key: cord-0703201-w0vjyycy
authors: Richardson, S. I.; Madzorera, V. S.; Spencer, H.; Manamela, N. P.; van der Mescht, M.; Lambson, B. E.; Oosthuysen, B.; Ayres, F.; Makhado, Z.; Moyo-Gwete, T.; Mzindle, N.; Motlou, T.; Strydom, A.; Mendes, A.; Tegally, H.; de Beer, Z.; de Villiers, T. R.; Bodenstein, A.; van den Berg, G.; Venter, M.; de Oliviera, T.; Ueckermann, V.; Rossouw, T. M.; Boswell, M. T.; Moore, P. L.
title: SARS-CoV-2 Omicron triggers cross-reactive neutralization and Fc effector functions in previously vaccinated, but not unvaccinated individuals
date: 2022-02-14
journal: nan
DOI: 10.1101/2022.02.10.22270789
sha: 9423a4d9e8e507584c4505382e515f931e1494c8
doc_id: 703201
cord_uid: w0vjyycy

The SARS-CoV-2 Omicron variant largely escapes neutralizing antibodies elicited by vaccines or infection. However, whether Omicron triggers humoral responses that are cross-reactive to other variants of concern (VOCs) remains largely unknown. We use plasma from 20 unvaccinated and seven vaccinated individuals infected during the Omicron wave in South Africa to test binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and neutralization against VOCs. In unvaccinated individuals, Fc effector function and binding antibodies target Omicron and other VOCs at comparable levels. However, Omicron-triggered neutralization is not extensively cross-reactive to VOCs, with 20 to 43-fold reductions in titer. In contrast, vaccination followed by breakthrough Omicron infection improved cross-neutralization of VOCs, with titers exceeding 1:2,900. This has important implications for the vulnerability of unvaccinated Omicron-infected individuals to reinfection by circulating and emerging VOCs. Further, while Omicron-based immunogens may be adequate boosters, they are unlikely to be superior to existing vaccines for priming in SARS-CoV-2 naive individuals.

Omicron is defined by multiple mutations across its genome, including more than 30 mutations in the spike protein, many of which are associated with immune evasion 1 . These mutations confer neutralization escape across multiple vaccine platforms and in donors previously infected with other variants 2-5 .

In contrast to neutralization, antibody binding to the Omicron variant is preserved, as previously observed for other VOCs 6, 7 . Although Fc receptor binding has been shown to be substantially reduced against Omicron 8 , functional Fc effector responses have not yet been reported. The ability of antibodies to bind the Omicron spike suggests that cytotoxic effector functions driven by antibodies may also be retained, as for earlier VOCs 9, 10 . This, along with the finding that T cells triggered by either infection or vaccination are cross-reactive for Omicron [11] [12] [13] , likely contributes to maintained vaccine effectiveness against severe disease following Omicron infection 14 .

While there is substantial data showing that Omicron evades neutralizing immune responses, little is known about the humoral response that Omicron infection itself triggers. Defining the capacity of Omicron to trigger cross-reactive binding, neutralizing and Fc effector responses antibodies will inform its potential to protect from reinfection by currently circulating or emerging VOCs. This is of particular relevance for developing countries such as South Africa, where vaccination levels are low and genomic surveillance indicates continued circulation of is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 14, 2022. ;  https://doi.org/10.1101/2022.02. 10 .22270789 doi: medRxiv preprint responsible for >90% of infections 1 (Table S1 ). Of these, seven plasma samples had matched nasal swabs available and all were confirmed as Omicron BA.1 infections by sequencing.

Twenty individuals were unvaccinated with no history of previous symptomatic infection. Seven individuals had previously been vaccinated with either one dose of Ad26.CoV2.S (n=2) or two doses of BNT162b2 (n=5) at least 56 days (56-163 days) prior to infection. Samples were taken a median of four days (1-10 days) after a positive PCR test.

The median ages of the vaccinated individuals were similar to those of the unvaccinated group (58 and 64 respectively), and infections ranged from mild to severe as determined by WHO scoring (Table S1 ).

We first compared binding antibody levels, as measured by enzyme-linked immunosorbent assay (ELISA) against the ancestral D614G, Beta, Delta and Omicron spikes. In unvaccinated individuals, binding antibody titers against Omicron were highest, as expected, and were detectable in all donors. Although we observed statistically significant 2.2, 1.8 and 1.7-fold decreases in binding to D614G, Beta and Delta respectively in this group, Omicron-triggered antibodies were fairly cross-reactive for all variants tested, losing activity against other VOCs in 10-25% of individuals ( Figure 1A , C). In previously vaccinated individuals who experienced breakthrough infection with Omicron, binding was substantially higher against Omicron than in unvaccinated individuals (geometric mean titer (GMT) of 2.92 versus 1.92) ( Figure 1B , C). Furthermore, antibodies from these vaccinated individuals exhibited higher levels of crossreactivity against all variants, and no significant fold losses were observed ( Figure 1B ).

As spike binding antibodies perform Fc effector functions known to contribute to reduced disease severity and vaccine efficacy 15,16 , we examined antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) in both groups. For unvaccinated individuals, ADCP against Omicron was detected in all 20 individuals with a geometric mean (GM) score of 32 ( Figure 1D ). Against VOCs, we observed less than two-fold reduction in activity across variants with 15%, 25% and 10% of individuals losing ADCP activity . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted February 14, 2022. ; https://doi.org/10.1101/2022.02.10.22270789 doi: medRxiv preprint against D614G, and Beta and Delta respectively. For vaccinated individuals, non-significant reductions against D614G, Beta and Delta were observed relative to Omicron and all donors exhibited activity against the panel of VOC tested here ( Figure 1E ). Compared to unvaccinated individuals, significantly higher levels of ADCP were observed in vaccinated individuals infected with Omicron, mirroring the binding antibodies ( Figure 1E , F).

In contrast to binding and ADCP, ADCC in unvaccinated individuals showed significant losses against D614G (3-fold loss) and Beta (4-fold loss). However, like ADCP and binding antibodies, ADCC activity against Delta was retained ( Figure 1G ). In this group, Omicrontriggered ADCC was undetectable against D614G and Beta in 25% and 30% of plasma samples, respectively. After previous vaccination, Omicron breakthrough infections resulted in overall preservation of ADCC against VOCs, with only one individual showing undetectable activity against Delta ( Figure 1H ). Levels of ADCC in previously vaccinated donors were significantly higher than those in unvaccinated individuals, with the exception of Delta, where similar ADCC activity was observed in both groups ( Figure 1I ).

Lastly, we measured neutralizing antibody responses to Omicron, and assessed their crossreactivity for VOCs. In addition to those variants tested for Fc effector function, we also tested C.1.2, a variant with several neutralization evasive mutations, which circulated at low levels during the third and fourth COVID-19 waves in South Africa 17 . Against Omicron, unvaccinated individuals showed potent neutralization, with a GMT of 4,288. However, against VOCs, we saw dramatic reductions of 27-, 43-, 23-and 20-fold in titer for D614G, Beta, C.1.2 and Delta, respectively, and knock out ranging from 27% to 45% of plasma tested (Figure 2A is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint We and others have previously shown that Fc effector function is largely preserved against VOCs in both convalescent and vaccine-elicited plasma 9, 10 . Also, as with neutralization, we have shown that Fc effector function triggered by Beta is substantially more cross-reactive compared to antibodies elicited by D614G, indicating that the spike sequence of the eliciting immunogen impacts the extent of ADCC cross-reactivity 9, 18 . Here, we show that Omicron infection similarly triggers differential ADCC cross-reactivity, with significantly decreased activity against D614G and Beta, but not Delta. This observation extended to vaccinated individuals, where even boosted ADCC was still significantly poorer against Beta. This differential targeting of ADCC-mediating antibodies indicates that they may preferentially bind sites that differ between Omicron and other VOCs. Alternatively, different VOCs may trigger antibodies with varied glycosylations and isotype, both of which are known to modulate Fc effector function 19 . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 14, 2022. ; https://doi.org/10.1101/2022.02.10.22270789 doi: medRxiv preprint This differential immune imprinting by VOCs was also confirmed for neutralization in this study in both unvaccinated and vaccinated Omicron-infected individuals. The highest titers in unvaccinated individuals were to the infection-matched Omicron. In contrast, in previously vaccinated individuals with breakthrough Omicron infections, high titers were observed against both D614G and Omicron; sequences that match the vaccine and infecting spikes to which these donors have been exposed. This is consistent with our previous studies indicating that the sequence of the infecting spike impacts the quality of the neutralization, suggesting imprinting of the immune response 20 .

We and others have shown that humoral function is significantly boosted in individuals with breakthrough infections after vaccination 21-23 . This study confirms that this is also true of Omicron breakthrough infections, with a 153-fold increase in titers to D614G in vaccinated compared to unvaccinated individuals, consistent with other studies 23-25 . We also extend our previous study, where we showed ADCC was boosted by breakthrough infection, to include ADCP, confirming that this applies to other Fc effector functions 21 .

In the absence of vaccination, Omicron-elicited humoral responses, while potent against the matched Omicron spike, show significantly less activity against VOCs. Thus, while highly immunogenic, Omicron does not elicit cross-neutralizing responses. This is consistent with a decrease in the ability of plasma from unvaccinated individuals to neutralize Delta compared to Omicron following Omicron infection 25 . This may result in risk of reinfection in this unvaccinated group with other variants that continued to circulate and evolve in South Africa at the time of this study, albeit at low levels including Beta, Delta and C.1.2.

Our data also have implications for the design of second-generation vaccines based on Omicron, suggesting that this may not trigger cross-reactive de novo responses in SARS-CoV-2 naive individuals. This is supported by immunogenicity studies, where Delta-infected mice elicited broadly protective antibodies, but Omicron-infected mice failed to mount responses . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted February 14, 2022. ; https://doi.org/10.1101/2022.02.10.22270789 doi: medRxiv preprint against other VOCs 23 . In addition, immunization of mice with a receptor binding domain (RBD)based Omicron mRNA vaccine only elicited strain-specific neutralization 26 . In support of this study and others 27 , our data also suggests that Omicron is highly immunogenic, eliciting comparable neutralization titers irrespective of vaccination status but only in terms of a strainspecific response. Given the significant boost we see against Omicron in vaccinated individuals, Omicron boosters may be effective in seropositive individuals, a group that exceeds 70% in South Africa 28 . However, in a comparison of mRNA-1273 vaccinated rhesus macaques boosted with either mRNA-Omicron or mRNA-1273, Omicron boosted animals showed lower titers than those with a homologous mRNA-1273 boost 29 . Overall, these data suggest that boosting individuals with or without immunity with vaccines specific for Omicron is unlikely to be superior to existing regimens.

Our study is limited by the fact that we cannot rule out prior asymptomatic infection, and that viral sequences confirming Omicron infection are available only for a subset of samples.

However, we note that Omicron overwhelmingly dominated infections during the wave in which these individuals were tested 1 . Further, the median age of individuals in this study is advanced (58 years) and may contribute to comprised cross-reactivity noted for unvaccinated individuals. Given the high prevalence of global seropositivity, through vaccination or previous infection, the ability to measure the response to Omicron infection in naive samples is limited.

As such our study offers valuable insights into the usefulness of Omicron as an immunogen, the potential risk of reinfection in unvaccinated individuals, and the observation that crossreactive responses are greatly improved by vaccination. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 14, 2022. sequencing. PLM conceptualized the study and wrote the manuscript.

All authors declare no competing interests. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

Further information and reasonable requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Penny Moore (pennym@nicd.ac.za). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

Materials will be made by request to Penny Moore (pennym@nicd.ac.za).

All data reported in this paper will be shared by the lead contact upon request.

· This paper does not report original code.

· Any additional information required to reanalyze the data reported in this paper is available from the Lead Contact upon request.

Samples infected in the fourth COVID-19 wave of infection in South Africa were collected from participants enrolled to the Pretoria COVID-19 study cohort. Participants were admitted to Tshwane District Hospital (Pretoria, South Africa) with mild to severe PCR confirmed SARS-CoV-2 infection between 25 November 2021-20 December 2021 (Table S1 ). Ethics approval was received from the University of Pretoria, Human Research Ethics Committee (Medical) (247/2020). All patients had PCR confirmed SARS-CoV-2 infection before blood collection which was done a median of 4 days post positive PCR test. Written informed consent was obtained from all participants.

Human embryo kidney HEK293T cells were cultured at 37°C, 5% CO2, in DMEM containing is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 14, 2022. ; https://doi.org/10.1101/2022.02.10.22270789 doi: medRxiv preprint were maintained in IMDM media with 10% heat-inactivated fetal bovine serum (Gibco, Gaithersburg, MD), 1% Penicillin Streptomycin (Gibco, Gaithersburg, MD) and 10 μg/ml of Blasticidin and 100 μg/ml of Zeocin was added to the growth medium every other passage.

THP-1 cells were used for both the ADCP and ADCT assays and obtained from the AIDS Reagent Program, Division of AIDS, NIAID, NIH contributed by Dr. Li Wu and Vineet N.

KewalRamani. Cells were cultured at 37°C, 5% CO2 in RPMI containing 10% heat-inactivated fetal bovine serum (Gibco, Gaithersburg, MD) with 1% Penicillin Streptomycin (Gibco, Gaithersburg, MD) and 2-mercaptoethanol to a final concentration of 0.05 mM and not allowed to exceed 4 x 10 5 cells/ml to prevent differentiation.

Sequencing of the spike was performed as previously described 30 using swabs obtained from Tshwane District Hospital patients of which 6 were available and confirmed to be Omicron (Table S1 ). RNA sequencing was performed as previously published. Briefly, extracted RNA was used to synthesize cDNA using the Superscript IV First Strand synthesis system (Life Technologies, Carlsbad, CA) and random hexamer primers. SARS-CoV-2 whole genome amplification was performed by multiplex PCR using primers designed on Primal Scheme is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F) proteins were expressed in Human Embryonic Kidney (HEK) 293F suspension cells by transfecting the cells with the respective expression plasmid. After incubating for six days at 37 °C, 70% humidity and 10% CO2, proteins were first purified using a nickel resin followed by size-exclusion chromatography. Relevant fractions were collected and frozen at -80 °C until use.

Two μg/ml of spike protein (D614G, Beta, Delta or Omicron) was used to coat 96-well, highbinding 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 1:100 starting 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 (Thermofisher Scientific). Upon stopping the reaction with 1 M H2SO4, absorbance was measured at a 450nm wavelength. In all instances, mAbs CR3022 and BD23 were used as positive controls and Palivizumab was used as a negative control.

The SARS-CoV-2 Wuhan-1 spike, cloned into pCDNA3.1 was mutated using the QuikChange is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

For the neutralization assay, plasma samples were heat-inactivated and clarified by centrifugation. Heat-inactivated plasma samples from vaccine recipients were incubated with the SARS-CoV-2 pseudotyped virus for 1 hour at 37°C, 5% CO2. Subsequently, 1x10 4 HEK293T cells engineered to over-express ACE-2 (293T/ACE2.MF)(kindly provided by M.

Farzan (Scripps Research)) were added and incubated at 37°C, 5% CO2 for 72 hours upon which the luminescence of the luciferase gene was measured. Titers were calculated as the reciprocal plasma dilution (ID50) causing 50% reduction of relative light units. CB6 and CA1 was used as positive controls for D614G, Beta and Delta. 084-7D, a mAb targeting K417N was used as a positive control for Omicron and Beta.

Avitagged SARS-CoV-2 spikes were biotinylated using the BirA biotin-protein ligase standard reaction kit (Avidity, LLC) and coated onto fluorescent neutravidin beads as previously is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint FACSAria II. Phagocytosis score was calculated as the percentage of THP-1 cells that engulfed fluorescent beads multiplied by the geometric mean fluorescence intensity of the population less the no antibody control. For this and all subsequent Fc effector assays, pooled plasma from 5 PCR-confirmed SARS-CoV-2 infected individuals and CR3022 were used as positive controls and plasma from 5 pre-pandemic healthy controls and Palivizumab were used as negative controls. In addition samples both waves were run head-to-head in the same experiment. ADCP scores for different spikes were normalised to each other and between runs using CR3022.

The ability of plasma antibodies to cross-link and signal through FcγRIIIa (CD16) and spike expressing cells or SARS-CoV-2 protein was measured as a proxy for ADCC. For spike assays, HEK293T cells were transfected with 5μg of SARS-CoV-2 spike plasmids using PEI-MAX 40,000 (Polysciences) and incubated for 2 days at 37°C. Expression of spike was confirmed by differential binding of CR3022 and P2B-2F6 and their detection by anti-IgG APC staining measured by flow cytometry. Subsequently, 1x10 5 spike transfected cells per well were incubated with heat inactivated plasma (1:100 final dilution) or monoclonal antibodies (final concentration of 100 μg/ml) in RPMI 1640 media supplemented with 10% FBS 1% Pen/Strep (Gibco, Gaithersburg, MD) for 1 hour at 37°C. Jurkat-Lucia™ NFAT-CD16 cells (Invivogen) (2x10 5 cells/well and 1x10 5 cells/well for spike and other protein respectively) were added and incubated for 24 hours at 37°C, 5% CO2. Twenty μl of supernatant was then transferred to a white 96-well plate with 50 μl of reconstituted QUANTI-Luc secreted luciferase and read immediately on a Victor 3 luminometer with 1s integration time. Relative light units (RLU) of a no antibody control was subtracted as background. Palivizumab was used as a negative control, while CR3022 was used as a positive control, and P2B-2F6 to differentiate the Beta from the D614G variant. 084-7D was used as a positive control for Omicron and Beta.

To induce the transgene 1x cell stimulation cocktail (Thermofisher Scientific, Oslo, Norway) and 2 μg/ml ionomycin in R10 was added as a positive control to confirm sufficient expression . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 14, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted February 14, 2022. ; https://doi.org/10.1101/2022.02.10.22270789 doi: medRxiv preprint

Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa

Antibodies elicited by SARS-CoV-2 infection or mRNA vaccines have reduced neutralizing activity against Beta and Omicron pseudoviruses

Plasma Neutralization of the SARS-CoV-2 Omicron Variant

Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization

mRNA-based COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 Omicron variant

Activity of convalescent and vaccine serum against SARS-CoV-2 Omicron

SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma

Preserved Omicron Spike specific antibody binding and Fc-recognition across COVID-19 vaccine platforms. medRxiv

SARS-CoV-2 Beta and Delta variants trigger Fc effector function with increased cross-reactivity

mRNA-1273 vaccine-induced antibodies maintain Fc effector functions across SARS-CoV-2 variants of concern

T cell responses to SARS-CoV-2 spike cross-recognize Omicron

Ancestral SARS-CoV-2-specific T cells cross-recognize the Omicron variant

SARS-CoV-2 vaccination induces immunological T cell memory able to cross-recognize variants from Alpha to Omicron

Effectiveness of BNT162b2 Vaccine against Omicron Variant in South Africa

DNA vaccine protection against SARS-CoV-2 in rhesus macaques

Compromised Humoral Functional Evolution Tracks with SARS-CoV-2 Mortality

Omicron-specific mRNA vaccine induced potent neutralizing antibody against Omicron but not other SARS-CoV-2 variants

Replicating RNA platform enables rapid response to the SARS-CoV-2 Omicron variant and elicits enhanced protection in naïve hamsters compared to ancestral vaccine

South African Population Immunity and Severe Covid-19 with Omicron Variant. medRxiv

mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits comparable B cell expansion, neutralizing antibodies and protection against Omicron

Detection of a SARS-CoV-2 variant of concern in South Africa

Nextstrain: real-time tracking of pathogen evolution

We acknowledge the participants who volunteered for this study and thank W van Hougenhouck-Tulleken for database support. We thank Rajiev Ramlall for assistance with participant recruitment at Tshwane District Hospital and Daniel Amoako and Jennifer Giandhari for sequencing support. The parental soluble spike was provided by J McLellan