key: cord-348455-vcxalkeo
authors: Graham, N. R.; Whitaker, A. N.; Strother, C. A.; Miles, A. K.; Grier, D.; McElvany, B. D.; Bruce, E. A.; Poynter, M. E.; Pierce, K. K.; Kirkpatrick, B. D.; Stapleton, R. D.; An, G.; Botten, J. W.; Crothers, J. W.; Diehl, S. A.
title: Kinetics and Isotype Assessment of Antibodies Targeting the Spike Protein Receptor Binding Domain of SARS-CoV-2 In COVID-19 Patients as a function of Age and Biological Sex.
date: 2020-07-22
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2020.07.15.20154443
sha: 
doc_id: 348455
cord_uid: vcxalkeo

SARS-CoV-2 is the newly emerged virus responsible for the global COVID-19 pandemic. There is an incomplete understanding of the host humoral immune response to SARS-CoV-2 during acute infection. Host factors such as age and sex as well the kinetics and functionality of antibody responses are important factors to consider as vaccine development proceeds. The receptor-binding domain of the CoV spike (RBD-S) protein is important in host cell recognition and infection and antibodies targeting this domain are often neutralizing. In a cross-sectional study of anti-RBD-S antibodies in COVID-19 patients we found equivalent levels in male and female patients and no age-related deficiencies even out to 93 years of age. The anti-RBD-S response was evident as little as 6 days after onset of symptoms and for at least 5 weeks after symptom onset. Anti-RBD-S IgG, IgM, and IgA responses were simultaneously induced within 10 days after onset, but isotype-specific kinetics differed such that anti-RBD-S IgG was most sustained over a 5-week period. The kinetics and magnitude of neutralizing antibody formation strongly correlated with that seen for anti-RBD-S antibodies. Our results suggest age- and sex- related disparities in COVID-19 fatalities are not explained by anti-RBD-S responses. The multi-isotype anti-RBD-S response induced by live virus infection could serve as a potential marker by which to monitor vaccine-induced responses.

and infection and antibodies targeting this domain are often neutralizing. In a cross-sectional 23 study of anti-RBD-S antibodies in COVID-19 patients we found equivalent levels in male and 24 female patients and no age-related deficiencies even out to 93 years of age. The anti-RBD-S 25 response was evident as little as 6 days after onset of symptoms and for at least 5 weeks after 26 symptom onset. Anti-RBD-S IgG, IgM, and IgA responses were simultaneously induced within 27 10 days after onset, but isotype-specific kinetics differed such that anti-RBD-S IgG was most 28 sustained over a 5-week period. The kinetics and magnitude of neutralizing antibody formation 29 strongly correlated with that seen for anti-RBD-S antibodies. Our results suggest age-and sex-30 related disparities in COVID-19 fatalities are not explained by anti-RBD-S responses. The multi-31 isotype anti-RBD-S response induced by live virus infection could serve as a potential marker by 32 which to monitor vaccine-induced responses. 33

Human pathogenic coronaviruses (CoV) such as severe acute respiratory syndrome (SARS)-37

CoV-1, middle east respiratory syndrome (MERS)-CoV, and SARS-CoV-2 (all b-CoVs) have 38 resulted from zoonoses and utilize cellular receptors to bind and access host cells for productive 39 infection (1-3). CoV spike (S) proteins are large (>200 kDa) glycosylated trimeric structures that 40 protrude from viral particles and enable binding of CoV to cellular receptors. SARS-CoV-2 41 interacts with angiotensin converting enzyme-2 (ACE2) via a flexible receptor-binding domain 42 (RBD) located on the distal tip of the S protein (4-7). After binding, several proteases act upon 43 S, priming it to adopt large conformational shifts that facilitate entry into host cells(8). First the 44 S1 domain (which contains RBD) is cleaved from the C-terminal S2 domain. For SARS-CoV-2 45 this process may involve furin in the host cell membrane due to a novel furin-recognition site in 46 the S1/S2 region (9-11). The S2 domain is further processed by other serine and cysteine-47 proteases such as trypsin, cathepsin, and TMPRSS2 to facilitate viral entry into the host cell (4, 48 12). 49

Neutralizing antibodies to SARS CoV-1 have been isolated and were found to target 50 RBD-S (13). One of these mAbs CR3022 was also found to bind SARS-CoV-2 RBD-S(14). At 51 the polyclonal level, the quantity of anti-RBD S IgG antibodies against SARS-CoV-2 correlate 52 well with neutralizing activity(15-18). Cross-neutralization amongst SARS viruses by RBD-S-53 targeting antibodies can occur (18-21). However, sequence homology for RBD-S is low for non-54 SARS b-CoVs (such as MERS) and for a-CoVs such as NL63, OC43, 229E, and HKU1(16, 17). 55

For these reasons serology for SARS-CoV-2 RBD-S is being used to help identify recovered 56 COVID-19 patients as plasma donors for passive immunotherapy (22). 57

There are several risk factors for COVID-19 mortality but whether two of these -age and 58 biological sex -are associated with the SARS-CoV-2 RBD-S immune response has to our 59 knowledge not been addressed in the peer-reviewed literature. Furthermore, most serology 60 studies have been done in the setting of severe COVID-19 disease and, save for one study 61 (17), without the benefit of detailed kinetics. Herein we tracked the kinetics and magnitude of 62 neutralizing and anti-SARS-CoV-2 S and RBD-S antibodies in a cross-sectional cohort of PCR-63 confirmed COVID-19 patients. 64 65 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We chose a two-step ELISA-based RBD-S-focused approach to serology in our study 67 population. Reagents and pre-print protocols were available in mid-March 2020, which indicated 68 that RBD-S screening and full-S confirmation could identify specific and functional antibodies 69 and be quickly operationalized. Using the established protocol (23) we confirmed the expected 70 protein size of mammalian-expressed RBD-S ( Figure 1A ) and trimerized spike ( Figure 1B ) 71 produced from DNA plasmids (gift from Florian Krammer, Mt Sinai School of Medicine). RBD-S 72 antibodies were specific and correlated with neutralization (15), findings that have been 73 validated using similar RBD-S-focused assays(16, 17). We confirmed RBD-S and S protein 74 conformation by binding of CR3022 human IgG1 (Figure 1C, D) . CR3022 was isolated as a 75 SARS-S1 domain-binding single chain antibody fragment by phage display and is neutralizing 76 as an IgG1(13). CR3022 binds adjacent to RBD-S in trimeric S of SARS-CoV-2 in a 77 glycosylation-sensitive manner(14). Mammalian expression of appropriate size proteins and 78 recognition by CR3022 together confirm that our protein preparations exhibited the expected 79 characteristics. 80

We first piloted our antigen preps for the RBD-S IgG screening assay using serum 81 samples from a PCR-confirmed severe COVID-19 patient (defined as admission to the Intensive 82 Care Unit, ICU) who was admitted to the hospital 10 days following symptom onset and based 83 on an early report suggesting that SARS-CoV-2 could trigger antibody responses in this 84 timeframe (24). We compared IgG reactivity in this sample to decreasing amounts of our RBD-S 85 antigen preparations against a fixed, recommended amount of commercially produced RBD-S 86 protein derived from the protocol we used (23). We found that a wide range of locally produced 87 RBD-S antigen yielded IgG reactivity equivalent to 100 ng of commercial antigen in an acute 88 serum sample from this COVID-19-positive patient ( Figure 1E) . No signal was observed in a 89

pre-2019 serum sample or in the absence of serum ( Figure 1E ). Using the standard 100 ng 90 amount hereafter, we found that RBD-S-binding IgM and IgG were present at 10-13 days after 91 All rights reserved. No reuse allowed without permission.

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

The copyright holder for this preprint this version posted July 22, 2020. . https://doi.org/10.1101/2020.07.15.20154443 doi: medRxiv preprint symptom onset. We did not detect any RBD-S-binding in healthy pre-2019 sera (Figure 1F) , in 92 agreement with extensive testing of this assay in pre-COVID-19 serum performed elsewhere 93 (15). Due to different secondary antibodies for IgM and IgG detection we cannot conclude 94 whether absolute levels of RBD-S IgG were higher than RBD-S IgM. Total IgG and IgM were 95 readily detected in both COVID-19 and in healthy non-COVID-19 serum ( Figure 1G) . 96

For a cross-sectional COVID-19 serological survey we collected serum samples from 32 97 patients that tested COVID-19 positive by nasopharyngeal swab RT-qPCR testing. All patients 98 had been admitted to the hospital and 13/32 (40%) were admitted to the ICU. Twenty-five 99 patients were subsequently discharged and 7 died. One to five serum samples were collected 100 from each patient with the first sample being taken within approximately 9 days after diagnosis, 101 in which diagnosis occurred around 5 days after symptom onset ( Table 1) . There was a 102 53%:47% male: female distribution and patients were on average 68 ± 14 years of age (range 103 30 -93 years) ( Table 1) . 104 A male bias in COVID-19 mortality was reported early during the pandemic (25-27) and 105 has been confirmed worldwide in a recent meta-analysis (28). One of the hypotheses to explain 106 this is differences in adaptive immunity between males and females. Although the mean serum 107 RBD-S IgG reactivity level appeared higher in male samples (O.D. = 1.8, n = 40) versus female 108 samples (O.D. = 1.0, n = 37) this difference was not significant and the same maximum 109 reactivity values were found in males and females (Figure 2A) . 110

Although not absolute, it appears that irrespective of comorbidities, there is a higher risk 111 of COVID-19 mortality and morbidity in older individuals (60 years of age and over) (29-31). We 112 therefore assessed RBD-S IgG antibodies by age. There was a broad range of RBD-S IgG 113 responses that did not differ as a function of age as assessed by correlation analysis (R 2 < 0.01, 114 Figure 2B ). Notably, one of the highest RBD-S IgG responses was from a 93-year old patient. A 115 serum sample from a 30-year old COVID-19 patient was negative for RBD-S IgG, but this 116 sample was taken just three days after symptom onset, which may be too early for induction of 117 All rights reserved. No reuse allowed without permission.

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

The copyright holder for this preprint this version posted July 22, 2020. . https://doi.org/10.1101/2020.07.15.20154443 doi: medRxiv preprint robust IgG responses. Taken together, we did not find evidence of biological sex-or age-related 118 deficiencies in RBD-S IgG responses in COVID-19 patients. 119 RDB-S-reactive serum IgG was detected in 5 of 12 (42%) samples that were taken 120 within 10 days of symptom onset ( Figure 2C ). After day 10 of symptoms, 98% of samples were 121 positive for RBD IgG ( Figure 2C ). There were small variations in positive threshold for RBD by 122 assay date ( Figure S1 ). We therefore confirmed each sample (whether RBD-positive or not) 123 with an endpoint titration and area under the curve calculation for reactivity against the full spike 124 ectodomain trimer (15). Samples that were RBD-S-negative were also low for spike total 125 reactivity (AUC) and titer (Figures 2D, E) . Furthermore, we found a very strong correlation 126 between RBD and spike IgG ( Figure In the patient-specific RBD IgG data ( Figure S2A ) we found several patterns: initial 134 seroconversion (e.g. patients 0003, and 0017), rapid increases (e.g. patients 0005, 0006, 0009, 135 0011, 0020, occurring between days 10-20), and plateaued responses (e.g. patients 0012 and 136 0021, occurring mainly after day 20). These responses were concordant with temporal patient-137 specific S IgG titers ( Figure S2B ). Anti-S titers in patients with a negative RBD-S test were 138 generally low and in RBD-positive samples, followed the same trends as RBD-reactivity, 139 providing further confirmation of robust serological responses to SARS-CoV-2 during acute 140 COVID-19. At the patient level, neutralizing activity was observed after as few as five days after 141 symptom onset and throughout the study period and was predominantly found in those samples 142 with positive RBD-S IgG ( Figure S3) . 143 All rights reserved. No reuse allowed without permission.

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To assess antibody isotype dynamics during acute SARS-CoV-2 we followed RBD-S 144 and full spike-specific IgM and IgA levels in the same samples for which RBD-S and spike IgG 145 was determined. At the patient level we found robust co-occurrence of IgM, IgG, and IgA 146 antibodies reactive to RBD-S in most samples, particularly in post-day 10 samples (Figure S4) . 147

Pooling all the data revealed that all pre-day 10 RBD-S responses for all isotypes were low. 148

Around day 10, IgM targeting RBD-S as well as the switched isotypes IgG and IgA 149 simultaneously rose. While RBD-reactive IgM and IgA responses tapered after 3 weeks post-150 onset (though remained higher than baseline), those for IgG continued to rise to a plateau that 151 was sustained up to 5 weeks after symptoms onset (the most protracted timepoint measured, 152 Figure 3A ). Similar patterns were obtained for full spike-reactive antibodies ( Figure 3B ). These 153 results suggesting that during acute infection COVID-19 patients undergo a seroconversion 154 across isotypes to SARS-CoV-2 rather than an expansion of pre-existing anti-CoV antibodies. 155

Lastly, we assessed anti-RBD-IgG responses by clinical severity. All the patients in this 156 study were hospitalized and 40% of were admitted to the intensive care unit. When we stratified 157 by ICU admission and compared RBS-S IgG levels, we found a trend towards higher levels in 158 those requiring ICU-level care (P = 0.09) ( Figure 4A) . Additionally, we observed a significant 159 association between RBD-S IgG and duration of ICU admission ( Figure 4B ). Lastly 7 of 32 160 (22%) patients succumbed to COVID-19. While a significant difference in the median RBD-S 161 IgG was not observed between survivors and decedents, a smaller range trending towards 162 higher RBD-S reactivity was observed in those patients that died ( Figure 4C ). Although we did 163 not have continuous monitoring of viral load in these patients during hospitalizations it is 164 possible that RBD-S IgG levels reflect ongoing viral replication during more severe disease and 165 in conjunction with other factors may allow for recovery. 166

Taken together, our results provide the first comprehensive survey of SARS-CoV-2 spike 167 RBD antibodies that accounts for two key risk factors for COVID-19. Neither RBD-S nor S 168 antibodies were significantly different as a function of biological sex. Anti-RBD-S and spike IgG 169 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted July 22, 2020. . https://doi.org/10.1101/2020.07.15.20154443 doi: medRxiv preprint responses were induced across 6 decades of age with robust responses found in several 170 samples from patients ≥ 80 years old. These results also extend kinetic analyses and confirm 171 the paucity of anti-SARS-CoV-2 anti-spike responses in very early blood samples taken prior to 172 day 10 after symptoms onset (17, 24). We also assessed protective anti-spike RBD responses 173 as a function of level of hospital care and disease severity and found that duration of ICU-level 174 care was associated with higher responses, possibly due to an extended period of SARS-CoV-2 175 replication during severe disease. A limitation of our study is that we only followed symptomatic 176 patients admitted to hospital; it is unclear whether antibody responses differ in asymptomatic or 177 mildly symptomatic patients. We also did not directly assess whether the RBD-specific 178 antibodies we studied were neutralizing at the clonal level, though we did observe a strong 179 association with polyclonal RBD-S IgG responses and SARS-CoV-2 neutralizing activity. This is 180 in agreement with other reports which confirm that RBD-S IgG levels correlate with neutralizing 181 activity and that the RBD of SARS-CoV-2 is a potent target for neutralizing antibodies (16-18, 182 20, 21, 33). It will be important to determine whether anti-RBD IgA or even IgM antibodies 183 contribute to blocking activity. 184 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted July 22, 2020. was also used as a positive control during assay set up and this reagent was produced in 208

HEK293T cells under HHSN272201400008C and obtained through BEI Resources, NIAID, NIH: 209 All rights reserved. No reuse allowed without permission.

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Wuhan-Hu-1, Recombinant from NR-52306. 211

CR3022 is a SARS-CoV S-specific antibody originally isolated by single chain variable region 213 phage display and then cloned as an IgG1/kappa monoclonal human IgG1/k (13). We received 214 CR3022 heavy chain (HC) and light chain (LC) cloned into pFUSEss-CHIg-hG1 and pFUSE2ss-215

CLIg-hK, respectively (Invivogen) from Florian Krammer spotted on filter paper. We (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted July 22, 2020. Graphics and Statistical testing. 285 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted July 22, 2020. . https://doi.org/10.1101/2020.07.15.20154443 doi: medRxiv preprint All statistics and graphics were performed using R version 3.6.1 using standard packages or 286 GraphPad Prism 8.4.3. Non-parametric LOESS (LOcal regrESSion) was used for smoothing. 287 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted July 22, 2020. Detection of serum IgG from a COVID-19 patient (left), but not from pre-2020 serum (center) or 421 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted July 22, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 22, 2020. . https://doi.org/10.1101/2020.07.15.20154443 doi: medRxiv preprint and 95% confidence intervals are shown for each isotype. 443 All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted July 22, 2020. Spearman's Rho coefficient (R 2 ), 95% confidence interval, and P-value are shown. (C) RBD-S 449 IgG in patients that were deceased or discharged were analyzed by student's t-test and P-value 450 is shown. Boxplots show the median, 95% confidence level, and all individual samples. 451 All rights reserved. No reuse allowed without permission.

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Genomic characterisation and 314 epidemiology of 2019 novel coronavirus: implications for virus origins and receptor 315 binding

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Receptor Recognition by the Novel 322 Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of 323 SARS Coronavirus

Site-specific glycan 325 analysis of the SARS-CoV-2 spike

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Coronavirus Spike Protein and Tropism 330 Changes

Cleavage Site in the Spike 332 Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

Phylogenetic Analysis 335 and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct 336 and Proteolytically Sensitive Activation Loop

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Characterization of spike glycoprotein of 340 SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

A highly conserved cryptic epitope 346 in the receptor binding domains of SARS-CoV-2 and SARS-CoV

349 A serological assay to detect SARS-CoV-2 seroconversion in humans

The receptor binding domain of the viral spike protein is an immunodominant and highly 353 specific target of antibodies in SARS-CoV-2 patients

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Disease and healthcare burden of 397 COVID-19 in the United States

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:8,100) and plotted against days of symptoms. (E) Spike IgG endpoint titer or (F) AUC 433 is plotted against RBD-IgG reactivity. (G) SARS-CoV-2 microneutralization titers are plotted 434 against RBD-S IgG reactivity. Cutoff values

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