key: cord-0713219-bbl7g1da authors: Annen, K.; Morrison, T. E.; DomBourian, M. G.; McCarthy, M. K.; Huey, L.; Merkel, P. A.; Andersen, G.; Schwartz, E.; Knight, V. title: Presence and short-term persistence of SARS-CoV-2 neutralizing antibodies in COVID-19 convalescent plasma donors. date: 2020-09-03 journal: nan DOI: 10.1101/2020.09.01.20185942 sha: 89d14aa9f7645d98e87132b277cc3c3ad0684fbd doc_id: 713219 cord_uid: bbl7g1da In March 2020, the FDA approved the use of COVID-19 convalescent plasma (CCP) as an investigational new drug for treatment of COVID-19. Since then, collection of CCP from COVID-19 recovered patients has been implemented in several donor centers across the country. Childrens Hospital Colorado rapidly put into practice a CCP collection protocol, necessitating the development and implementation of assays to evaluate SARS-CoV-2 antibodies in CCP units. We evaluated 87 separate units of CCP collected from 36 donors over two to four sequential donations using both antigen-binding assays for SARS-CoV-2 nucleoprotein and spike antigens, and a live virus focus reduction neutralization test (FRNT50). Our data shows that the majority of donors (83 percent) had a FRNT50 titer of 1/80 or greater, and 61 percent had a titer greater than or equal to 1/160, which meet the FDA criteria for acceptable CCP units. Additionally, our data indicates that analysis of antibodies to a single SARS-CoV-2 antigen is likely to miss a percentage of seroconverters. These individuals, however, tend to have neutralizing antibody titers of less than 1/80. Of note, there was considerable variability in the short term, sustained antibody response, measured by neutralizing antibody titers, among our donor population. including CHCO, allowed repeat convalescent plasma donors as frequently as every 7 40 days. However, the impact on the donors anti-SARS-CoV-2 antibody levels with this 41 frequency of donation, or any frequency, is unknown, as is the pattern of decline or 42 retention of antibodies to SARS-CoV-2. Here, we compare two ELISA assays, both currently implemented in clinical laboratories 58 for clinical diagnostics and for screening of CCP, with a SARS-CoV-2 virus 59 neutralization assay in our CCP donor population. As CCP donors are currently 60 permitted to return for relatively frequent plasma donations, we have additionally 61 The Euroimmun ELISA assay utilizes the S1 domain, which includes the receptor 85 binding domain (RBD) of the SARS-CoV-2 spike protein (7). For this assay, a kit-86 specific calibrator, positive and negative controls and samples, were diluted 1:101 with 87 the kit-specific dilution buffer and added to pre-coated wells. Following a 1 h incubation 88 at 37°C, plates were washed three times with kit-specific wash buffer. Anti-human IgG-89 HRP conjugated detection antibody was added and plates were incubated for 30 min at 90 37°C followed by three washes. TMB was added and absorbance read at 450 nm within 91 10 min of halting the reaction. 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) The copyright holder for this preprint this version posted September 3, 2020. For OD450 values and S1-RBD 128 ratios, the mean and 95% confidence intervals were calculated using GraphPad Prism's 129 statistical analysis package. Significant differences between groups were calculated 130 . 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) The copyright holder for this preprint this version posted September 3, 2020. intervals between sequential plasma donations ranged from 7 to 24 days. Comparison of N and S1-RBD antibody detection with virus neutralizing activity. 141 To determine if qualitative IgG antibody detection by ELISA, whether against the N or 142 the S1-RBD antigen, correlated with virus neutralizing activity, samples were analyzed 143 for the presence of anti-N IgG antibodies using the Epitope Diagnostics ELISA, anti-S1-144 RBD IgG antibodies using the Euroimmun ELISA, and neutralizing activity using a live 145 virus focus reduction assay. Samples with a neutralizing antibody titer of 1:80 or greater 146 had a positive or borderline-positive result for both N and S1-RBD IgG antibodies, with 147 the exception of sample 018-D (neutralizing titer of 1:85), which was positive for N and 148 negative for S1-RBD, and sample 023-D (neutralizing titer of 1:91) which was negative 149 for N and was not analyzed for S1-RBD antibodies (Table 2 ). More variability between 150 anti-N and anti-S1-RBD IgG ELISA results was noted for samples with lower 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 September 3, 2020. . https://doi.org/10.1101/2020.09.01.20185942 doi: medRxiv preprint were either negative or borderline-positive for anti-S1-RBD IgG, and one was positive. Of note, donor 019-D (samples 1 and 2) remained persistently negative for anti-N IgG, 155 had a marginal increase in anti-S1-RBD IgG on the second CCP donation, and had very 156 low FRNT50 titers. To determine whether OD450 values for anti-N IgG antibodies or the ratio for anti-S1-158 RBD IgG antibodies was predictive of neutralizing antibody titers, we compared the . 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. The copyright holder for this preprint this version posted September 3, 2020. . https://doi.org/10.1101/2020.09.01.20185942 doi: medRxiv preprint that the numerical values obtained are not in the linear range, and, therefore, these 177 results affect correlations with neutralizing antibody titers as the FRNT50 is a 178 quantitative assay. Nevertheless, an anti-N IgG OD450 of 0.4 and above correlated well 179 with a neutralizing titer of ≥1:80 in 90% of the samples, and an anti-S1-RBD IgG ratio of Figure 3A ). Neutralizing antibody titers were, in general, between 1/80 and 1/500 for the majority of 194 samples tested (52%). Approximately 20% had titers greater than 1/500 and very few (7 195 of the 87 tested) had neutralizing antibody titers of >1:1000 ( Figure 3A ). Sustainability of the antibody response. Because analysis of N and S1-RBD IgG 197 antibodies by single dilution ELISA is qualitative at best, we chose to analyze the 198 . 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. The copyright holder for this preprint this version posted September 3, 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. The copyright holder for this preprint this version posted September 3, 2020. Such information is typically generated from biological assays such as the FRNT50, 280 described in our study, that examines the ability of CCP to neutralize viral replication in 281 permissive cell lines. We found that close to 80% of the population of donor samples we 282 tested had a neutralizing antibody titer of ≥1:80, and 60% ≥1:160, both of which meet 283 the FDA's criteria for eligible CCP units. The longevity of the antibody response is a critical part of potential protection against re-285 infection, although such information continues to be gathered. Analysis of the longevity 286 of the antibody response to SARS1 indicates that anti-SARS1 antibodies were 287 detectable two to three years following infection in one study (25), and, in a second 288 study, detectable for close to a year following infection but declined over the course 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. The copyright holder for this preprint this version posted September 3, 2020. Although the Signal-to-Cutoff ratio was provided for these reports, a correlation to 304 neutralizing antibody titer thresholds in the prior FDA requirement of >1:80 titer with 305 preference for ≥1:160 titer was not provided. This incites the question of the adequacy 306 of the minimum threshold for CCP treatment and may impact future collections if the 307 threshold is increased for therapeutic efficacy. Our data suggests that a majority of donors (67%) had a neutralizing antibody 309 response that was either sustained or increased over the short period of approximately 310 three weeks to two months following a positive SARS-CoV-2 PCR result, and a smaller 311 percentage (33%) showed a decrease in neutralizing antibody titer over sequential 312 donations. Notably, repeat donations did not appear to affect antibody titer for the 313 . 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. The copyright holder for this preprint this version posted September 3, 2020. . https://doi.org/10.1101/2020.09.01.20185942 doi: medRxiv preprint majority (67%) of donors. A drawback of our dataset is that for the majority of donors, 314 we were able to test only two time points (7-24 days apart) following a positive SARS- CoV-2 PCR test, making it challenging to comment on longer term sustainability of the 316 response. The longevity of the SARS-CoV-2 antibody response and the level of 317 protection it will provide for reinfection is yet to be determined. to higher titers). Eighty-five samples were compared for correlation among anti-N, anti-346 S1-RBD and neutralizing antibody titers. Two samples with the highest FRNT50 titers 347 (red circles), also had the highest S1-RBD ratios and moderately high levels of anti-N1. 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. The copyright holder for this preprint this version posted September 3, 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. The copyright holder for this preprint this version posted September 3, 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) The copyright holder for this preprint this version posted September 3, 2020. . https://doi.org/10.1101/2020.09.01.20185942 doi: medRxiv preprint . 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) The copyright holder for this preprint this version posted September 3, 2020. . https://doi.org/10.1101/2020.09.01.20185942 doi: medRxiv preprint Table 3 : Correlation of >1:80 or <1:80 neutralizing antibody titers with anti-N or anti-S1-RBD level of positivity. Anti-S1-RBD IgG <3.0 Anti-S1-RBD IgG >3.0 Total anti-N IgG >0.4 Anti-N IgG <0.4 3 (4.2%) 4 (5.6%) Anti-N IgG > 0.4 10 (14.1%) 54 (76.1%) 90.2% Total anti-S-RBD IgG > 3.0 81.7% Neutralizing titer <1:80 Anti-S1-RBD IgG <3.0 Anti-S1-RBD IgG >3.0 Total anti-N IgG >0.4 Anti-N IgG <0.4 10 (71.4%) 0 (0%) Anti-N IgG > 0.4 3 (21.4%) 1 (7%) 28.4% Total anti-S-RBD IgG > 3.0 7% Effectiveness of convalescent 404 plasma therapy in severe COVID-19 patients COVID-19) Patients with Convalescent 408 Plasma Treatment of 5 Critically Ill 410 Patients With COVID-19 With Convalescent Plasma Treatment with 412 convalescent plasma for COVID-19 patients in Wuhan Plasma Therapy on Viral Shedding and Survival in Patients With Coronavirus Disease 415 2019 Effect of Convalescent Plasma on Mortality among Hospitalized Patients with COVID-418 19: Initial Three-Month Experience. medRxiv Neutralizing Antibody Responses in COVID-19 Convalescent Sera *ND = not done. Anti-S1-RBD FRNT50 Anti-N Anti-S1-RBD FRNT50 Anti-N Anti-S1-RBD FRNT50