key: cord-0979801-35aiokr6
authors: Richardson, Simone I.; Manamela, Nelia P.; Motsoeneng, Boitumelo M.; Kaldine, Haajira; Ayres, Frances; Makhado, Zanele; Mennen, Mathilda; Skelem, Sango; Williams, Noleen; Sullivan, Nancy J.; Misasi, John; Gray, Glenda G.; Bekker, Linda-Gail; Ueckermann, Veronica; Rossouw, Theresa M.; Boswell, Michael T.; Ntusi, Ntobeko A.B.; Burgers, Wendy A.; Moore, Penny L.
title: SARS-CoV-2 Beta and Delta variants trigger Fc effector function with increased cross-reactivity
date: 2022-01-17
journal: Cell Rep Med
DOI: 10.1016/j.xcrm.2022.100510
sha: fe24b5e2114c710797fd66be72c9ced33f7968ce
doc_id: 979801
cord_uid: 35aiokr6

SARS-CoV-2 variants of concern (VOCs) exhibit escape from neutralizing antibodies, causing concern about vaccine effectiveness. However, while non-neutralizing cytotoxic functions of antibodies are associated with improved disease outcome and vaccine protection, Fc effector function escape from VOCs is poorly defined. Furthermore, whether VOCs trigger Fc functions with altered specificity, as has been reported for neutralization, is unknown. Here, we demonstrate that the Beta VOC partially evades Fc effector activity in individuals infected with the original (D614G) variant. However, not all functions are equivalently affected, suggesting differential targeting by antibodies mediating distinct Fc functions. Furthermore, Beta and Delta infection trigger responses with significantly improved Fc cross-reactivity against global VOCs compared to D614G-infected or Ad26.COV2.S vaccinated individuals. This suggests that, as for neutralization, the infecting spike sequence impacts Fc effector function. These data have important implications for vaccine strategies that incorporate VOCs, suggesting these may induce broader Fc effector responses.

Continued SARS-CoV-2 transmission worldwide through inadequate vaccine coverage has resulted in the emergence of viral variants of concern (VOCs) including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2). These VOCs are able to evade neutralizing responses in vaccinee and convalescent sera [1] [2] [3] [4] [5] [6] , though T cell function and spike binding antibody levels retain activity 4, [7] [8] [9] . In addition to mediating neutralization, antibodies drive effector functions through their ability to engage cellular receptors via their Fc portion, including antibody-dependent cellular cytotoxicity (ADCC), cellular phagocytosis (ADCP), cellular trogocytosis (ADCT) or cell membrane nibbling and complement deposition (ADCD).

Cross-reactive binding antibodies are consistent with preserved Fc effector function in convalescent sera and after vaccination, and that several vaccines maintain effectiveness against VOCs 2,7,10 . For example, the Ad26.COV.2S vaccine maintained efficacy against severe COVID-19 illness caused by Beta despite reduced neutralization titers 2, 4, 5, 11 .

Most antibodies elicited by infection are non-neutralizing 12 . As mutations in VOCs occur primarily in the RBD and the N-terminal domains targeted by neutralizing antibodies, antibodies able to bind outside of these sites and mediate potent antiviral function may confer protection from severe disease. As for other diseases, Fc effector function is associated with reduced COVID-19 severity and mortality, suggesting an important early role for these functions in disease outcome 13, 14 . Furthermore, isolated antibodies from convalescent donors require Fc function for optimal protection and therapeutic efficacy 15, 16 . Fc functions persist beyond neutralizing responses following SARS-CoV-2 infection, and may be important for vaccine design 17, 18 .

Fc effector function correlated with protection through vaccination in non-human primates 10, 19, 20 and is elicited by vaccination in humans 2, 7, 21, 22 . Beyond this, nuances in magnitude and breadth of Fc receptor binding responses from convalescent donors and J o u r n a l P r e -p r o o f different vaccine regimens suggest these responses vary by specific antigens, formulations or doses 23 .

For neutralization, the sequence of the infecting virus impacts the breadth of the resulting neutralizing antibodies 3, 9, 24 . Neutralizing antibodies triggered by VOCs show varying patterns of breadth compared to the original D614G and one another, suggesting that spikes with different genotypes differentially impact the repertoire of triggered antibodies. However, similar studies characterizing Fc effector functions in infections by VOC have not been conducted.

Since March 2020, South Africa experienced three distinct waves of COVID-19 infection, each dominated by a different variant. We leveraged these virologically distinct waves to define Fc effector response escape from VOCs, and to describe Fc responses to VOCs. We used convalescent sera from individuals infected with D614G to show that Beta partially evades several Fc effector functions. However, individuals infected with Beta developed Fc effector function with improved cross reactivity for all VOCs. Lastly, we show that Fc effector function elicited by the Ad26.COV.2.S vaccine is largely retained across VOCs, but is not as crossreactive as those elicited by Beta. Therefore, VOCs differentially trigger Fc effector functions, with implications for vaccination.

South Africa's first wave peaked in mid-July 2020, the second in January 2021 and the third in August 2021 ( Figure 1A ). The first wave was dominated by Wuhan D614G, the second by the Beta variant, and the third wave by Delta. We used convalescent plasma from the first two waves (first wave n = 27; second wave n = 21) to determine its ability to bind and neutralize original (D614G) or Beta. First and second wave participants were hospitalized patients, matched in age with a median of 52 years (range 27-72) and 55 years (range 24-73), respectively. Samples were collected a median of 10 days (range 7-33) and 13 days (range J o u r n a l P r e -p r o o f 2-29) after a positive SARS-CoV-2 PCR test (Table S1 ). Although wave 1 viral sequences were not obtained, these samples were collected several months prior to the emergence of Beta and were assumed to have been D614G infections ( Figure 1A ). Wave 2 samples were collected when Beta accounted for >90% of infections in the region, with sequences from nasal swabs of all 8 samples available from 21 patients confirmed as Beta (Table S1) , as described previously 24 .

Comparison of IgA and IgG binding from Wave 1 plasma to the original (D614G) or Beta spikes showed a significant decrease in binding to Beta ( Figure 1B and C). In contrast, wave 2 plasma from Beta infections showed a significant increase in both IgA and IgG binding to Beta.

However, the differences in geometric mean titer against the original and Beta spike for wave 1 and 2 plasma were less than 2-fold for both IgG and IgA. In contrast, neutralization titers in plasma from wave 1 decreased 14.7 fold against Beta, while wave 2 titers of Beta were 5.1 fold higher than those of D614G ( Figure 1C ), as we previously show 6, 24 . This difference in reduction of binding versus neutralizing antibodies confirms the ability of convalescent plasma to target epitopes beyond the neutralizing epitopes mutated in VOCs.

We next measured whether Fc effector functions elicited by the original D614G variant or the Beta variant were equivalent in magnitude and cross-reactivity. We measured spike-specific Fc responses using spike protein coated on to fluorescent beads (for ADCP and ADCD) or spike expressed on the surface of target cells (ADCC and ADCT). To validate these assays, we tested monoclonal antibodies previously characterized for cell surface spike binding to D614G and Beta 25 . A23-58.1, B1-182.1, A19-46.1 and A19-61.1 have previously been shown to bind D614G and Beta equivalently, and showed similar ADCP, ADCC, ADCD and ADCT activity against both D614G and Beta ( Figure S1 ). Similarly, Class 4 mAb CR3022, which binds to regions of the RBD that exclude sites mutated in Beta 26 , showed similar Fc effector J o u r n a l P r e -p r o o f function for both variants indicating that these assays were comparable across variants. For Class 2 mAbs BD23, LY-CoV555 and P2B-2F6, which are unable to bind or neutralize Beta 6,27 , Fc effector function against Beta was also reduced.

Plasma from wave 1 participants showed a significantly decreased ability to mediate ADCP, ADCC, ADCD and ADCT of Beta, compared to the original D614G spike, though all retained some activity against Beta (Figure 2A As Fc effector function is modulated by Fc receptor binding, we examined the ability of antibodies from wave 1 and 2 to crosslink dimeric Fc receptors FcRIIa or FcRIIIa (which modulate ADCP and ADCC, respectively) and the original or Beta spike protein by ELISA. As expected, Spearman's correlations >0.5 were noted between FcRIIa binding and ADCP score, and between FcRIIIa binding and ADCC against original D614G spike ( Figure S2A and B). Similar to the functional readout, wave 1 samples showed significant decreases Beta specific FcRIIa ( Figure S2C) and FcRIIIa cross-linking ( Figure S2D ), while no significant differences were noted for wave 2 samples ( Figure S2E ).

We considered the possibility that differences in Fc effector function simply reflected varying IgG levels between waves, although the samples had been matched for age, severity and time since PCR test. Wave 1 and wave 2 samples showed no significant difference in IgG binding titers and ADCT activity to the autologous infecting spike (original or Beta respectively) ( Figure   1C and 2A; Figure S2F ). However, despite being run in head-to head assays with wave 1 plasma, the wave 2 plasma showed significantly lower neutralization, ADCP and ADCC activity and enhanced ability to deposit complement protein compared to wave 1 plasma against autologous spike ( Figure S2F ). This shows that Fc effector VOC cross-reactivity is not a result of binding titer. Overall, preserved Fc effector function, but substantial loss in neutralization against Beta in wave 1 samples ( Figure 2B ) suggests targeting by Fc effector function is distinct from that of neutralization.

Although Fc effector function elicited by the original D614G virus was not completely abrogated against Beta, the significant decrease in activity suggests that NTD and RBD, mutated in Beta, are substantial targets. Given the significant decrease of ADCC observed against Beta by wave 1 plasma (Figure 2A ) we mapped these responses. We determined the contribution to ADCC of antibodies to NTD and RBD by measuring FcRIIIa signaling as a result of crosslinking to NTD or RBD proteins from the D614G and Beta variants.

We confirmed our ability to map these responses using monoclonal antibodies. As for full spike ( Figure S1 ), CR3022 ADCC against the RBD was unaffected by Beta RBD mutations (K417N, sites are targeted, they do not account for the majority of ADCC activity against RBD ( Figure   3C ). Beta-elicited ADCC did not show significant differences between the original or Beta RBD ( Figure 3C ), suggesting broader tolerance of RBD mutations in wave 2 plasma, as with neutralization. Similarly, ADCC was detected against the original NTD protein with a 3 fold decrease (median 677 to 204) against Beta in Wave 1 plasma ( Figure 3C ). This may indicate that ADCC antibodies more frequently target NTD sites mutated in Beta (L18, D80, D215), or are less able to tolerate the conformation change of the NTD that may result from the 242-244 deletion. Conversely, as for RBD, ADCC elicited by Beta was not significantly different against the original or Beta NTD ( Figure 3C ). Therefore, NTD and RBD are targets of ADCC responses in convalescent plasma, but mutations in these regions that confer neutralization escape in original (D614G) infections, only slightly affect ADCC.

As Beta-elicited plasma showed enhanced cross-reactivity for the original variants, we assessed a larger panel of VOCs (D614G and Alpha, Beta, Gamma, Delta and SARS-1).

Samples and VOC were run head-to-head and normalized by CR3022 ADCC activity. 

To assess whether Delta, which dominated the third wave, also triggered ADCC with increased cross-reactivity, we tested 22 samples from the third wave. Of these, sequences were available from 9, and all were Delta (Table S1 ). Delta specific ADCC showed the highest level (median 320), followed by the original, Alpha and Gamma (median 206, 191 and 180 respectively). However, wave 3 plasma ADCC was significantly reduced against Beta (median 86) ( Figure 4C ) and SARS-1. Thus, Delta triggers ADCC that is more cross-reactive that the original variant but less cross-reactive than that triggered by Beta. Responses to SARS-1 were significantly lower.

Vaccine and wave 1 ADCC against VOCs were similar, indicating comparable levels of cross reactivity ( Figure 4E ). Strikingly though, vaccine-elicited plasma showed decreased capacity to perform ADCC against Beta compared to wave 1 plasma ( Figure 4E ). For Beta, Delta and Gamma, the fold differences for wave 2 sera were consistently >1, and significantly different from wave 1 and Ad26.COV2.S, while Alpha showed no difference ( Figure 4E Here, we confirm overall preservation of Fc effector function against VOCs in infection and vaccination, but show that the magnitude of Fc effector activity against Beta is reduced. While Fc effector functions against Beta were only slightly lower, loss of activity was not equivalent for all functions, with ADCD most affected. RBD is a major target for complement binding in both vaccinated and convalescent individuals 28 , which may explain why ADCD has greater loss against Beta, which has RBD mutations. In contrast to ADCD, ADCC activity against Beta was less reduced. Epitope mapping data indicates that Beta RBD and NTD are not the predominant targets of this function. Therefore preservation of ADCC may be the result of antibodies targeting epitopes or sites beyond those commonly mutated in VOCs, such as J o u r n a l P r e -p r o o f the S2 region. Differential epitope targeting has also been suggested for other functions, with RBD depletion greatly decreasing ADNP activity in contrast to ADCP, which remained unaffected in convalescent plasma 23 . In addition to antibody targeting that varied by function, we also show that individuals infected by different variants have unique Fc effector profiles despite similar levels of binding antibodies. Not all Fc effector functions were affected in the same way; ADCD was higher in wave 2 samples while ADCC was substantially higher in wave 1. This likely reflects the impact of varying spike sequence on the ability to affect different Fc effector functions, with regions mutated in Beta being dominant targets of ADCD, perhaps as a result of altered steric constraints at the Fc-complement interface. Different VOCs and vaccine platforms may also trigger antibodies with varying glycosylation and/or isotypes 23, 29 .

These data suggest differential targeting of individual Fc functions in response to VOC, and future studies should include detailed mapping of Fc effector function targets.

We also show subtle differences in the ability of antibodies elicited by either the original D614G or the Ad26.COV2.S vaccine to perform ADCC against Beta. This was despite the fact that the sequence of immunodominant regions of the eliciting immunogens were the same, with only the single D614G mutation differing between them. Similar findings have been reported for ADCP, where RBD is targeted to varying levels in different vaccine modalities and convalescent plasma 23 . This suggests that beyond sequence, nuanced differences in antigen stability and presentation affect functional responses to vaccination or infection.

We have previously shown that Beta infection imprints a cross-reactive neutralizing response 3, 24 . Here we show that this also extends to Fc effector function and other VOCs including Delta, suggesting that features intrinsic to each spike shape the antibody repertoire. These data suggests that the spike sequence of the priming immunogen is likely to determine unique Fc effector function profiles, allowing for their potential modulation in future vaccine design. However this should be considered in the context of neutralization for which the choice of immunogen sequence is far more constrained and alongside which Fc effector function is likely to play a supporting role in protection. While current vaccines provide sufficient protection against severe disease, vaccination strategies may be improved by a spike immunogen associated with a more balanced and broad response.

While we have used the national database of infection, and our clinical data to excluded unknown infection, we cannot rule out the possibility of asymptomatic prior infections. 

PLM is a member of the advisory board for Cell Reports Medicine. All other authors declare no competing interests. Statistical differences between waves and vaccine responses were calculated using the Kruskal-Wallis test with Dunn's multiple test comparisons. *p<0.05; ***p<0.001; ****p<0.0001 and ns = non-significant.

For wave 2 and 3 samples, sequencing of the spike was performed as previously described 24 using swabs obtained from randomly collected Groote Schuur Hospital patients of which eight were included and confirmed as Beta for wave 2 and 9 were included and confirmed as Delta for wave 3 in this study (Table S1 ). RNA sequencing was performed as previously 

Two μg/ml of spike protein (Original or Beta) 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 1:100 starting dilution in a blocking buffer and added to the plates. IgG or IgA secondary antibody was diluted to 1:3000 or 1:1000 respectively 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 

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 a positive control.

SARS-CoV-2 original or Beta spike was biotinylated using EZ link Sulfo-NHS-LC-Biotin kit (ThermoFisher) and coated on to fluorescent neutravidin beads as previously described 33 .

Briefly, beads were incubated for two hours with monoclonal antibodies at a starting concentration of 2 μg/ml and titrated five-fold or plasma at a single 1 in 100 dilution. Opsonized beads were incubated with the monocytic THP-1 cell line overnight, fixed and interrogated on the 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 original and Beta 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. score was calculated as the percentage of C3b-FITC positive beads multiplied by the geometric mean fluorescent intensity of FITC in this population less the no antibody or heat inactivated controls. ADCD scores for original and Beta spikes were normalised to each other and between runs using CR3022. Both wave 1 and wave 2 samples were run head to head using the same batch of bead preparation.

ADCT was performed as described in and modified from a previously described study 35 .

HEK293T cells transfected with a SARS-CoV-2 spike pcDNA vector as above were surface biotinylated with EZ-Link Sulfo-NHS-LC-Biotin as recommended by the manufacturer. Fiftythousand cells per well were incubated with 5-fold titration of mAb starting at 25 μg/ml or single 1 in 100 dilution for 30 minutes. Following a RPMI media wash, these were then incubated with carboxyfluorescein succinimidyl ester (CFSE) stained THP-1 cells (5 X10 4 cells per well) for 1 hour and washed with 15mM EDTA/PBS followed by PBS. Cells were then stained for biotin using Streptavidin-PE and read on a FACSAria II. Trogocytosis score was determined as the proportion of CFSE positive THP-1 cells also positive for streptavidin-PE less the no antibody control with waves run head-to-head.

High-binding 96 well ELISA plates were coated with 1 ug/ml spike protein in PBS overnight at 4°C. Three wells on each plate were directly coated with 5 ug/ml IgG, isolated from healthy donors, and signals from these wells were used to normalize the Fc receptor activity of the Fc effector functions are preserved against SARS-CoV-2 variants of concern. · Complement deposition against VOCs is reduced more than other functions. · VOC infection triggers improved Fc cross-reactivity compared to vaccination. ·

The sequence of the infecting virus determines the breadth of the Fc response.

Beyond neutralization, antibodies trigger cytotoxic functions associated with SARS-CoV-2 vaccine protection. Richardson et al. show these functions are retained against variants of concern (VOC), and that infection by VOCs triggers cross-reactive cytotoxic antibodies. This suggests that SARS-CoV-2 VOC could be used as the basis of vaccines triggering enhanced immune breadth.

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We thank Dr B Lambson, D Mhlanga and B Oosthuysen for generating viral variants of concern. We thank Drs M Madzivhandila and T Moyo-Gwete for initial wave 2 neutralization and binding data. We thank E du Toit, A Ngomti, R Baguma, R Keeton, M van der Mescht, Z van der Walt, T de Villiers, F Abdullah, P Rheeder, A Malan, W van Hougenhouck-Tulleken,