key: cord-0764970-s7x5czmj authors: Lee, W. T.; Girardin, R. C.; Dupuis, A. P.; Kulas, K. E.; Payne, A. F.; Wong, S. J.; Arinsburg, S.; Nguyen, F. T.; Mendu, D. R.; Firpo-Betancourt, A.; Jhang, J.; Wajnberg, A.; Krammer, F.; Cordon-Cardo, C.; Amler, S.; Montecalvo, M. A.; Hutton, B.; Taylor, J.; McDonough, K. A. title: Neutralizing Antibody Responses in COVID-19 Convalescent Sera date: 2020-07-11 journal: nan DOI: 10.1101/2020.07.10.20150557 sha: 942f3203f2535a91ee837a28ae4bfe529f83e25a doc_id: 764970 cord_uid: s7x5czmj Passive transfer of antibodies from COVID-19 convalescent patients is being used as an experimental treatment for eligible patients with SARS-CoV-2 infections. The United States Food and Drug Administration's (FDA) guidelines for convalescent plasma recommends target antibody titers of 160. We evaluated SARS-CoV-2 neutralizing antibodies in sera from recovered COVID-19 patients using plaque reduction neutralization tests (PRNT) at low (PRNT50) and high (PRNT90) stringency thresholds. We found that neutralizing activity increased with time post symptom onset (PSO), reaching a peak at 31-35 days PSO. At this point, the number of sera having neutralizing titers of at least 160 was ~93% (PRNT50) and ~54% (PRNT90). Sera with high SARS-CoV-2 antibody levels ([≥]960 ELISA titers) showed maximal activity, but not all high titer sera contained neutralizing antibody at FDA recommended levels, particularly at high stringency. These results underscore the value of serum characterization for neutralization activity. 1. Competing interests -Mount Sinai Hospital is in the process of licensing assays 24 to commercial entities based on the assays described here and has filed for 25 patent protection. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.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 11, 2020. CoV-2 receptor binding domain (RBD) and the entire spike protein ectodomain. For 151 most of the sera described in this report, specimens were screened initially at a 1:50 152 dilution using the SARS-CoV-2 RBD as the target antigen and specific IgG was 153 detected. The endpoint titers of the "presumptive screen positive" sera were then 154 determined by ELISA using the whole recombinant spike ectodomain as the target 155 antigen. 156 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 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.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 11, 2020. . https://doi.org/10.1101/2020.07.10.20150557 doi: medRxiv preprint elsewhere [18] . For all specimens included in this study, the MIA measured reactivity to 159 the SARS-CoV-1 nucleoprotein (N) antigen that had been coupled to polystyrene 160 microspheres. Total antigen-specific immunoglobulin was measured using a biotinylated 161 goat anti-human Ig (reactive with IgM, IgA, and IgG), followed by detection with 162 phycoerythrin-conjugated streptavidin. Anti-SARS-CoV-2 N Ab are strongly identified 163 due to extensive amino acid identity between SARS-CoV-1 and SARS-CoV-2 (~90%), 164 and ongoing studies show that the substitution of the SARS-CoV-2 N protein provides (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 11, 2020. . https://doi.org/10.1101/2020.07.10.20150557 doi: medRxiv preprint forming units (PFUs) of SARS-CoV-2, isolate USA-WA1/2020 (BEI Resources, NR-180 52281) and incubated at 37°C in an incubator with 5% CO2 for one hour. 100 ul of 181 virus:serum mixture was added to Vero E6 cells (C1008, ATCC CRL-1586) and 182 adsorption proceeded at 37ºC in an incubator with 5% CO2 for one hour, after which a (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 11, 2020. In the MSH cohort, the titers of SARS-CoV-2 Abs were determined for those specimens 206 that were positive for RBD binding at a greater than 1:50 dilution in the initial screen. Over the three-week course of the study, a higher proportion of sera showed 208 increasingly higher titers, presumably reflecting maturation of the immune response. Supplementary Table 2 shows distribution of serum titers of samples received during 210 the final 9 days of the study, when most individuals would be expected to be > one 211 month post symptom onset (PSO). As indicated, 89.9% of the "screen positive" sera 212 had SARS-CoV-2 titers that exceeded a 320 threshold. We noted that 10% of the Ab 213 positive sera failed to meet a 320 titer threshold for strong Ab responses even three 214 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 11, 2020. . https://doi.org/10.1101/2020.07.10.20150557 doi: medRxiv preprint weeks from initial symptom onset. In sera that were collected from the WCDH cohort of 215 convalescent individuals who were at least 21 days PSO, the MIA showed reactivity for 216 that 83.2% (6 SD) or 90.6% (3 SD) of the specimens. A composite analysis of Ab levels, 217 as indicated by differences in MIA signal intensities is shown in Figure 1 . Our objective in this portion of the study was to identify those sera that met the 226 recommended (160) or minimal (80) titers for convalescent plasma proposed by the 227 FDA at both the 50% (PRNT50) and 90% (PRNT90) neutralization levels. 228 We examined the relationship between ELISA and PRNT titers using sera collected by 229 MSH to determine whether a direct correlation could be made between Ab reactivity in 230 the ELISA and neutralizing activity. Analysis of 159 sera showed that, as expected, 231 there was a general trend toward more neutralization with higher Ab titers (Table 1) . 232 Approximately half (52.9%) of the samples had a ≥160 neutralizing titer PRNT50 level, 233 including 84.1% of sera with an ELISA titer of 2880. However, only 50% of the sera at 234 the highest ELISA Ab titer (2880) had neutralizing titers of 160 or greater when a more 235 stringent (PRNT90) determination for neutralization was used. Figure 2 shows a 236 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 11, 2020. . https://doi.org/10.1101/2020.07.10.20150557 doi: medRxiv preprint graphical representation of these data, except that results are grouped to show the 237 frequencies of specimens that met the minimal FDA recommended 80 and 160 titers for 238 convalescent plasma use ("Medium") in addition to frequencies of specimens that either 239 failed to meet that recommendation ("Low") or exceeded the recommended titers 240 ("High"). In general, samples with high Ab titers by ELISA also had high MIA MFIs, consistent 242 with overall agreement between the two Ab detection assays. Likewise, we found that 243 specimens with higher amounts of Ab detected using the MIA, had higher levels of 244 neutralizing Ab activity (Figure 3 ). Although more overall Ab led to more neutralizing Ab, 245 we did not find a specific MFI value to be an absolute predictor of neutralizing activity. (Table 2) , where ~93% of 256 the sera had ≥ 160 PRNT50 titers, while ~54% of the sera had ≥ 160 titers using the 257 more stringent PRNT90 evaluation. Beyond 35 days, the number of sera which had 258 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 11, 2020. Figure 4) . (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 11, 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 11, 2020. including a smaller study that evaluated serial samples from 12 COVID-19 patients [29] 305 and a larger study using a pseudo-typed virus neutralization assay [28] . A recent study 306 using the MSH ELISA and an optimized and sensitive MN assay recently also found 307 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 11, 2020. . https://doi.org/10.1101/2020.07.10.20150557 doi: medRxiv preprint high IC50 neutralization titers and excellent correlation between ELISA and MN assay 308 [30]. Our results indicate that high level Ab quantitation is a useful, but imperfect, guide 309 to donor suitability, depending on the required level of neutralization. Additional factors, 310 including the specific target antigen used to measure antibody levels, should also be 311 considered. As expected, we found that timing post infection had a major impact on the degree of (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 11, 2020. . https://doi.org/10.1101/2020.07.10.20150557 doi: medRxiv preprint PRNT50 titers [35]. However, the optimal level of neutralizing Abs for plasma therapy is 331 likely to differ from that required for an individual's own immune status, as donor plasma 332 will be diluted as much as ten-fold upon transfer into a recipient and protection from 333 viruses after infection or vaccination is not just limited to neutralizing antibodies [36] . The mechanisms for potential immunity to SARS-CoV-2 have yet to be determined. (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 11, 2020. Symptoms. Graphical representation of the data shown in Figure 1 and 487 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 11, 2020. . https://doi.org/10.1101/2020.07.10.20150557 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 11, 2020. . https://doi.org/10.1101/2020.07.10.20150557 doi: medRxiv preprint Table 1 Serum specimens from COVID-19 convalescent patients that screened Ab positive to SARS-CoV-2 RBD, were titered by ELISA with respect to reactivity to SARS-CoV-2 whole spike protein. Sera with ELISA titers ≥160 were tested for their ability to neutralize SARS-CoV-2 infection of Vero E6 cells. The neutralization titer is the inverse endpoint dilution of sera that could neutralize 50% (top) or 90% (bottom) of viral plaque formation. % Neutralizers at FDA recommended level have a neutralizing titer of 160 or greater. % Neutralizers at FDA minimal level have a neutralizing titer of 80 or greater. Ab positive serum specimens from COVID-19 convalescent patients with onset of symptom and blood collection information were tested. All of the specimens were assessed for either SARS-CoV RBD (Mount Sinai) and SARS-CoV N or SARS-CoV N plus RBD (Mount Sinai, Westchester) reactivity using a MIA. An interactive web-based dashboard to track COVID-19 in 371 real time Clinical course and risk factors for mortality of adult 376 inpatients with COVID-19 in Wuhan, China: a retrospective cohort study COVID-19: What proportion are asymptomatic?