key: cord-0865856-wkz2ammt
authors: Anderson, Elizabeth M.; Goodwin, Eileen C.; Verma, Anurag; Arevalo, Claudia P.; Bolton, Marcus J.; Weirick, Madison E.; Gouma, Sigrid; McAllister, Christopher M.; Christensen, Shannon R.; Weaver, JoEllen; Hicks, Philip; Manzoni, Tomaz B.; Oniyide, Oluwatosin; Ramage, Holly; Mathew, Divij; Baxter, Amy E.; Oldridge, Derek A.; Greenplate, Allison R.; Wu, Jennifer E.; Alanio, Cécile; D’Andrea, Kurt; Kuthuru, Oliva; Dougherty, Jeanette; Pattekar, Ajinkya; Kim, Justin; Han, Nicholas; Apostolidis, Sokratis A.; Huang, Alex C.; Vella, Laura A.; Kuri-Cervantes, Leticia; Pampena, M. Betina; Betts, Michael R.; Wherry, E. John; Meyer, Nuala J.; Cherry, Sara; Bates, Paul; Rader, Daniel J.; Hensley, Scott E.
title: Seasonal human coronavirus antibodies are boosted upon SARS-CoV-2 infection but not associated with protection
date: 2021-02-09
journal: Cell
DOI: 10.1016/j.cell.2021.02.010
sha: 252bc9551dc3ee58a6bea9e5079a7217cef810b4
doc_id: 865856
cord_uid: wkz2ammt

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread within the human population. Although SARS-CoV-2 is a novel coronavirus, most humans had been previously exposed to other antigenically distinct common seasonal human coronaviruses (hCoVs) before the COVID-19 pandemic. Here, we quantified levels of SARS-CoV-2-reactive antibodies and hCoV-reactive antibodies in serum samples collected from 431 humans before the COVID-19 pandemic. We then quantified pre-pandemic antibody levels in serum from a separate cohort of 251 individuals who became PCR-confirmed infected with SARS-CoV-2. Finally, we longitudinally measured hCoV and SARS-CoV-2 antibodies in the serum of hospitalized COVID-19 patients. Our studies indicate that most individuals possessed hCoV-reactive antibodies before the COVID-19 pandemic. We determined that ∼20% of these individuals possessed non-neutralizing antibodies that cross-reacted with SARS-CoV-2 spike and nucleocapsid proteins. These antibodies were not associated with protection against SARS-CoV-2 infections or hospitalizations, but they were boosted upon SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread within the 2 human population. Although SARS-CoV-2 is a novel coronavirus, most humans had been 3 previously exposed to other antigenically distinct common seasonal human coronaviruses 4 (hCoVs) before the COVID-19 pandemic. Here, we quantified levels of SARS-CoV-2-reactive 5 antibodies and hCoV-reactive antibodies in serum samples collected from 431 humans before the 6 COVID-19 pandemic. We then quantified pre-pandemic antibody levels in serum from a 7 separate cohort of 251 individuals who became PCR-confirmed infected with SARS-CoV-2. 8

Finally, we longitudinally measured hCoV and SARS-CoV-2 antibodies in the serum of 9 hospitalized COVID-19 patients. Our studies indicate that most individuals possessed hCoV-10 reactive antibodies before the COVID-19 pandemic. We determined that ~20% of these 11 individuals possessed non-neutralizing antibodies that cross-reacted with SARS-CoV-2 spike 12 and nucleocapsid proteins. These antibodies were not associated with protection against SARS-13 CoV-2 infections or hospitalizations, but they were boosted upon SARS-CoV-2 infection. Coronaviruses commonly infect humans (Dijkman et al., 2012 , Friedman et al., 2018 , Gaunt et 20 al., 2010 , Killerby et al., 2018 . The severe acute respiratory syndrome coronavirus 2 (SARS-21

CoV-2) emerged at the end of 2019 and has rapidly spread among humans, many of whom have 22 been previously exposed to common seasonal human coronaviruses (hCoVs) (Edridge et al., 23 2020) . Common seasonal hCoVs include the betacoronaviruses HKU1 and OC43 and the 24 alphacoronaviruses 229E and NL63 (Pfefferle et al., 2009 , Pyrc et al., 2006 , Vijgen et al., 2006 , 25 Woo et al., 2005 . SARS-CoV-2 belongs to the betacoronavirus genus and is more closely 26 related to HKU1 and OC43 compared to the alphacoronaviruses 229E and NL63 (Jaimes et al., 27 2020 , Okba et al., 2020 . A recent study examining electronic medical records suggested that 28 recent hCoV infections are not associated with decreased SARS-CoV-2 infections, but are 29 associated with reducing the severity of Coronavirus Disease 2019 (COVID-19) (Sagar et al., 30 2020) . It is unclear if this apparent cross-protection is mediated by antigen-specific cellular or 31 humoral immunity or if it is due to short-term general cross-protection similar to what has been 32 recently reported with rhinovirus and influenza virus infections . It is unknown 33 if prior hCoV exposures elicit antibodies that prevent or alter the outcomes of SARS-CoV-2 34 infections. Further, it is unknown if different aged individuals have distinct hCoV immune 35 histories that can affect SARS-CoV-2 susceptibility. To address this, we completed a serological 36 survey using serum samples collected from different aged humans prior to the COVID-19 37 pandemic. We quantified levels of antibodies reactive to viral proteins from hCoVs and 38 determined if these antibodies were associated with SARS-CoV-2 protection. Finally, we 39 completed a series of studies using serum collected from COVID-19 patients to determine if 40 antibodies reactive to hCoVs are boosted upon SARS-CoV-2 infections. 41 J o u r n a l P r e -p r o o f 7 antibodies reactive to the OC43 S protein and found no differences among samples from 111 individuals who did or did not become infected with SARS-CoV-2 (Figure 2A ; p=0.90 and 112 Table S1 and Table S2 ). Among those with PCR-confirmed SARS-CoV-2 infections, we found 113 no relationship between SARS-CoV-2 and OC43 antibody titers and hospitalization or disease 114 severity among hospitalized patients ( Table S1 and Table S2 ). We found no relationship 115 between SARS-CoV-2 and OC43 antibody titers and the need for respiratory support and 116 admittance into the ICU following SARS-CoV-2 infection ( Table S1 and Table S2) . 117

Previous studies indicated that immunity to hCoV can be short-lived (Huang et al., 2020) 118 and a recent study documented that antibody titers against hCoV can fluctuate over time 119 (Edridge et al., 2020) , presumably due to repetitive hCoV exposures. In our study, pre-pandemic 120 serum samples were collected from 2013-2020 and therefore it is possible that antibody levels in 121 some of the samples collected several years prior to 2020 do not accurately reflect antibody 122 levels present during the COVID-19 pandemic. To address this, we compared SARS-CoV-2 and 123 OC43 IgG antibody titers in the serum of individuals in our cohort who had samples collected 124

within one year of the pandemic (between April 2019 and March 2020). Using this smaller 125 cohort (n=39 SARS-CoV-2 cases and n=57 controls), we still found no differences in levels of 126 antibodies reactive to the SARS-CoV-2 S protein, S-RBD protein, N protein, or OC43 S protein 127 ( Figure 2B ). Taken together, our data suggest that a subset of humans possessed non-128 neutralizing cross-reactive antibodies against SARS-CoV-2 S and N proteins prior to the 129 COVID-19 pandemic, but these antibodies were not associated with protection from SARS-CoV-130 8 Recent studies indicate that COVID-19 recovered donors possess higher levels of 134 antibodies against seasonal betacoronaviruses (Nguyen-Contant et al., 2020) . To determine if 135 antibodies against the S protein of hCoVs are boosted upon SARS-CoV-2 infection, we 136 measured 229E, NL63, OC43, and SARS-CoV-2 S IgG antibody levels in sera collected 137 longitudinally from 27 hospitalized COVID-19 patients. Samples from a subset of the 138 hospitalized patients (10 of 27) were tested using an extended respiratory pathogen viral panel to 139 confirm that they were not simultaneously co-infected with SARS-CoV-2 and a different 140 coronavirus. Serum IgG antibodies reactive to the S protein of the 229E and NL63 141 alphacoronaviruses did not change over 7 days of hospitalization ( Figure 3A-B) . Conversely, 142 serum antibodies reactive to the S protein of OC43 and SARS-CoV-2 betacoronaviruses 143 significantly increased over the course of hospitalization (Figure 3A-B) . We found that boosted 144 antibodies in hospitalized patients primarily targeted the S2 domain, and not the S1 domain, of 145 the OC43 S protein (Figure S6A-B) . Overall OC43 IgG antibody titers ( Figure 3C ) and the 146 magnitude of OC43 S antibody boosts ( Figure 3D ) were not associated with outcome of disease. 147

These data indicate that cross-reactive antibodies elicited by previous hCoV infections are not 148 associated with protection from SARS-CoV-2 infections, but are boosted following infection 149 with SARS-CoV-2. 150

Our study demonstrates that ~20% of individuals possessed SARS-CoV-2 cross-reactive 153 serum antibodies prior to the COVID-19 pandemic. Using samples collected in 2017, we found 154 that pre-pandemic cross-reactive antibodies directed against the SARS-CoV-2 N protein were 155 more prevalent compared to those directed against the SARS-CoV-2 S protein (16.2% 156 9 seropositive versus 4.2% seropositive). We found that most individuals possessed pre-pandemic 157 serum antibodies reactive to the S proteins of 229E, NL63, and OC43 ( Figure S1) ; however, 158 pre-pandemic samples with detectable levels of SARS-CoV-2 antibodies had higher levels of 159 antibodies against the OC43 S protein ( Figure 1H ). Although our data suggest that prior 160 infections with seasonal human betacoronaviruses (such as OC43) likely elicit antibodies that 161 cross-react with SARS-CoV-2 proteins, in is unclear why only a subset of OC43 seropositive 162 individuals possessed antibodies reactive to SARS-CoV-2 prior to the pandemic. Further studies 163 will be needed to determine the temporal relationship between seasonal human betacoronavirus 164 infections and the induction of SARS-CoV-2 cross-reactive antibodies. Further studies 165 investigating the relationship of pre-pandemic antibodies against other betacoronaviruses, such 166 as HKU1, with pre-pandemic SARS-CoV-2 cross-reactive antibodies are also needed. 167

Our study is consistent with a recent manuscript demonstrating a lack of SARS-CoV-2 168 neutralizing activity in pre-pandemic sera (Poston et al., 2020) . In contrast, a different study 169 reported that pre-pandemic serum from young children possess SARS-CoV-2 neutralizing 170 antibodies (Ng et al., 2020) . It is unclear if these differences are due to the specific assays used in 171 each study or other factors such as geographic differences in sampling. For example, the Ng et. 172 al. study (Ng et al., 2020) used a pseudotyped neutralization assay will cells that lack ACE2, 173 which is the cellular receptor for SARS-CoV-2. Our study is unique in that we were able to 174 directly assess whether pre-pandemic antibodies were associated with protection from SARS-175

CoV-2 infections and hospitalizations. While we found no differences in pre-pandemic antibody 176 levels against SARS-CoV-2 and OC43 among those infected and not infected with SARS-CoV-2 177 Further studies also need to be completed to determine how immune history affects de 184 novo immune responses following SARS-CoV-2 infection. We find that individuals infected 185 with SARS-CoV-2 produce antibodies reactive to both the SARS-CoV-2 S protein and OC43 S 186 protein (Figure 3 ). In the case of influenza viruses, sequential infections with antigenically 187 distinct strains can elicit antibodies against conserved epitopes between the strains and it is 188 unclear if these cross-reactive antibodies inhibit de novo immune responses or affect disease 189 severity (Cobey and Hensley, 2017) . Our studies suggest that SARS-CoV-2 infection boosts 190 antibodies reactive to the S2 domain of the OC43 S protein. Further studies are needed to 191 precisely map the footprints of these antibodies and additional studies need to be completed to 192 determine if these antibodies help resolve infections or if they enhance disease in Given that our data suggest that pre-pandemic non-neutralizing antibodies elicited by 195 hCoVs do not provide SARS-CoV-2 protection, special attention should be directed towards 196 evaluating if T cell responses primed against hCoV infections provide partial protection against 197 SARS-CoV-2 infections. Recent studies have clearly shown that some individuals possessed 198 SARS-CoV-2-specific CD4+ and CD8+ T cells prior to the COVID-19 pandemic (Braun et al., 199 2020 , Le Bert et al., 2020 , Sette and Crotty, 2020 , 200 Schulien et al., 2020 , and it is possible that pre-existing cellular immunity might play an 201 important protective role in the context of pandemic viruses that only share non-neutralizing 202 antibody epitopes with previously circulating viral strains. 203

The data presented here show that pre-pandemic serum antibodies that cross-react with 206 SARS-CoV-2 do not correlate with protection against SARS-CoV-2 infections and severity of 207 COVID-19. We generated data using pre-pandemic samples that were collected from individuals 208 who became PCR+ confirmed infected with SARS-CoV-2. We compared antibody levels in 209 these samples to antibody levels in pre-pandemic samples from individuals who did not get 210 infected with SARS-CoV-2. For these studies, we included samples that were collected from 211

August 2013 to March 2020 (Figure 2A) . Since immunity to hCoVs can be short-lived (Huang 212 et al., 2020) and fluctuate over time (Edridge et al., 2020) , we also directly compared antibody 213 titers in samples that were collected within one year of the pandemic ( Figure 2B ). Using both 214 datasets, we found no correlation between pre-pandemic antibody levels and SARS-CoV Vanderbeck) for sample procurement, processing, and logistics. We thank the staff of the PMBB. 236

We thank F. Krammer (Mt. Sinai) for sending us the SARS-CoV-2 spike RBD expression 237 plasmids. We thank David Anderson for assistance with the graphical abstract. previously (Hoffmann et al., 2020) . All cell lines were cultured using manufacturer's guidelines 405 and used as described in Method Details below. 406 407 408

Serum antibody titers against SARS-CoV-2 and other human coronavirus (hCoV) antigens were 411 quantified by enzyme-linked immunosorbent assays (ELISA) as previously described (Flannery 412 et al., 2020) . For some experiments, we purified IgG from sera samples before completing ELISAs. IgG was 443 purified from sera samples using PureProteome Protein G magnetic beads (Millipore, Darmstadt, 444 Germany: cat. LSKMAGG02 ) as previously described . Sera samples were 445 diluted in PBS and incubated with 100 µL of washed magnetic beads for 1 hour at room 446 temperature with constant mixing. Unbound fractions were removed using the magnetic stand 447 and beads were washed with PBS. Bound IgG was eluted with the addition of 100 µL of 0.2 M 448 glycine, pH 2.5 followed by 5 minute incubation at room temperature. The eluant containing 449 purified IgG was neutralized with 10 µL of 1.0 M Tris, pH 8.8 prior to being run in ELISA. 450 451

SARS-CoV-2 pseudotypes were generated with a previously described vesicular stomatitis virus 453 (VSV) pseudotype platform (Anderson et al., 2020) . Briefly, pseudotyped VSV virions with 454 SARS-CoV-2 Spike were produced through transfection of 293T with 35µg of pCG1 SARS-455

CoV-2 S delta18 expression plasmid encoding a codon optimized SARS-CoV-2 S gene with an 456 18-residue truncation in the cytoplasmic tail (kindly provided by Stefan Pohlmann) (Hoffmann et 457 al., 2020) . 30 hours post transfection, the SARS-CoV-2 spike expressing cells were infected for 458 2-4 hours with VSV-G pseudotyped VSV∆G-RFP at a multiplicity of infection (MOI) of ~1-3. 459

Then, the cells were washed twice with media to remove unbound virus. 28-30 hours after 460 infection, the media containing the VSV∆G-RFP SARS-CoV-2 pseudotypes were harvested and 461 clarified by centrifugation two times at 6000xg. SARS-CoV-2 pseudotypes were aliquoted and 462 stored at -80°C until used for antibody neutralization analysis. 463 464

Serum SARS-CoV-2 neutralizing antibodies were measured as previously described (Anderson 466 et al., 2020) . Vero E6 cells stably expressing TMPRSS2 were seeded in 100µl at 2. 1920 1930 1940 1950 1960 1970 1980 1990 2000 1920 1930 1940 1950 1960 1970 1980 1990 2000 1920 1930 1940 1950 1960 1970 1980 1990 2000 1920 1930 1940 1950 1960 1970 1980 1990 2000 1920 1930 1940 1950 1960 1970 1980 1990 2000 1920 1930 1940 1950 1960 1970 1980 1990 2000 1920 1930 1940 1950 1960 1970 1980 1990 2000 J o u r n a l P r e -p r o o f 

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washed, fixed with 4% paraformaldehyde, and visualized on an S6 FluoroSpot Analyzer (CTL, 473 Shaker Heights OH) and individual infected foci were enumerated. The focus reduction 474 neutralization titer 50% (FRNT 50 ) was measured as the greatest serum dilution at which focus 475 count was reduced by at least 50% relative to control cells that were infected with pseudotype 476 virus in the absence of human serum. FRNT 50 titers for each sample were measured in at least 477 two technical replicates performed on separate days. Reciprocal serum dilution antibody titers were log2 transformed for statistical analysis. ELISA 498 antibody titers below the limit of detection (LOD; reciprocal titer <50) were set to a reciprocal 499 titer of 25. Log2 transformed antibody titers were compared with unpaired t-tests and statistical 500 significance was set to p-value <0.05. Linear regressions were also performed using log2 501 transform titers and untransformed data from the other variables. We compared antibody titers in 502 pre-pandemic serum samples from individuals who did and did not have a subsequent PCR-503 J o u r n a l P r e -p r o o f 24 confirmed SARS-CoV-2 infection. For these analyses we selected serum sample from 504 individuals with RT-PCR negative results matching sex, age, and race for each SARS-CoV-2 505 PCR-confirmed case (RT-PCR positive) to define controls for our cohort. In instances we did not 506 find matched controls, we randomly selected patients with RT-PCR negative test results. We also 507 compared antibody titers in pre-pandemic serum samples among SARS-CoV-2 PCR-confirmed 508 individuals in relationship to hospitalization or need for respiratory support due to Multivariate logistic regression was used to compare the antibody differences for these studies. 510All the models were adjusted by sex, age, race, and analyses were performed in R (R Core Team, 511 2016). We compared Log2 transformed antibody titers in COVID-19 hospitalized patients at day 512 0 and day 7. We also compared the fold change in titer by day 7. We compared the fold change 513 in OC43 titers between patients who survived and patients who died by day 28 of hospitalization. 514 515 516 J o u r n a l P r e -p r o o f