key: cord-0996422-dxnxfnr1
authors: Lee, W. S.; Selva, K. J.; Davis, S. K.; Wines, B. D.; Reynaldi, A.; Esterbauer, R.; Kelly, H. G.; Haycroft, E. R.; Tan, H.-X.; Juno, J. A.; Wheatley, A. K.; Hogarth, P. M.; Cromer, D.; Davenport, M. P.; Chung, A. W.; Kent, S. J.
title: Decay of Fc-dependent antibody functions after mild to moderate COVID-19
date: 2020-12-14
journal: nan
DOI: 10.1101/2020.12.13.20248143
sha: df85cfec0166860bf73a4d5bc8203fc271d301e0
doc_id: 996422
cord_uid: dxnxfnr1

The capacity of antibodies to engage with innate and adaptive immune cells via the Fc region is important in preventing and controlling many infectious diseases, and is likely critical in SARS-CoV-2 infection. The evolution of such antibodies during convalescence from COVID-19 is largely unknown. We developed novel assays to measure Fc-dependent antibody functions against SARS-CoV-2 spike (S)-expressing cells in serial samples from a cohort of 53 subjects primarily with mild-moderate COVID-19, out to a maximum of 149 days post-infection. We found that S-specific antibodies capable of engaging dimeric Fc{gamma}RIIa and Fc{gamma}RIIIa decayed linearly over time. S-specific antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADP) activity within plasma declined linearly as well, in line with the decay of S-specific IgG. Although there was significant decay in S-specific plasma ADCC and ADP activity, they remained readily detectable by all assays in 94% of our cohort at the last timepoint studied, in contrast with neutralisation activity which was only detectable in 70% of our cohort by the last timepoint. Our results suggest that Fc effector functions such as ADCC and ADP could contribute to the durability of SARS-CoV-2 immunity, particularly late in convalescence when neutralising antibodies have waned. Understanding the protective potential of antibody Fc effector functions is critical for defining the durability of immunity generated by infection or vaccination.

Fc region is important in preventing and controlling many infectious diseases, and is 23 likely critical in SARS-CoV-2 infection. The evolution of such antibodies during 24 convalescence from COVID-19 is largely unknown. We developed novel assays to 25 measure Fc-dependent antibody functions against SARS-CoV-2 spike (S)-expressing 26 cells in serial samples from a cohort of 53 subjects primarily with mild-moderate 27 COVID-19, out to a maximum of 149 days post-infection. We found that S-specific 28 antibodies capable of engaging dimeric FcγRIIa and FcγRIIIa decayed linearly over 29 time. S-specific antibody-dependent cellular cytotoxicity (ADCC) and antibody-30 dependent phagocytosis (ADP) activity within plasma declined linearly as well, in line 31 with the decay of S-specific IgG. Although there was significant decay in S-specific 32 plasma ADCC and ADP activity, they remained readily detectable by all assays in 94% 33 of our cohort at the last timepoint studied, in contrast with neutralisation activity which 34 was only detectable in 70% of our cohort by the last timepoint. Our results suggest 35 that Fc effector functions such as ADCC and ADP could contribute to the durability of 36 SARS-CoV-2 immunity, particularly late in convalescence when neutralising 37 antibodies have waned. Understanding the protective potential of antibody Fc effector 38 functions is critical for defining the durability of immunity generated by infection or 39

Introduction 66 We previously reported that binding antibodies to SARS-CoV-2 S exhibit substantially 67 longer half-lives than the neutralising antibody response (8), suggesting that Fc-68 mediated antibody function may extend the protective window beyond that inferred 69 from neutralising activity alone. At present, analyses of Fc-mediated functions of 70 SARS-CoV-2 antibodies within COVID-19 convalescent subjects have focussed upon 71 cross-sectional analyses or short-term longitudinal studies up to 1-2 months post-72 symptom onset (15, 18, 19) . We extend these findings and analyse Fc effector 73 functions mediated by S-specific antibodies in a cohort of 53 convalescent individuals 74 up to 149 days post-symptom onset. We developed novel functional assays using 75 SARS-CoV-2 S-expressing cells to comprehensively analyse plasma ADCC and ADP 76 activity against SARS-CoV-2 S. Our results show that plasma ADCC and ADP activity 77 decay over the first 4 months post-infection, mirroring the decline in S-specific IgG 78 titres. Importantly, however, S-specific antibodies capable of Fc-mediated antiviral 79 activity remain readily detectable in almost all donors out to 4 months post-infection, 80 even in donors whose neutralising antibody responses have waned to undetectable 81 levels. Consequently, S-specific IgG could potentially mediate Fc-dependent effector 82 functions that contribute to protection from SARS-CoV-2 infection even in the absence 83 of plasma neutralising activity. 84

We collected repeated (2-4) longitudinal samples from a cohort of 53 subjects after 87 recovery from COVID-19 ( Fig 1A, Table S1 ). The first sample was collected at a 88 SARS-CoV-2 S antigens (trimeric S, S1 or S2 subunits or the RBD; Table S2 ). Using 96 mixed-effects modelling, we assessed the fit of single-phase or two-phase decay in 97

FcγR-binding between the timepoints analysed. We found that dimeric FcγRIIIa 98 (V158)-binding antibodies against SARS-CoV-2 trimeric S and RBD both had single-99 phase decay kinetics with half-lives (t1/2) of 175 and 95 days respectively ( Fig. 1B-C) . 100

Dimeric FcγRIIa (H131) binding-antibodies against SARS-CoV-2 trimeric S and RBD 101 also decayed constantly with t1/2 of 175 and 74 days respectively. Kinetics of decay 102 for dimeric FcγR-binding antibodies against S and RBD for the lower affinity 103 polymorphisms of FcγRIIIa (F158) and FcγRIIa (R131) were broadly similar to their 104 higher affinity counterparts (Fig. S1A) , with dimeric FcγR-binding antibodies against 105 RBD decaying faster than for S. Consistent with our previous report that S1-specific 106 IgG decays faster than S2-specific IgG(8), FcγR binding activity with antibodies 107 against the S1 subunit decayed faster than that of S2 (FcγRIIIa, t1/2 of 84 vs 227 days; 108 FcγRIIa, t1/2 of 65 vs 317 days; Fig. S1B ). 109 110 Decay of S-specific ADCC 111 ADCC could play a role in eliminating cells infected with SARS-CoV-2. We generated 112

Ramos-and A549-derived cell lines as model target cells that stably express 113 membrane-localised S with either mOrange2 or luciferase reporters (Fig. S2A-B) . The 114 capacity of plasma IgG to recognise S was measured in 36 subjects in our cohort who 115 had at least 60 days between the first and last visits (median of 89 days between first 116 and last visits; Table S1 ) and 8 seronegative controls. Using a Ramos cell line 117 expressing high levels of S (Ramos S-Orange) (Fig. S2C ), we find IgG binding to cell-118 surface displayed S proteins decayed significantly between the first and last visits 119 (p<0.0001; Fig. S2C ) with a half-life of 97 days (Fig. S3 ). These results are consistent 120 with the decay of S-specific IgG titres we observed previously (8) and the decay of 121 dimeric FcγR-binding antibodies against S in Fig 1B.  122 123 As a surrogate measure of ADCC, we next used FcγRIIIa reporter cells to quantify the 124 capacity of S-specific antibodies in plasma to engage cell surface FcγRIIIa and 125 activate downstream NF-κB signalling (measured by induced nano-luciferase 126 expression in the FcγRIIIa reporter cells) ( Fig. 2A, Fig. S4A ). FcγRIIIa activity decayed 127 significantly over time (p<0.0001; Fig. 2C ) with a half-life of 119 days (Fig. S3) , and 128 was correlated with S-specific IgG titres measured using stably transduced cells or by 129 binding to dimeric FcγRIIIa (Fig. 2D ). To confirm antibody recognition could mediate 130 killing of S-expressing cells, we quantified the loss of cellular luciferase signal in 131 (p<0.0001; Fig. 2E ) with a half-life of 105 days (Fig. S3 ), and correlated with both cell-134 associated S-specific IgG and dimeric FcγRIIIa-binding antibodies against S (Fig. 2F) . 135 136 Decay of S-specific ADP 137

As has been suggested for SARS-CoV, ADP could play a role in eliminating antibody-138 opsonised virions (22). We first used a well-established ADP assay (23) to measure 139 antibody-mediated uptake of S-conjugated fluorescent beads into THP-1 monocytes 140 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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Cross-reactive antibodies between endemic human coronaviruses (HCoV) and SARS-161

CoV-2 have been widely reported (27, 28), suggesting past exposure to HCoVs may 162 prime ADCC and ADP immunity against SARS-CoV-2. In addition, several studies 163 have shown back-boosting of antibodies against endemic human coronaviruses 164 (HCoV) following infection with SARS-CoV-2 (29, 30), likely due to the recall of pre-165 existing B cell responses against conserved regions of S. We thus determined whether 166

IgG antibodies against S from four HCoV strains (OC43, HKU1, 229E and NL63) 167 (Table S2) were boosted in COVID-19 convalescent subjects compared to uninfected 168 healthy controls. We found that COVID-19 convalescent subjects had increased IgG 169 antibodies against S from the betacoronaviruses OC43 and HKU1 (that are more 170 closely related to SARS-CoV-2) at the first timepoint sampled compared to uninfected 171 controls (Fig S7) , while there was no difference in IgG levels against S from the 172 alphacoronaviruses 229E and NL63. Correspondingly, the elevated IgG against OC43 173 and HKU1 S decayed over time while IgG against 229E and NL63 S remained stable 174 ( Fig 4A) . We then measured whether dimeric FcγR-binding antibodies against HCoV 175 S antigens in COVID-19 convalescent individuals declined over time. Dimeric FcγR-176 binding antibodies against OC43 and HKU1 S antigens were much higher in COVID-177 19 convalescent individuals than in healthy controls and decayed more rapidly over 178 time compared to that against 229E and NL63 (Fig. 4A, Fig S8A-C) . While there was 179 an overall decay of dimeric FcγR-binding antibodies against OC43 S (FcγRIIIa t1/2 = 180 224, FcγRIIa t1/2 = 171 days), this was largely due to a decay in antibodies against the 181 more conserved S2 subunit (FcγRIIIa t1/2 = 229, FcγRIIa t1/2 = 179 days) as FcγR-182 binding antibodies against the S1 subunit were not boosted and did not change 183 . 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 December 14, 2020. ; https://doi.org/10.1101/2020.12.13.20248143 doi: medRxiv preprint substantially over time ( Fig. 4B-C) . This was also the case for HKU1, where dimeric 184

FcγR-binding antibodies against S decayed over time but antibodies against the S1 185 subunit did not change ( Fig S8A) . 186 187 Decay kinetics of S-specific antibodies, neutralisation and Fc effector functions 188

To compare the decay kinetics of S-specific antibodies, neutralisation and Fc effector 189 functions, we plotted the best fit decay slopes over time as a percentage of the 190 response measured at timepoint 1 (Fig. 5A) . The best-fit decay slopes of S-specific 191

IgG and plasma neutralisation titres were obtained from a previous dataset that 192 encompass the same subjects analysed for dimeric FcγR-binding antibodies and Fc 193 effector functions (8) (Fig. S3 ). The general decline in plasma S-specific IgG titres and 194 dimeric FcγR-binding activity was similarly reflected in reductions in Fc effector 195 functions during convalescence from COVID-19. Importantly, Fc effector functions at 196 the last timepoint sampled were still readily detectable above baseline activity 197 observed in uninfected controls (97% for FcγRIIIa activation, 94% for ADCC, 100% for 198 ADP and 100% for THP-1 association). This contrasted with plasma neutralisation 199 activity, which was detectable above background for only 70% of subjects (Fig. 5B) . 200

The longer persistence of S-specific IgG and dimeric FcγR-binding antibodies against 201 S has important implications as they may contribute to protection from SARS-CoV-2 202 infection following the decline of neutralising antibodies. 203 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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Using a multiplex bead array and novel assays measuring Fc effector functions against 205 SARS-CoV-2 S, we find that FcγR-binding, ADCC and ADP activities of S-specific 206 antibodies decay during convalescence from COVID-19. The decline of plasma ADCC 207 and ADP activity correlated with the decay of S-specific IgG and FcγR-binding 208 antibodies. Importantly, Fc effector functions were readily detectable above uninfected 209 controls in 94% of subjects for all assays at the last timepoint sampled, in sharp 210 contrast with neutralisation activity, which remained detectable above background for 211 only 70% of subjects. While neutralising antibodies are likely to form a correlate of 212 protection for SARS-CoV-2 (7), several studies find that neutralising antibodies in is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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The copyright holder for this this version posted December 14, 2020. ; https://doi.org/10.1101/2020.12.13.20248143 doi: medRxiv preprint after initial neutralising antibodies have waned but non-neutralising antibodies remain. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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The copyright holder for this this version posted December 14, 2020. ; https://doi.org/10.1101/2020.12.13.20248143 doi: medRxiv preprint covalently coupled to magnetic carboxylated beads (Bio Rad) using a two-step 293 carbodiimide reaction and blocked with 0.1% BSA, before being resuspended and 294 stored in PBS 0.05% sodium azide till use. 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 December 14, 2020. ; https://doi.org/10.1101/2020.12.13.20248143 doi: medRxiv preprint while S-orange cells were bulk sorted on high S-and mOrange2-expression. Following 318 a week of outgrowth, the bulk sorted cells were single-cell sorted to obtain clonal 319 populations of S-orange and S-luciferase cells (Fig. S2B) . The Ramos cell lines were 320 grown in complete RPMI medium (10% fetal calf serum (FCS) with 1% penicillin 321 strepytomycin glutamine (PSG)) while the A549 cell lines were grown in complete 322 DMEM medium (10% FCS with 1% PSG). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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The copyright holder for this this version posted December 14, 2020. ; https://doi.org/10.1101/2020.12.13.20248143 doi: medRxiv preprint was read using a FLUOstar Omega microplate reader (BMG Labtech). The relative 368 light units (RLU) measured were used to calculate %ADCC with the following formula: 369 ("no plasma control" -"plasma sample") ÷ "target cell only control" × 100. For each 370 plasma sample, %ADCC was plotted against log10(plasma dilution -1 ) and the area 371 under curve (AUC) was calculated using Graphpad Prism. Fig. S6 for optimisation). THP-1 monocytes (10,000/well) were then 384 added to opsonized beads and incubated for 16 hours under cell culture conditions. 385

Cells were fixed with 2% formaldehyde and acquired on a BD LSR Fortessa with a 386 HTS. The data was analyzed using FlowJo 10.7.1 (see Fig. S5 for gating strategy) and 387 a phagocytosis score was calculated as previously described (51) using the formula: 388 (%bead-positive cells × mean fluorescent intensity)/10 3 . To account for non-specific 389 uptake of S-conjugated beads, the phagocytosis scores for each plasma sample were 390 subtracted with that of the "no plasma" control. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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To assess the capacity of THP-1 monocytes to associate with S-expressing target 394 cells via Ab-FcγR interactions, an assay using THP-1 cells as effectors and Ramos S-395 orange cells as targets was performed. THP-1 monocytes were first stained with 396 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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The parameter & is a constant (intercept), and &" is a subject-specific adjustment to 417 the overall intercept. The slope parameter + is a fixed effect to capture the decay 418 slope before & (as a fixed parameter, 70 days); which also has a subject-specific 419 random effect +" . To fit a model with two different decay rates, an extra parameter -420 (with a subject-specific random effect -" ) was added to represent the difference 421 between the two slopes. Assay variability between replicates (only for HCoV response 422 variables) was modelled as a single fixed effect ) , in which we coded the replicate as 423 a binary categorical variable "# . The random effect was assumed to be normally 424 distributed with zero mean and variance . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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were performed using the Wilcoxon signed-rank test. Comparisons between 443 uninfected individuals and COVID-19 convalescent individuals were performed using 444 the Mann-Whitney test. 445 . 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 December 14, 2020. ; https://doi.org/10.1101/2020.12.13.20248143 doi: medRxiv preprint an early convalescent COVID-19 patient, are essential for the optimal 544 therapeutic efficacy of the antibody. bioRxiv, 2020.2010.2026.355107 (2020). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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The copyright holder for this this version posted December 14, 2020. 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 December 14, 2020. ; https://doi.org/10.1101/2020.12.13.20248143 doi: medRxiv preprint 795 796 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

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measured as the loss of cellular luciferase. (C) S-specific FcγRIIIa-activating plasma 762 antibodies in COVID-19 convalescent individuals in the first (T1; filled) and last

open) timepoints available. (D) Correlation of S-specific FcγRIIIa-activating antibodies 764 to cell-associated S-specific IgG and S-specific dimeric FcγRIIIa-binding antibodies

S-specific ADCC mediated by plasma antibodies from COVID-19 convalescent 766 individuals in the first (T1; filled) and last (T2; open) timepoints available

Correlation of S-specific ADCC to cell-associated S-specific IgG and S-specific 768

dimeric FcγRIIIa-binding antibodies. Red lines indicate the median responses of 769

COVID-19 convalescent individuals (N=36) while dashed lines indicate median 770 responses of uninfected controls (N=8). Statistical analyses were performed with the 771

Wilcoxon signed-rank test (****, p<0.0001). Correlations were performed with the non-772 parametric Spearman test

COVID-19 convalescent individuals in the first (T1) and last (T2) timepoints available

Correlation of ADP to cell-associated S-specific IgG and S-specific dimeric 785

FcγRIIa-binding antibodies. (E) FcγR-dependent association of THP-1 cells with 786

Ramos S-orange cells mediated by plasma antibodies from COVID-19 convalescent 787 individuals in the first (T1) and last (T2) timepoints available. (F) Correlation of 788 association of THP-1 cells with Ramos S-orange cells to cell

Red lines indicate the median 790 responses of COVID-19 convalescent individuals (N=36) while dashed lines indicate 791 median responses of uninfected controls (N=8)

 722 We thank the cohort participants for generously providing samples. We thank 723