key: cord-279584-9x1d1kp1
authors: Anderson, E. M.; Goodwin, E. C.; Verma, A.; Arevalo, C. P.; Bolton, M. J.; Weirick, M. E.; Gouma, S.; McAllister, C. M.; Christensen, S. R.; Weaver, J.; Hicks, P.; Manzoni, T. B.; Oniyide, O.; Ramage, H.; Mathew, D.; Baxter, A. E.; Oldridge, D. A.; Greenplate, A. R.; Wu, J. E.; Alanio, C.; D'Andrea, K.; Kuthuru, O.; Dougherty, J.; Pattekar, A.; Kim, J.; Han, N.; Apostolidis, S. A.; Huang, A. C.; Vella, L.; The UPenn COVID Processing Unit,; Wherry, E. J.; Meyer, N. J.; Cherry, S.; Bates, P.; Rader, D. J.; Hensley, S. E.
title: Seasonal human coronavirus antibodies are boosted upon SARS-CoV-2 infection but not associated with protection
date: 2020-11-10
journal: nan
DOI: 10.1101/2020.11.06.20227215
sha: 
doc_id: 279584
cord_uid: 9x1d1kp1

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 204 humans before the COVID-19 pandemic. We then quantified pre-pandemic antibody levels in serum from a separate cohort of 252 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 ~23% 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 paradoxically these hCoV cross-reactive antibodies were boosted upon SARS-CoV-2 infection.

Coronaviruses commonly infect humans [1] [2] [3] [4] . The severe acute respiratory syndrome coronavirus 2 22

(SARS-CoV-2) emerged at the end of 2019 and has rapidly spread among humans, many of 23 whom have been previously exposed to common seasonal human coronaviruses (hCoVs) 5 . 24

Common seasonal hCoVs include the betacoronaviruses HKU1 and OC43 and the 25 alphacoronaviruses 229E and NL63 [6] [7] [8] [9] . SARS-CoV-2 belongs to the betacoronavirus genus and is 26 more closely related to HKU1 and OC43 compared to the alphacoronaviruses 229E and NL63 10 . 27

A recent study examining electronic medical records concluded that recent hCoV infections are 28 not associated with decreased SARS-CoV-2 infections, but are associated with reducing the 29 severity of Coronavirus Disease 2019 (COVID- 19) 11 . It is unknown if prior hCoV exposures 30 elicit antibodies that prevent or alter the outcomes of SARS-CoV-2 infections. Further, it is 31 unknown if different aged individuals have distinct hCoV immune histories that can affect 32 SARS-CoV-2 susceptibility. To address this, we completed a serological survey using serum 33 samples collected from different aged humans prior to the COVID-19 pandemic. We quantified 34 levels of antibodies reactive to viral proteins from hCoVs and determined if these antibodies 35

were associated with SARS-CoV-2 protection. Finally, we completed a series of studies using 36 serum collected from COVID-19 patients to determine if antibodies reactive to hCoVs are 37 boosted upon SARS-CoV-2 infections. 38

We completed ELISAs to quantify levels of pre-pandemic SARS-CoV-2-reactive IgG 42 antibodies in 204 human serum samples collected in 2017. We tested serum samples collected 43

We completed ELISAs to quantify levels of pre-pandemic hCoV-reactive IgG antibodies 69 in all 204 human serum samples collected in 2017. Most serum samples possessed antibodies 70 that reacted to the S protein of 229E and NL63 (both alphacoronaviruses), as well as OC43 (a 71 betacoronavirus) (Figure S1d-f ). There were no major differences in levels of these antibodies 72 among individuals with different birth years, however serum from very young children possessed 73 lower levels of antibodies reactive to the 229E and NL63 S proteins (Figure S1d-f). We 74 completed full antibody titrations to directly compared levels of hCoV antibodies in a subset of 75 pre-pandemic samples from individuals who either did (n=12) or did not (n=51) possess cross-76 reactive SARS-CoV-2 antibodies (Figure 1f-h) . Pre-pandemic antibody levels against the 229E 77 and NL63 alphacoronavirus S proteins were similar among individuals with and without SARS-78

CoV-2 reactive antibodies (Figure 1f-g) . In contrast, antibody levels against the betacoronavirus 79 OC43 S protein were higher in individuals with SARS-CoV-2 reactive antibodies compared to 80 individuals who did not possess pre-pandemic SARS-CoV-2 reactive antibodies (Figure 1h) . 81

These data suggest that pre-pandemic SARS-CoV-2 reactive antibodies were likely elicited by 82 previously circulating betacoronavirus strains, such as OC43. 83 84

It is unknown if antibodies elicited by prior hCoV infections protect against SARS-CoV-87 2 infections and/or prevent severe COVID-19. To address this, we measured SARS-CoV-2 IgG 88 antibodies in pre-pandemic serum samples from 251 individuals who subsequently went on to 89 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227215 doi: medRxiv preprint become PCR-confirmed infected with SARS-CoV-2 and in a control group of pre-pandemic 90 samples from 251 matched individuals who did not become infected with SARS-CoV-2. Pre-91 pandemic samples were collected by the Penn Medicine BioBank from August 2013 to March 92 2020 and PCR-confirmed SARS-CoV-2 infections were identified by nasopharyngeal swab PCR 93 testing results in electronic health records. We found that 2.2% samples possessed pre-pandemic 94 antibodies reactive to the SARS-CoV-2 full length S protein, 0.6% samples possessed pre- Table S1 and Table S2 ). We also measured antibodies reactive to 100 the OC43 S protein and found no differences among samples from individuals who did or did not 101 become infected with SARS-CoV-2 (Figure 2a ; p=0.90 and Table S1 and Table S2 ). Among 102 those with PCR-confirmed SARS-CoV-2 infections, we found no relationship between antibody 103 titers and hospitalization or disease severity among hospitalized patients (Table S1 and Table  104 S2). We found no relationship between antibody titers and the need for respiratory support and 105 admittance into the ICU following SARS-CoV-2 infection (Table S1 and Table S2) . 106

Previous studies indicated that immunity to hCoV can be short-lived 14 and a recent study 107 documented that antibody titers against hCoV can fluctuate over time 5 , presumably due to 108 repetitive hCoV exposures. In our study, pre-pandemic serum samples were collected from 2013-109 2020 and therefore it is possible that antibody levels in some of the samples collected several 110 years prior to 2020 do not accurately reflect antibody levels present during the COVID-19 111

pandemic. To address this, we compared SARS-CoV-2 and OC43 IgG antibody titers in the 112 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227215 doi: medRxiv preprint serum of individuals in our cohort who had samples collected within one year of the pandemic 113 (between April 2019 and March 2020). Using this smaller cohort (n=39 SARS-CoV-2 cases and 114 n=57 controls), we still found no differences in levels of antibodies reactive to the SARS-CoV-2 115 S protein, S-RBD protein, N protein, or OC43 S protein ( Figure 2B) . Taken together, our data 116 suggest that a subset of humans possessed non-neutralizing cross-reactive antibodies against 117 SARS-CoV-2 S and N proteins prior to the COVID-19 pandemic, but these antibodies were not 118 associated with protection from SARS-CoV-2 infections or reducing hospitalizations upon 119 SARS-CoV-2 infections. 120

Recent studies indicate that COVID-19 recovered donors possess higher levels of CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227215 doi: medRxiv preprint

Our study demonstrates that ~23% of individuals possessed SARS-CoV-2 cross-reactive 137 serum antibodies prior to the COVID-19 pandemic. Using samples collected in 2017, we found 138 that pre-pandemic cross-reactive antibodies directed against the SARS-CoV-2 N protein were 139 more prevalent compared to those directed against the SARS-CoV-2 S protein (18.6% 140 seropositive versus 5.4% seropositive). We found that most individuals possessed pre-pandemic 141 serum antibodies reactive to the S proteins of 229E, NL63, and OC43 ( Figure S2) ; however, 142 pre-pandemic samples with detectable levels of SARS-CoV-2 antibodies had higher levels of 143 antibodies against the OC43 S protein ( Figure 1H ). Although our data suggest that prior 144 infections with seasonal human betacoronaviruses (such as OC43) likely elicit antibodies that 145 cross-react with SARS-CoV-2 proteins, in is unclear why only a subset of OC43 seropositive 146 individuals possessed antibodies reactive to SARS-CoV-2 prior to the pandemic. Further studies 147 will be needed to determine the temporal relationship between seasonal human betacoronavirus 148 infections and the induction of SARS-CoV-2 cross-reactive antibodies. Further studies 149

investigating the relationship of pre-pandemic antibodies against other betacoronaviruses, such 150 as HKU1, with pre-pandemic SARS-CoV-2 cross-reactive antibodies are also needed. 151

We show that pre-pandemic SARS-CoV-2 cross-reactive antibodies are non-neutralizing 152 and are not associated with reducing SARS-CoV-2 infections and hospitalizations. We compared 153 serum from individuals who were and were not hospitalized after SARS-CoV-2 infections and 154 found no differences in pre-pandemic antibody levels against SARS-CoV-2 and OC43 ( Figure  155 2). We evaluated the need for respiratory support and admittance into the ICU as a proxy for 156 COVID-19 severity (Table S2) ; however, larger cohorts including individuals with a large range 157 of different clinically-defined disease severities will be required to determine if pre-pandemic 158 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227215 doi: medRxiv preprint levels of antibodies are associated with reducing some aspects of severe COVID-19. Additional 159 studies need to be completed to determine if neutralizing antibodies elicited by SARS-CoV-2 160 infections protect against subsequent reinfections with SARS-CoV-2. 161

Further studies also need to be completed to determine how immune history affects de 162 novo immune responses following SARS-CoV-2 infection. We find that individuals infected 163 with SARS-CoV-2 produce antibodies reactive to both the SARS-CoV-2 S protein and OC43 S 164 protein (Figure 3 ). In the case of influenza viruses, sequential infections with antigenically 165 distinct strains can elicit antibodies against conserved epitopes between the strains and it is 166 unclear if these cross-reactive antibodies inhibit de novo immune responses or affect disease 167 CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 10, 2020. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227215 doi: medRxiv preprint

Further information and requests for resources and reagents should be directed to and will be 182 fulfilled by the Lead Contact, Scott E. Hensley (hensley@pennmedicine.upenn.edu). 183 184

All unique reagents generated in this study will be available from the Lead Contact upon 186 reasonable request. 187

188

Data and Code Availability 189

The published article includes all data generated or analyzed during this study. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227215 doi: medRxiv preprint

Serum SARS-CoV-2 neutralizing antibodies were measured as previously described 20 Reciprocal serum dilution antibody titers were log2 transformed for statistical analysis. ELISA 292 antibody titers below the limit of detection (LOD; reciprocal titer <50) were set to a reciprocal 293 titer of 25. Log2 transformed antibody titers were compared with unpaired t-tests and statistical 294 significance was set to p-value <0.05. Linear regressions were also performed using log2 295 transform titers and untransformed data from the other variables. We compared antibody titers in 296 pre-pandemic serum samples from individuals who did and did not have a subsequent PCR-297 confirmed SARS-CoV-2 infection. For these analyses we selected serum sample from 298 individuals with RT-PCR negative results matching sex, age, and race for each SARS-CoV-2 299 PCR-confirmed case (RT-PCR positive) to define controls for our cohort. In instances we did not 300 find matched controls, we randomly selected patients with RT-PCR negative test results. We also 301 compared antibody titers in pre-pandemic serum samples among SARS-CoV-2 PCR-confirmed 302 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. 304 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.06.20227215 doi: medRxiv preprint All the models were adjusted by sex, age, race, and analyses were performed in R 21 . We 305 compared Log2 transformed antibody titers in COVID-19 hospitalized patients at day 0 and day 306 7. We also compared the fold change in titer by day 7. We compared the fold change in OC43 307 titers between patients who survived and patients who died by day 28 of hospitalization. for sample procurement, processing, and logistics. We thank the staff of the PMBB. We thank F. ELISAs were completed to quantify levels of serum antibodies binding to the full length S 341 proteins from 229E, NL63, and OC43 using pre-pandemic serum samples with (n=12) and 342 without (n=51). Unpaired t-tests of log2 transformed antibody titers ****p<0.0001 and 343 **p=0.0027. Horizontal lines indicate geometric mean and error bars represent standard 344 deviation. 345 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

S . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Fold-Change OC43 titer (day 7/day 0)

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 10, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint

The copyright holder for this this version posted November 10, 2020.

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