key: cord-0812912-fr1kha6v
authors: Lee, Wen Shi; Selva, Kevin John; Davis, Samantha K.; Wines, Bruce D.; Reynaldi, Arnold; Esterbauer, Robyn; Kelly, Hannah G.; Haycroft, Ebene R.; Tan, Hyon-Xhi; Juno, Jennifer A.; Wheatley, Adam K.; Hogarth, P. Mark; Cromer, Deborah; Davenport, Miles P.; Chung, Amy W.; Kent, Stephen J.
title: Decay of Fc-dependent antibody functions after mild to moderate COVID-19
date: 2021-05-09
journal: Cell Rep Med
DOI: 10.1016/j.xcrm.2021.100296
sha: 12d8de0772d457977d5bfd761e9b1e8799ddb370
doc_id: 812912
cord_uid: fr1kha6v

The capacity of antibodies to engage with immune cells via the Fc region is important in preventing and controlling many infectious diseases. The evolution of such antibodies during convalescence from COVID-19 is largely unknown. We develop assays to measure Fc-dependent antibody functions against SARS-CoV-2 spike (S)-expressing cells in serial samples from subjects primarily with mild-moderate COVID-19, up to 149 days post-infection. We find that S-specific antibodies capable of engaging Fcγ receptors decay over time, with S-specific antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADP) activity within plasma declining accordingly. Although there is significant decay in ADCC and ADP activity, they remain readily detectable in almost all subjects at the last timepoint studied (94%) in contrast with neutralisation activity (70%). While it remains unclear the degree to which Fc effector functions contribute to protection against SARS-CoV-2 re-infection, our results indicate that antibodies with Fc effector functions persist longer than neutralising antibodies.

Most individuals who recover from COVID-19 develop binding and neutralising 49 antibody responses against SARS-CoV-2 spike (S) protein 1, 2 , with neutralising 50 antibody responses generally targeted to the receptor-binding domain (RBD) of S 3 . 51

Passive transfer of neutralising monoclonal antibodies (mAbs) can protect animal 52 models from subsequent SARS-CoV-2 challenge 4-6 , suggesting neutralisation is 53 likely to be a correlate of protection in humans 7 . However, the duration of protection 54 from re-infection in humans conferred by neutralising antibodies is not known. 55

Several studies now show neutralising antibodies decline rapidly during early 56 convalescence 2, 8, 9 , with the magnitude of the antibody response positively 57 correlating with disease severity 10, 11 . Following mild COVID-19, many subjects 58 mount modest neutralising antibody responses that decline to undetectable levels 59 within 60 days, despite the maintenance of S-and RBD-specific IgG binding 60 antibodies 10 . Given that reported cases of SARS-CoV-2 re-infection have been rare 61 to date, it is likely that immune responses beyond neutralisation, including T cell 62 responses 12 , contribute to SARS-CoV-2 protective immunity. Apart from direct virus 63 neutralisation, antibodies can also mediate antiviral activity such as antibody- 

We collected repeated (2-4) longitudinal samples from a cohort of 53 subjects after 96 recovery from COVID-19 ( Figure 1A , Table S1 ). The first sample was collected at a 97 antibodies specific for SARS-CoV-2 S antigens (trimeric S, S1 or S2 subunits or the 105 RBD; Table S2 ) with a multiplex bead array. Using mixed-effects modelling, we 106 assessed the fit of single-phase or two-phase decay in FcγR-binding between the 107 timepoints analysed. We found that dimeric FcγRIIIa (V158)-binding antibodies 108 against SARS-CoV-2 trimeric S and RBD both had single-phase decay kinetics with 109 half-lives (t 1/2 ) of 175 and 95 days respectively (Figure 1B-C). Dimeric FcγRIIa (H131) 110 binding-antibodies against SARS-CoV-2 trimeric S and RBD also decayed constantly 111 with t 1/2 of 175 and 74 days respectively. Kinetics of decay for dimeric FcγR-binding 112 antibodies against S and RBD for the lower affinity polymorphisms of FcγRIIIa (F158) 113 and FcγRIIa (R131) were broadly similar to their higher affinity counterparts ( Figure  114 S1A), with dimeric FcγR-binding antibodies against RBD decaying faster than for S. 115 Consistent with our previous report that S1-specific IgG decays faster than S2-116 specific IgG 8 , FcγR binding activity with antibodies against the S1 subunit decayed 117 J o u r n a l P r e -p r o o f 6 faster than that of S2 (FcγRIIIa, t 1/2 of 84 vs 227 days; FcγRIIa, t 1/2 of 65 vs 317 days; 118 Figure S1B ). 119 120 Decay of S-specific ADCC 121 ADCC could play a role in eliminating cells infected with SARS-CoV-2. We 122 generated Ramos-and A549-derived cell lines as model target cells that stably 123 express membrane-localised S with either mOrange2 or luciferase reporters ( Figure  124 S2A-B). The capacity of plasma IgG to recognise S was measured in 36 subjects in 125 our cohort who had at least 60 days between the first and last visits (median of 89 126 days between first and last visits; Table S1 ) and 8 seronegative controls. Using a 127

Ramos cell line expressing high levels of S (Ramos S-Orange), we find IgG binding 128 to cell-surface displayed S proteins decayed significantly between the first and last 129 visits (p<0.0001; Figure S2D ; gating in Figure IgG titres measured using stably transduced cells or by binding to dimeric FcγRIIIa 141 ( Figure 2D ). Since FcγRIIIa-crosslinking and activation may not necessarily reflect 142 J o u r n a l P r e -p r o o f downstream target lysis, we next performed an ADCC assay to confirm antibody 143 recognition could mediate killing of S-expressing cells. We quantified the loss of 144 cellular luciferase signal in Ramos S-luciferase target cells in the presence of 145 convalescent plasma and primary human NK cells ( Figure 2E , Figure S3B ). S-146 specific ADCC decayed significantly over time (p<0.0001; Figure 2F (Table S2) were higher in COVID-19 convalescent subjects 196 compared to uninfected healthy controls. Using a multiplex bead array, we found that 197 COVID-19 convalescent subjects had increased IgG antibodies against S from the 198 betacoronaviruses OC43 and HKU1 (which are more closely related to SARS-CoV-2) 199 at the first timepoint sampled compared to uninfected controls ( Figure S6 FcγRIIIa t 1/2 = 224, FcγRIIa t 1/2 = 171 days), this was largely due to a decay in 210 antibodies against the more conserved S2 subunit (FcγRIIIa t 1/2 = 229, FcγRIIa t 1/2 = 211 179 days). FcγR-binding antibodies against the S1 subunit were not increased 212 compared to healthy controls and did not change over time ( Figure 5B ). This was 213 also the case for HKU1, where dimeric FcγR-binding antibodies against S decayed 214 over time but antibodies against the S1 subunit did not change ( Figure 5C ). 215

To compare the decay kinetics of S-specific antibodies, neutralisation and Fc effector 219 functions, we plotted the best fit decay slopes over time as a percentage of the 220 response measured at timepoint 1 ( Figure 6A ). The best-fit decay slopes of S-221 specific IgG and plasma neutralisation titres ( Figure S7 ) were obtained from a 222 previous dataset that encompass the same subjects analysed for dimeric FcγR-223 binding antibodies and Fc effector functions 8 . The general decline in plasma S-224 specific IgG titres and dimeric FcγR-binding activity was similarly reflected in 225 reductions in Fc effector functions during convalescence from COVID-19. Importantly, 226

Fc effector functions at the last timepoint sampled were still readily detectable above 227 baseline activity observed in uninfected controls (97% for FcγRIIIa activation, 94% 228 for ADCC, 100% for ADP and 100% for THP-1 association). This contrasted with 229 plasma neutralisation activity, which was detectable above background for only 70% 230 of subjects ( Figure 6B ). The longer persistence of S-specific IgG and dimeric FcγR-231 binding antibodies against S has important implications for the durability of SARS-232

CoV-2 immunity following the decline of neutralising antibodies. 233

Using a multiplex bead array and assays measuring Fc effector functions against 235 SARS-CoV-2 S, we find that FcγR-binding, ADCC and ADP activities of S-specific 236 antibodies decay during convalescence from COVID-19. The decline of plasma 237 ADCC and ADP activity correlated with the decay of S-specific IgG and FcγR-binding 238 antibodies. Importantly, Fc effector functions were readily detectable above 239 uninfected controls in 94% of subjects for all assays at the last timepoint sampled, in 240 contrast with neutralisation activity, which remained detectable above background for 241 only 70% of subjects. While neutralising antibodies are likely to form a correlate of 242 protection for SARS-CoV-2 7, 32 , several studies find that neutralising antibodies in 243 convalescent donors with mild COVID-19 wane rapidly 2, 8, 9 . The rapid decline of 244 plasma neutralisation activity in the early weeks following infection is likely in part 245 explained by the rapid decline of plasma IgM and IgA titres against S and RBD 20, 33 , 246 which substantially contribute to neutralisation of SARS-CoV-2 34-36 . Given the 247 relative scarcity of re-infection cases reported to date, it is likely that immune 248 responses beyond neutralisation, including antibody Fc effector functions and T cell 249 responses, contribute to long-term protection from SARS-CoV-2. Indeed, a recent 250 study demonstrated that cellular immunity in convalescent macaques, mainly CD8 + T 251 cells, contribute to protection against re-challenge after neutralising antibodies have 252 waned 37 . 253

Our results demonstrate that FcγR-binding antibodies against betacoronaviruses 255 OC43 and HKU1 are much higher in COVID-19 convalescent individuals compared 256 to uninfected controls. This could either be due to the back-boosting of pre-existing 257

HCoV antibodies that are cross-reactive with SARS-CoV-2 28, 29 , or the de novo 258 generation of SARS-CoV-2 antibodies that are cross-reactive with conserved HCoV 259 epitopes. Cross-reactive S antibodies were largely directed against the more 260 conserved S2 subunit, in line with other reports 28, 29 , which also likely explains the 261 longer half-life of S2 antibodies that we observed relative to S1 antibodies. A recent 262 study found cross-reactive binding and neutralising antibodies against SARS-CoV-2 263 S2 in uninfected children and adolescents 28 , suggesting prior infections with OC43 or 264 HKU1 can elicit cross-reactive antibodies against the S2 subunit of SARS-CoV-2 S. Luminex bead-based multiplex assay 520 A custom bead array was designed using SARS-CoV-2 S trimer, S1 subunit (Sino 521 Biological), S2 subunit (ACRO Biosystems) and RBD (BEI Resources), as well as 522

HCoV (OC43, HKU1, 229E, NL63) S and S1 subunit (Sino Biological) (as described 523

in Table S2 ) 52 . Tetanus toxoid (Sigma-Aldrich), influenza hemagglutinin (H1Cal2009; 524

Sino Biological) and SIV gp120 (Sino Biological) were also included in the array as 525 positive and negative controls respectively. These antigens were covalently coupled 526 to magnetic carboxylated beads (Bio Rad) using a two-step carbodiimide reaction 527 and blocked with 0.1% BSA, before being resuspended and stored in PBS 0.05% 528 sodium azide till use. Each condition was tested in duplicate and "no plasma" and "target cell only" controls 567 were included. Cells were centrifuged at 250g for 4 min prior to a 4-hour incubation 568 at 37ºC with 5% CO 2 . Cells were then washed with PBS and lysed with 25µl of 569 passive lysis buffer (Promega). Cell lysates (20µl) were transferred to a white flat-570 bottom plate and developed with 30µl of britelite plus luciferase reagent (Perkin 571 Elmer). Luminescence was read using a FLUOstar Omega microplate reader (BMG 572 Labtech). The relative light units (RLU) measured were used to calculate %ADCC 573 with the following formula: ("no plasma control" -"plasma sample") ÷ "target cell only 574 control" × 100. For each plasma sample, %ADCC was plotted against log 10 (plasma 575 dilution -1 ) and the area under curve (AUC) was calculated using Graphpad Prism. 576 577 Bead-based THP-1 ADP assay 578

To examine ADP mediated by COVID-19 convalescent plasma, a previously 579 published bead-based ADP assay was adapted for use in the context of SARS-CoV-580 581 kit (Thermo Scientific) with 20mmol excess according to manufacturer's instructions 582 and buffer exchanged using 30kDa Amicon centrifugal filters (EMD millipore) to 583 remove free biotin. The binding sites of 1µm fluorescent NeutrAvidin Fluospheres 584 beads (Invitrogen) were coated with biotinylated S at a 1:3 ratio overnight at 4°C. S-585 conjugated beads were washed four times with 2% BSA/PBS to remove excess 586 antigen and incubated with plasma (1:100 dilution) for 2 hours at 37ºC in a 96-well 587 U-bottom plate (see Figure S5 for optimisation). THP-1 monocytes (10,000/well) 588

were then added to opsonized beads and incubated for 16 hours under cell culture 589

conditions. Cells were fixed with 2% formaldehyde and acquired on a BD LSR 590

Fortessa with a HTS. The data was analyzed using FlowJo 10.7.1 (see Figure S4 for 591 gating strategy) and a phagocytosis score was calculated as previously described 53 592 using the formula: (%bead-positive cells × mean fluorescent intensity)/10 3 . To 593 account for non-specific uptake of S-conjugated beads, the phagocytosis scores for 594 each plasma sample were subtracted with that of the "no plasma" control. 595 596 Cell-based THP-1 association assay 597

To assess the capacity of THP-1 monocytes to associate with S-expressing target 598 cells via Ab-FcγR interactions, an assay using THP-1 cells as effectors and Ramos 599 S-orange cells as targets was performed. THP-1 monocytes were first stained with 600 CellTrace TM Violet (CTV) (Life Technologies) as per manufacturer's instructions. In a 601 96-well V-bottom cell culture plate, Ramos S-orange cells (10,000/well) were 602 incubated with plasma from convalescent or uninfected donors (1:2700 dilution) for 603 30 minutes (see Figure S5 for optimisation). Opsonised Ramos S-orange cells were 604 then washed prior to co-culture with CTV-stained THP-1 monocytes (10,000/well) for 605 1 hour at 37ºC with 5% CO 2 . After the incubation, cells were washed with PBS, fixed 606 with 2% formaldehyde and acquired using the BD LSR Fortessa with a high-607 throughput sampler attachment (HTS). The data was analyzed using FlowJo 10.7.1 608 (see Figure S4 for gating strategy). The percentage of Ramos S-orange cells 609 associated with THP-1 monocytes (% association) was measured for each plasma 610 sample and background-subtracted with the "no plasma" control. The parameter is a constant (intercept), and is a subject-specific adjustment to 636 the overall intercept. The slope parameter is a fixed effect to capture the decay 637 slope before (as a fixed parameter, 70 days); which also has a subject-specific 638 random effect

. To fit a model with two different decay rates, an extra parameter 639 (with a subject-specific random effect ) was added to represent the difference 640 between the two slopes. Assay variability between replicates (only for HCoV 641 response variables) was modelled as a single fixed effect , in which we coded the 642 replicate as a binary categorical variable . The random effect was assumed to be 643 normally distributed with zero mean and variance . 644 645 We fitted the model to log-transformed data of various response variables (assuming 646 exponential decay), and we censored the data from below if it was less than the 647 threshold for detection. The data for each subject included either two or three 

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