Global Health, University of Washington, Seattle, USA.15Max Planck Institute for
Infection Biology, Berlin, Germany.

26* Corresponding author. Email: alex.sigal@ahri.org

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Omicron has been shown to be highly transmissible and have extensive evasion of neutralizing
antibody immunity elicited by vaccination and previous SARS-CoV-2 infection. Omicron infections
are rapidly expanding worldwide often in the face of high levels of Delta infections. Here wecharacterized developing immunity to Omicron and investigated whether neutralizing immunity
elicited by Omicron also enhances neutralizing immunity of the Delta variant. We enrolled bothpreviously vaccinated and unvaccinated individuals who were infected with SARS-CoV-2 in the
Omicron infection wave in South Africa soon after symptom onset. We then measured their ability
to neutralize both Omicron and Delta virus at enrollment versus a median of 14 days after
enrollment. Neutralization of Omicron increased 14-fold over this time, showing a developing
antibody response to the variant. Importantly, there was an enhancement of Delta virus
neutralization, which increased 4.4-fold. The increase in Delta variant neutralization in individuals
infected with Omicron may result in decreased ability of Delta to re-infect those individuals. Alongwith emerging data indicating that Omicron, at this time in the pandemic, is less pathogenic than
Delta, such an outcome may have positive implications in terms of decreasing the Covid-19 burdenof severe disease.

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The Omicron variant of SARS-CoV-2, first identified in November 2021 in South Africa and Botswana,has been shown by us1 and others2-7 to have extensive but incomplete escape from immunity elicitedby vaccines and previous infection, with boosted individuals showing effective neutralization, eventhoughvaccineandbooster
efficacy
may
wane
over
time
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(https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/fil
e/1043807/technical-briefing-33.pdf). In South Africa Omicron infections led to a lower incidence ofsevere disease relative to other variants8, although this can be at least partly explained by pre-existing
immunity1. While Omicron infections are rising steeply, many countries still have high levels of
infection with the Delta variant. How Delta and Omicron will interact is still unclear, and one possibility
is that Omicron will curtail the spread of Delta by eliciting a neutralizing immune response against
Delta in people infected by Omicron.

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We investigated whether Omicron infection elicits neutralizing immunity to the Delta variant. We
isolated Omicron virus without the R346K mutation from an infection in South Africa. This virus had
similar neutralization escape (Fig S1) as a previous Omicron isolate with the R346K mutation1. We
neutralized this isolate with plasma from the blood of 15 participants enrolled during the Omicron
infection wave in South Africa, with each participant having a confirmed diagnosis of SARS-CoV-2 by
qPCR. To quantify neutralization, we used a live virus neutralization assay and calculated the focus
reduction neutralization test (FRNT50) value, the inverse of the plasma dilution required for 50%
reduction in infection foci. The majority infecting viruses from the enrolled participants weresuccessfully sequenced and all of these were Omicron (Table S1).

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Eleven out of 15 participants were admitted to hospital because of Covid-19 symptoms, but none
required supplemental oxygen. Participants were sampled at enrollment, which was a median of 4days post-symptom onset and again at a median of 14 days post-enrollment. Two participants did notdetectably neutralize Omicron at either timepoint and were excluded from the analysis. Two of theremaining 13 participants did not have detectable SARS-CoV-2 at enrollment, indicating that infection
was already cleared, and therefore that these participants were sampled later post-infection. Out of
the 13 participants, 7 were vaccinated, 3 with two doses of Pfizer-BNT162b2 and 4 with Johnson andJohnson Ad26.CoV2.S (Table S1) with one of the Ad26.CoV2.S vaccines being boosted with a second
Ad26.CoV2.S dose.

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We measured neutralization at enrollment and the later visit and observed that Omicron
neutralization increased from a low geometric mean (GMT) FRNT50 of 20 to 285, a 14.4-fold increase(95% CI 5.5-37.4, Fig 1A). Importantly, neutralization of Delta increased during this period 4.4-fold(95% CI 2.1-9.2), from FRNT50 of 80 to 354 (Fig 1B). The two participants who were likely sampled at alonger time post-infection showed relatively high neutralization values at enrollment both against
Omicron and Delta virus, and these did not appreciably increase with time, indicating thatneutralization capacity plateaued before enrollment. Comparing Omicron and Delta neutralization at
the last available timepoint showed that vaccinated participants were able to mount a better
neutralizing response against Delta virus, while the response in unvaccinated participates was morevariable (Fig 1C).

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The ability of one variant to elicit immunity which can cross-neutralize another variant varies by
variant9-11. Immunity elicited by Delta infection does not cross-neutralize Beta virus and Beta elicitedimmunity does not cross-neutralize Delta well12,13. However, participants in this study have likely beenpreviously infected, and more than half were vaccinated. Therefore, it is unclear if what we observe
is effective cross-neutralization of Delta virus by Omicron elicited antibodies, or activation of antibody
immunity from previous infection and/or vaccination.

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These results are consistent with Omicron displacing the Delta variant, since it can elicit immunity
which neutralizes Delta making re-infection with Delta less likely. In contrast, Omicron escapes
neutralizing immunity elicited by Delta6 and therefore may re-infect Delta infected individuals. The
implications of such displacement would depend on whether Omicron is indeed less pathogenic than
Delta. If so, then the incidence of Covid-19 severe disease would be reduced and the infection may
shift to become less disruptive to individuals and society.

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Materials and methods

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Informed consent and ethical statement

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Blood samples were obtained after written informed consent from adults with PCR-confirmed SARSCoV-2 infection 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 ofresidual swab sample was approved by the University of the Witwatersrand Human Research Ethics
Committee (HREC) (ref. M210752).

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Data availability statement

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Sequence of outgrown virus has been deposited in GISAID with accession EPI_ISL_7886688. Raw
images of the data are available upon reasonable request.

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Code availability

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Image analysis and curve fitting scripts in MATLAB v.2019b are available on GitHub(https://github.com/sigallab/NatureMarch2021).

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Whole-genome sequencing, genome assembly and phylogenetic analysis

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RNA was extracted on an automated Chemagic 360 instrument, using the CMG-1049 kit (Perkin Elmer,
Hamburg, Germany). The RNA was stored at −80◦C prior to use. Libraries for whole genome
sequencing were prepared using either the Oxford Nanopore Midnight protocol with Rapid Barcodingor the Illumina COVIDseq Assay. For the Illumina COVIDseq assay, the libraries were preparedaccording 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 anddenatured with 0.2 N sodium acetate. A 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 Inc, USA), an amplicon-based nextgeneration sequencing approach. The first strand synthesis was carried using random hexamers
primers from Illumina and the synthesized cDNA underwent two separate multiplex PCR reactions.
The pooled PCR amplified products were processed for tagmentation and adapter ligation using IDT
for Illumina Nextera UD Indexes. Further enrichment and cleanup was performed as per protocols
provided by the manufacturer (Illumina Inc). Pooled samples were quantified using Qubit 3.0 or 4.0
fluorometer (Invitrogen Inc.) using the Qubit dsDNA High Sensitivity assay according to manufacturer’s
instructions. The fragment sizes were analyzed using TapeStation 4200 (Invitrogen). The pooled
libraries were further normalized to 4nM concentration and 25 μL of each normalized pool containing
unique index adapter sets were combined in a new tube. The final library pool was denatured andneutralized with 0.2N sodium hydroxide and 200 mM Tris-HCL (pH7), respectively. 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, San Diego, CA, USA). For Oxford
Nanopore sequencing, the Midnight primer kit was used as described by Freed and Silander55. 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 which produce 1200bp
amplicons which overlap to cover the 30-kb SARS-CoV-2 genome. Amplicons from each pool werepooled 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 clean-up,
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
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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 Tool56. For Illumina assembly, 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) nearsequencing/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 ofreads, was increased. In addition, we also used the wf_artic (ARTIC SARS-CoV-2) pipeline as built using
the nextflow workflow framework57. In some instances, mutations were confirmed visually with .bamfiles using Geneious software V2020.1.2 (Biomatters). The reference genome used throughout theassembly process was NC_045512.2 (numbering equivalent to MN908947.3). For lineageclassification, we used the widespread dynamic lineage classification method from the ‘Phylogenetic
Assignment of Named Global Outbreak Lineages’ (PANGOLIN) software suite(https://github.com/hCoV-2019/pangolin)19. P2 stock was sequenced and confirmed Omicron withthefollowing
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:S371L,S:S373P,S:S375F,S:K417N,S:N440K,S:G446S,S:S477N,S:T478K,S:E4
84A,S:Q493R,S:G496S,S:Q498R,S:N501Y,S:Y505H,S:T547K,S:D614G,S:H655Y,S:N679K,S:P681H,S:N76
4K,S:D796Y,S:N856K,S:Q954H,S:N969K,S:L981F. Sequence was deposited in GISAID, accession:
EPI_ISL_7886688.

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Cells

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Vero E6 cells (ATCC CRL-1586, obtained from Cellonex in South Africa) were propagated in complete
growth medium consisting of Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine
serum (Hyclone) containing 10mM of HEPES, 1mM sodium pyruvate, 2mM L-glutamine and 0.1mM
nonessential amino acids (Sigma-Aldrich). Vero E6 cells were passaged every 3–4 days. H1299 cell lines
were propagated in growth medium consisting of complete Roswell Park Memorial Institute (RPMI)
1640 medium with 10% fetal bovine serum containing 10mM of HEPES, 1mM sodium pyruvate, 2mM
L-glutamine and 0.1mM nonessential amino acids. H1299 cells were passaged every second day. The
H1299-E3 (H1299-ACE2, clone E3) cell line was derived from H1299 (CRL-5803) as described in ourprevious work9 and Figure S1. Briefly, vesicular stomatitis virus G glycoprotein (VSVG) pseudotyped
lentivirus containing hACE2 was used to spinfect H1299 cells. ACE-2 transduced H1299 cells(containing an endogenously yellow fluorescent protein labelled histone H2AZ gene14) were thensubcloned at the single cell density in 96-well plates (Eppendorf) in conditioned media derived fromconfluent cells. After 3 weeks, wells were detached using a 0.25% trypsin-EDTA solution (Gibco) andplated in two replicate plates, where the first plate was used to determine infectivity and the second
was stock. The first plate was screened for the fraction of mCherry positive cells per cell clone upon
infection with a SARS-CoV-2 mCherry expressing spike pseudotyped lentiviral vector. Screening was
performed using a Metamorph-controlled (Molecular Devices, Sunnyvale, CA) Nikon TiE motorized
microscope (Nikon Corporation, Tokyo, Japan) with a 20x, 0.75 NA phase objective, 561 nm laser line,
and 607 nm emission filter (Semrock, Rochester, NY). Images were captured using an 888 EMCCD
camera (Andor). The clone with the highest fraction of mCherry expression was expanded from thestock plate and denoted H1299-E3. Infectivity was confirmed with mCherry expressing lentivirus byflow cytometry using a BD Fortessa instrument and analyzed using BD FACSDiva Software (BD
Biosciences). This clone was used in the outgrowth and focus forming assay. Cell lines have not beenauthenticated. The cell lines have been tested for mycoplasma contamination and are mycoplasma
negative.

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Virus expansion

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All work with live virus was performed in Biosafety Level 3 containment using protocols for SARS-CoV2 approved by the Africa Health Research Institute 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 (completion of passage
1 (P1)), cells were trypsinized, centrifuged at 300 rcf for 3 min and resuspended in 4 mL growth
medium. Then all infected cells were added to Vero E6 cells that had been seeded at 2 × 105 cells per
mL, 20mL total, 18–20 h earlier in a T75 flask 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 20 mL of complete
growth medium and incubated for 4 days. The viral supernatant (passage 2 (P2) stock) was used forexperiments.

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Live virus neutralization assay

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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 andstored at −80 °C. Aliquots of plasma samples were heat-inactivated at 56 °C for 30 min and clarified bycentrifugation 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 (SigmaAldrich) 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 withprimary antibody overnight at 4 °C, then washed with wash buffer containing 0.05% Tween-20 in PBS.
Secondary goat anti-rabbit HRP conjugated antibody (Abcam ab205718) was added at 1 μg/mL andincubated for 2 h at room temperature with shaking. TrueBlue peroxidase substrate (SeraCare 55100030) was then added at 50 μL per well and incubated for 20 min at room temperature. Plates wereimaged in an ImmunoSpot Ultra-V S6-02-6140 Analyzer ELISPOT instrument with BioSpot Professionalbuilt-in image analysis (C.T.L).

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Statistics and fitting

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All statistics and fitting were performed using custom code in MATLAB v.2019b. Neutralization data
were fit to:222

Tx=1/1+(D/ID50).

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Here Tx is the number of foci normalized to the number of foci in the absence of plasma on the sameplate 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. We note that the most concentrated
plasma dilution was 1:25 and therefore FRNT50 < 25 were extrapolated. We have marked these values
in Figure 1C and calculate the fold-change FRNT50 either for the raw values or for values where FRNT50> 25 in Figure 1D.

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Acknowledgements

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This study was supported by the Bill and Melinda Gates award INV-018944 (AS), National Institutes of
Health award R01 AI138546 (AS), and South African Medical Research Council awards (AS, TdO, PLM)
and the UK Foreign, Commonwealth and Development Office and Wellcome Trust (Grant no221003/Z/20/Z, PLM). PLM is also supported by the South African Research Chairs Initiative of the
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Department of Science and Innovation and the NRF (Grant No 98341). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.

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A
20

285

FRNT50103

Omicron
14.4x
***

102

VaccinatedUnvaccinatedUndetectable Ct

101
100
Enrollment

B30

FRNT50104

Follow-up
Delta
4.4x
*

354

103
102
101
100
Enrollment

Follow-up

C
285

1.2x
*

354

FRNT50

104
103
102
101
100

OmicronDelta

Figure 1: Enhancement of Delta neutralization by Omicron infection. (A) Omicron (A) or Delta (B)virus neutralization by blood plasma from n=13 participants infected in the Omicron infection wave at enrollment (median 4 days post-symptom onset) and at follow-up (median 14 days post-enrollment). (C) Comparisonof neutralization activity against Omicron and Delta virus at follow-up. Participants were either previously vaccinated (green) or not (orange). Two participants (unvaccinated) with undetectable SARS-CoV-2 at enrollment
are marked in purple. Numbers are geometric mean titers (GMT) of the reciprocal plasma dilution (FRNT50 )resulting in 50% reduction in the number of infection foci. Red horizontal line is most concentrated plasma used.
p=3.6 ×10−4 for (A), p=0.016 for (B), and p=0.045 for (C) as determined by the Wilcoxon rank sum test.

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3190

18.8x
***

170

FRNT50

105
104
103
102
101
100

D614G

OmicronFigure S1: Neutralization of Omicron without R346K by Pfizer BNT162b2. Neutralization Omicron
virus compared to D614G ancestral virus in participants vaccinated with BNT162b2.Samples were tested fromn=19 participants, where n=6 were vaccinated and n=13 were vaccinated and previously infected, as describedin Cele et al., Nature doi: https://doi.org/10.1038/d41586-021-03824-5. Numbers in black above each virus
strain are GMT FRNT50 . Red horizontal line is most concentrated plasma used. p=2.3 ×10−4 by the Wilcoxon
rank sum test.

Table S1: Participant details
Participant
1
2
3
4
5
6
7
8
9
10
11
12
13

Age
30-40
30-40
50-60
30-40
30-40
20-30
30-40
30-40
20-30
40-50
20-30
20-30
20-30

Sex
M
M
F
M
F
F
F
M
F
F
F
M
F

Vaccine
J&J
J&J
Pfizer
None
J&J
None
J&J
PfizerNone
None
Pfizer
None
None

Symptoms date
DecemberNovember
December
December
December
December
DecemberNovemberNovember
December
December
December
DecemberCt enrollment
25
14
17
18
31
28
24
32
UND
32
23
30
UND

Seq. confirmed
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes

Ct: Cycle threshold by qPCR. Seq. confirmed: Verified Omicron by sequencing

2

Yes
Yes
Yes