key: cord-275894-puwaty70 authors: Wajnberg, A.; Amanat, F.; Firpo, A.; Altman, D.; Bailey, M.; Mansour, M.; McMahon, M.; Meade, P.; Mendu, D. R.; Muellers, K.; Stadlbauer, D.; Stone, K.; Strohmeier, S.; Aberg, J.; Reich, D.; Krammer, F.; Cordon-Cardo, C. title: SARS-CoV-2 infection induces robust, neutralizing antibody responses that are stable for at least three months date: 2020-07-17 journal: nan DOI: 10.1101/2020.07.14.20151126 sha: doc_id: 275894 cord_uid: puwaty70 SARS-CoV-2 has caused a global pandemic with millions infected and numerous fatalities. Questions regarding the robustness, functionality and longevity of the antibody response to the virus remain unanswered. Here we report that the vast majority of infected individuals with mild-to-moderate COVID-19 experience robust IgG antibody responses against the viral spike protein, based on a dataset of 19,860 individuals screened at Mount Sinai Health System in New York City. We also show that titers are stable for at least a period approximating three months, and that anti-spike binding titers significantly correlate with neutralization of authentic SARS-CoV-2. Our data suggests that more than 90% of seroconverters make detectible neutralizing antibody responses and that these titers are stable for at least the near-term future. titer of 1:80, 943 (4.77%) of 1:160, 4391 (22.23%) of 1:320, 6272 (31.75%) of 1:960 and 7641 92 (38.68%) of 1:2880 (Figure 1) . Therefore, the vast majority of positive individuals have moderate 93 to high titers of anti-spike antibodies. Of course, the argument could be made that we could be 94 missing a number of individuals that had been infected with SARS-CoV-2 and did not produce 95 antibodies, since many individuals included in our data set had never been tested by a nucleic 96 acid amplification test (NAAT) for the virus. An earlier analysis performed with a smaller subset 97 of 568 PCR-confirmed individuals using the same ELISA showed that >99% of them developed 98 an anti-spike antibody response (9). In a later dataset of 2,347 patients who self-reported positive 99 PCR, 95% of them had positive antibody titers, indicating we are not missing large numbers of 100 patients and confirming our prior sensitivity findings. We therefore report here that the rate of 101 individuals who do not seroconvert after SARS-CoV-2 infection is low, although such individuals 102 may exist, and the majority of responders mount titers of 1:320 or higher. 103 Binding antibody titers tell us how robust the immune response to a certain virus or antigen is. In order to determine if antibodies induced against the spike protein exert neutralizing activity, we 111 performed a well-established, quantitative microneutralization assay (18) based on authentic 112 SARS-CoV-2 with 120 samples of known ELISA titers ranging from 'negative' to 1:2880. 113 Neutralization titers significantly correlated (Spearman r= 0.87, p<0.0001) with spike-binding titers 114 (Figure 2A) . While there was some variability, sera with 1:320 ELISA titers had a geometric mean 115 50% inhibitory dilution (ID50) of approximately 1:30, for the 1:960 titer, it was approximately 1:75 116 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 17, 2020. . https://doi.org/10.1101/2020.07.14.20151126 doi: medRxiv preprint and for the 1:2880 titer it was approximately 1:550. Considering any neutralizing activity above 117 background, approximately 50% of sera in the1:80-1:160 titer range had neutralizing activity, 90% 118 in the 1:320 range had neutralizing activity, and all sera in the 1:960-1:2880 range had neutralizing 119 activity ( Figure 2B) . This is encouraging, and further validates our use of antibody titers to refer 120 for plasma donation and for further investigation into protective effects at the various titer levels. 121 Another important question is longevity of the antibody response to the spike. To assess the 122 medium-range stability of serum antibody titers against the spike protein, we recalled 121 plasma 123 donors at a variety of titer levels that had initially been screened at approximately day 30 post 124 symptom onset. The mean interval between the initial titer measurement and the second was 52 125 days (range 33-67 days), setting the second time point at a mean of 82 days post symptom onset 126 (range 52-104 days). When we compared overall titers we saw a slight drop from a geometric 127 mean titer (GMT) of 670 to a GMT of 642 ( Figure 3A ). In the higher titer range of 1:2880 and 128 1:960, we also observed a slight drop in titer between the two time points (Figure 3B and C) . 129 Surprisingly, but in agreement with earlier observations from our group that seroconversion in 130 mild COVID-19 cases might take longer time to mount (9), we saw an increase in individuals who 131 had an initial titer of 1:320, 1:160 or 1:80 ( Figure 3D-F) . The exception was one individual that 132 dropped from a 1:80 titer to being negative. The initial serum antibody titer was likely produced 133 by plasmablasts, and plasmablast-produced antibody peaks 2-3 weeks post symptom onset. antibodies measured in their paper were targeting the NP plus a single linear spike epitope. Much 140 more in agreement with our data, the same paper also reports relatively stable (slightly declining) 141 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 17, 2020. . https://doi.org/10.1101/2020.07.14.20151126 doi: medRxiv preprint neutralizing antibody titers. The stability of the antibody response over time might therefore also 142 depend on the target antigen. The titers we measure here do correlate with neutralization as 143 discussed above. significantly. For some viruses/vaccines, the kinetics of the antibody response is also known, 151 allowing for an accurate prediction of how long protection will last (22). 152 long. We know from work with common human coronaviruses that neutralizing antibodies are 154 induced and these antibodies can last for years and provide protection from re-infection or 155 attenuate disease, even if individuals get re-infected (23). Furthermore, we now know from non-156 human primate models that infection with SARS-CoV-2 does protect from re-infection for at least 157 some time (24, 25). This protection is, in some cases, more pronounced in the lower respiratory 158 tract than in the upper respiratory tract (25). We also know that transferring serum, for example 159 from infected hamsters into naïve hamsters, reduces virus replication significantly when the naïve responses. Antibody binding titers to the spike protein correlate significantly with neutralization 166 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 17, 2020. . https://doi.org/10.1101/2020.07.14.20151126 doi: medRxiv preprint with authentic SARS-CoV-2 virus, and the vast majority of individuals with antibody titers of 1:320 167 or higher show neutralizing activity in their serum. Consistent with data for human coronaviruses, 168 SARS-CoV-1 and Middle Eastern respiratory syndrome-CoV (23), we also find stable antibody 169 titers over a period of at least 3 months, and we plan to follow this cohort over longer intervals of 170 time. While this cannot provide conclusive evidence that these antibody responses protect from 171 re-infection, we believe it is very likely that they will decrease the odds ratio of getting re-infected, (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 17, 2020. . https://doi.org/10.1101/2020.07.14.20151126 doi: medRxiv preprint contract 75N93019C00051 (FK), and the generous support of the JPB foundation, the Open 247 Philanthropy Project (#2020-215611) and other philanthropic donations. 248 249 Mount Sinai has licensed serological assays to commercial entities and has filed for patent 251 protection for serological assays. (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 17, 2020. . https://doi.org/10.1101/2020.07.14.20151126 doi: medRxiv preprint (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 17, 2020. . https://doi.org/10.1101/2020.07.14.20151126 doi: medRxiv preprint (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 17, 2020. . https://doi.org/10.1101/2020.07.14.20151126 doi: medRxiv preprint 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 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 17, 2020. . https://doi.org/10.1101/2020.07.14.20151126 doi: medRxiv preprint Supplemental CoV-2, Roche Diagnostics, Indiana). Due to the lack of PCR testing in the New York area prior 330 to mid-March, 2020, we also included people for antibody testing and screening if they were 331 symptomatic with suspected SARS-CoV-2 symptoms after February 1, 2020, if they had a high 332 risk exposure to someone with a positive SARS-CoV-2 PCR test, or were healthcare workers. 333 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 17, 2020. . https://doi.org/10.1101/2020.07.14.20151126 doi: medRxiv preprint Additionally, only participants who were asymptomatic at time of survey were eligible to come in 334 for antibody testing. Respondents self-reported date of symptom onset, date of positive SARS-335 CoV-2 test (if applicable), and last date of symptoms. 336 During the first week of serum antibody testing, participants were brought in ten or more days 337 after they had a confirmed/suspected diagnosis and had been asymptomatic for at least three 338 days. In week 2, as we identified more potential donors and learned more about our antibody 339 assay, we extended our timeline to 14 days after symptoms onset, with at least three days 340 asymptomatic. We saw quickly that this was a little early for serum conversion, and by week 3, 341 we included participants 21 days or more after symptom onset, who had been completely 342 asymptomatic for at least 14 days. We referred donors for plasma donation to the New York Blood 343 Center if their antibodies were ≥1:320. All interested participants with antibody titers >1:320 and 344 negative SARS-CoV-2 PCR swabs were screened by the New York Blood Center using standard 345 criteria for plasma donation and included as donors in our convalescent plasma study if eligible 346 as per CFR Title 21. Studies were reviewed and approved by our institutional review board. 347 It is important to note that as we identified greater numbers of potential donors and built capacity 348 for testing in our clinical lab, we offered testing to wider groups including family and friends of 349 indivduals with positive PCR or antibodies, essential workers, patients in our health system, 350 members of our community, and finally to all employees of the Mount Sinai Health System. The Mount Sinai Hospital (MSH) enzyme linked immunosorbent assay (ELISA) is an orthogonal 358 immune assay specific for IgG anti SARS-CoV-2 spike protein in serum or plasma and measures 359 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 17, 2020. . https://doi.org/10.1101/2020.07.14.20151126 doi: medRxiv preprint onto the cells. The cells were incubated for an hour at 37°C. After an hour, the virus-serum mixture 386 was removed and 100 uls of 1X MEM and 100 uls of each respective serum dilution was added 387 to the cells. The cells were kept in the 37°C incubator for 2 days. After 48 hours, the media from 388 the cells was removed and 150 uls of 10% formaldehyde (Polysciences) was added to the cells 389 to inactivate the virus for 24 hours. The next day, cells were permeabilized and stained using an 390 anti-NP antibody. Percent inhibition of virus was calculated for each dilution. This protocol has 391 been earlier published in much greater detail (18). 392 393 Correlation analysis was performed using Spearman's rank test. A paired t-test was used to 395 compare longitudinal titers. A p>0.05 was considered significant. Analysis was performed in 396 GraphPad Prism. 397 398 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 17, 2020. . https://doi.org/10.1101/2020.07.14.20151126 doi: medRxiv preprint Serology assays to manage COVID-19 High neutralizing antibody titer in intensive care unit patients with COVID-19 Antibody responses to SARS-CoV-2 in patients with COVID-19 Vaccine efficacy in senescent mice challenged with recombinant SARS-CoV 185 bearing epidemic and zoonotic spike variants Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Functional assessment of cell entry and receptor usage for SARS-189 CoV-2 and other lineage B betacoronaviruses SARS-CoV-2 Vaccines: Status Report A serological assay to detect SARS-CoV-2 seroconversion in humans Humoral immune response and prolonged PCR positivity in a cohort of 1343 194 SARS-CoV 2 patients in the New York City region. medRxiv Seroprevalence of SARS-CoV-2 among children visiting a hospital during the 196 initial Seattle outbreak. medRxiv Asymptomatic Seroconversion of Immunoglobulins to SARS-CoV-2 in a 198 Pediatric Dialysis Unit Seroconversion of a city: Longitudinal monitoring of SARS-CoV-2 200 seroprevalence in New York City. medRxiv Antigen Production, and Test Setup Convalescent plasma treatment of severe COVID-19: A matched control study. 204 medRxiv The Role of Fc Gamma Receptors in Broad Protection against Influenza 206 Viruses A Role for Fc Function in Therapeutic Monoclonal Antibody-Mediated 208 Protection against Ebola Virus Antibody-mediated protection 210 against Ebola virus An In Vitro Microneutralization Assay for SARS-CoV-2 Serology and Drug 212 Screening Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections Meeting report and review: 216 Immunological assays and correlates of protection for next-generation influenza vaccines. 217 Influenza Other Respir Viruses Correlates of protection induced by vaccination Inactivated hepatitis A vaccine-induced antibodies: follow-up and 221 estimates of long-term persistence A systematic review of antibody mediated immunity to coronaviruses