key: cord-0773213-1bmu7tis authors: Weisberg, S. P.; Connors, T.; Zhu, Y.; Baldwin, M.; Lin, W.-H.; Wontakal, S.; Szabo, P. A.; Wells, S. B.; Dogra, P.; Gray, J. I.; Idzikowski, E.; Bovier, F.; Davis-Porada, J.; Matsumoto, R.; Li Poon, M. M.; Chait, M. P.; Mathieu, C.; Horvat, B.; Decimo, D.; Bitan, Z. C.; La Carpia, F.; Ferrara, S. A.; Mace, E.; Milner, J.; Moscona, A.; Hod, E. A.; Porotto, M.; Farber, D. L. title: Antibody responses to SARS-CoV2 are distinct in children with MIS-C compared to adults with COVID-19 date: 2020-07-14 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2020.07.12.20151068 sha: a1093a75a3734a5e9de029c5e68b6b90ef2aa2db doc_id: 773213 cord_uid: 1bmu7tis Clinical manifestations of COVID-19 caused by the novel coronavirus SARS-CoV-2 are associated with age. While children are largely spared from severe respiratory disease, they can present with a SARS-CoV-2-associated multisystem inflammatory syndrome (MIS-C) similar to Kawasaki's disease. Here, we show distinct antibody (Ab) responses in children with MIS-C compared to adults with severe COVID-19 causing acute respiratory distress syndrome (ARDS), and those who recovered from mild disease. There was a reduced breadth and specificity of anti-SARS-CoV-2-specific antibodies in MIS-C patients compared to the COVID patient groups; MIS-C predominantly generated IgG Abs specific for the Spike (S) protein but not for the nucleocapsid (N) protein, while both COVID-19 cohorts had anti-S IgG, IgM and IgA Abs, as well as anti-N IgG Abs. Moreover, MIS-C patients had reduced neutralizing activity compared to COVID-19 cohorts, indicating a reduced protective serological response. These results suggest a distinct infection course and immune response in children and adults who develop severe disease, with implications for optimizing treatments based on symptom and age. A new clinical manifestation of SARS-CoV-2 infection has been identified in children, designated Multisystem Inflammatory Syndrome in Children (MIS-C), characterized by a systemic inflammatory response with similarities to Kawasaki disease and toxic shock syndrome but lacking respiratory illness that is a hallmark of COVID-19 in adults 1, 2 . The nature of the immune response in MIS-C relative to the more common forms of COVID-19 is not well understood. The generation of virus-specific antibodies which neutralize or block infectivity is the most consistent correlate of protective immunity for multiple infections and vaccines 3, 4 . Antibodies specific for the Spike (S) protein of SARS-Cov-2 which binds the cellular receptor for viral entry, have been detected in actively infected patients and in patients with mild disease who recovered [5] [6] [7] [8] . Anti-S antibodies can exhibit neutralizing activity and are currently being pursued as a therapeutic option for infusion into patients during severe disease and for targeted generation in vaccines 9-11 12 . In this study, we investigated the specificity and functionality of the antibody response and its protective capacity in three patient cohorts seen at New York Presbyterian hospital during the height of the pandemic from March-June, 2020 1, 10, 13, 14 , including patients with mild COVID-19 respiratory disease who recovered and were recruited as convalescent plasma donors (CPD), hospitalized patients with the severest form of respiratory disease (COVID-ARDS), and a cohort of children hospitalized with MIS-C (See Table 1 for clinical characteristics). The COVID-19 patient cohorts represented a broad age range (14-84 years) while MIS-C ages were younger (4-17yrs) ( Table 1) . Co-morbidities were relatively common in adult subjects with COVID-ARDS, but not in MIS-C. Samples were obtained from hospitalized patients within 24-36hrs of being admitted or intubated, which represented a shorter time post-symptom onset in MIS-C compared to COVID ARDS patients, respectively. Both MIS-C and COVID-ARDS subjects exhibited markers of systemic inflammation including highly elevated levels of 4 IL-6 and C-reactive protein (CRP), while ferritin and lactate dehydrogenase (LDH), were significantly increased in COVID-ARDS compared to MIS-C subjects ( Table 1) . None of the MIS-C subjects developed respiratory failure or ARDS, and had reduced overall organ damage when compared to the COVID ARDS group, indicating distinct inflammatory and systemic responses in these two disease manifestations. We initially established that MIS-C and COVID-19 in our patients are associated with a specific immune response to SARS-CoV-2, by testing binding of plasma IgG to recombinant S proteins expressed on the cell surface. Plasma from all patient samples but not pre-pandemic control samples bound SARS-CoV-2 S protein and the common circulating D614G S protein variant 15 but not to S protein from SARS-CoV-1 or MERS coronaviruses (Extended Data, Fig. 1 ). We then quantitated the Ab response in each cohort in terms of specificity and Ab class, including IgM generated initially in a primary response and IgG and IgA classes prominent in serum and secretions, respectively. Anti-S antibodies of the IgM, IgG and IgA classes were significantly elevated in COVID and CPD donors relative to negative control plasma obtained from pre-pandemic samples; COVID-ARDS patients had the highest levels of anti-S Abs for all classes (Fig. 1a-c) . By contrast, anti-S antibodies in patients with MIS-C were predominantly IgG and to a lesser extent IgA-both at similar levels as the CPD donors while anti-S IgM levels in MIS-C were at the same low level as negative control plasma ( Fig. 1a-c, left) . Moreover, the ratio of anti-S IgG to IgM was significantly higher in MIS-C patients compared to the COVID-ARDS and CPD subjects (MIS-C anti-S IgG/IgM ratio 3.35 vs COVID ARDS 1.48, p<0.0001 and vs CPD 1.95, p=0.001) indicating skewed production of anti-S IgG in MIS-C. No significant correlation was found between any of the anti-S Ig isotypes and patient age (Fig. 1a-c, right) . We also assessed the presence of antibodies specific for the SARS-CoV-2 nucleocapsid (N) protein, which complexes with viral RNA, and is involved in viral replication during active infection 16 . The MIS-C group showed significantly lower anti-N IgG levels compared to both the COVID ARDS and CPD groups (Fig. 1d, left) . In addition, a significant correlation of anti-N IgG levels with subject age was observed within the CPD group but not the COVID ARDS and MIS-C groups (Fig. 1d, right) . These results show that MIS-C patients generate mostly anti-S IgG antibodies, while COVID-19 adult patient groups had a broader antibody response in terms of isotype and specificities with COVID ARDS patients exhibiting the highest titers and antibody levels of all patient groups. The ability of antibodies to provide protection is correlated with their neutralizing activity in blocking virus infection. We developed a cell-based pseudovirus assay based on a system previously reported 17, 18 in which multi-cycle infection of RFP-expressing Vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV-2 S protein is measured in the presence of serially diluted plasma samples (see methods). We validated this assay in two ways: First, we compared neutralizing activity of plasma samples tested in the pseudovirus assay to activity measured in live virus microneutralization assay based on inhibition of cytopathic effect 19 , and found a direct correlation in neutralizing activity calculated from the pseudovirus and live virus assay over a wide range of neutralizing activity (r 2 =0.54, slope=0.95, p=0.0044) (Extended Data Fig. 2a) . Second, we compared neutralizing activity of samples with known anti-S IgG positivity by ELISA (n=18) to samples that tested negative (n=30), and found significantly increased neutralizing activity in the ELISA-positive compared to ELISA-negative samples ( Fig. S1 ), demonstrating both specificity and sensitivity of the SARS-CoV-2 pseudovirus assay. We measured SARS-CoV-2 neutralizing activity in the different patient groups using the pseudovirus assay. Overall, the CPD and COVID ARDS plasma samples exhibited greater neutralizing activity compared to plasma from MIS-C patients (Fig. 2a) ; plasma from COVID ARDS patients show the highest neutralizing potency of the three groups across the dilution series (Extended Data Fig. 2b ). Within the MIS-C group, 4/15 (26.7%) patients had neutralizing activity > 2 SD above the mean of 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 14, 2020. . https://doi.org/10.1101/2020.07.12.20151068 doi: medRxiv preprint 6 negative controls compared to 11/19 (57.9%) in the CPD group and 13/14 (92.9%) in the COVID ARDS group. These variations in neutralizing activity between groups did not correlate with patient age (Fig. 2b) . MIS-C patients also maintained the same levels of anti-S IgG and neutralizing activity 2-4 weeks after hospital discharge based on paired analysis of the follow-up compared to the retested primary sample in 10/15 (66.7%) of the patients (Fig. 2c) . These results suggest that the distinct SARS-CoV-2 antibody profile in MIS-C remains stable during recovery. Only a small fraction of antibodies raised against viral antigens will have neutralizing activity against the virus, which correlates with protective capacity 20 . By linear regression, there was significant correlation between the level of anti-S IgG and neutralizing activity within each group, albeit with a lower elevation and y intercept for MIS-C group relative to the COVID-ARDS and CPD groups (Fig. 2d ). Comparing the ratio of plasma neutralizing activity to anti-S IgG level (Neu:IgG) for each sample reveals a significantly higher Neu:IgG for the CPD group compared to the MIS-C and COVID ARDS groups Fig. 2e ). This analysis suggests that the anti-SARS-CoV-2 immune response in the most critically ill patients was less targeted to protection. It remains to be determined whether the distinct serological responses observed in the MIS-C patients is a characteristic of the pediatric immune response to SARS-CoV-2 or specific for this syndrome. Plasma samples from a large cohort of otherwise healthy children who recovered uneventfully from SARS-CoV-2 infection could not be obtained due to restrictions on clinical research needed to slow the spread of the pandemic. Comparison of samples from 3 pediatric COVID, non-MIS-C patients -two of which had pre-existing hematologic or oncologic disease -showed similar anti-S IgG and neutralizing activity to the follow-up samples from recovered MIS-C patients (Table S1 ). Our results show clear quantitative and qualitative differences in the functional SARS-CoV-2specific antibody response in children with MIS-C compared to adults with mild and severe COVID-19. 7 Children with MIS-C showed the least extensive antibody response that was largely limited to IgG anti-S antibodies with the lowest overall level of neutralizing activity, while patients with the most severe form of COVID ARDS had the highest overall levels and breadth of anti-SARS-CoV2 antibodies and the highest overall neutralizing activity. Despite MIS-C patients having the shortest interval among the groups between symptom onset and sample collection, their SARS-CoV-2-specific antibody response is strongly biased towards IgG vs IgM production, suggesting that MIS-C may arise during a late stage after initial infection. SARS-CoV-2 is a novel virus and as such, both adults and children lack adaptive immunologic memory and are experiencing primary exposures. The less robust antibody response in children with MIS-C could result from a less productive infection in children, also supported by the lack of anti-Nspecific antibodies in MIS-C that requires lysis of virally infected cells. Interestingly, we found that among the CPD group, there was an age-dependence in production of anti-N antibodies, indicating that reduced anti-N antibodies may be a feature of infection in younger individuals. While a small cohort of non-MIS-C patients showed a similar Ab response as MIS-C, studies in larger cohorts of healthy children who were exposed to or developed COVID-19 are needed to assess the pediatric immune response to SARS-CoV-2. The presence of SARS-CoV-2-specific T cells in the peripheral blood of recovered and COVID-ARDS adult patients has been recently reported 21, 22 , though the protective capacity of these T cells is unclear. For respiratory viruses, T cells which localize and establish residency in the lung mediate protective immunity, as shown in mouse models [23] [24] [25] . Because younger individuals have increased number of naïve T cells in different sites to respond to new pathogens 26 , it is possible that a robust T cell response efficaciously clears infection in the lung preventing severe respiratory disease in children, and a low level, persistent infection in other sites may build up over time 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 14, 2020. 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 14, 2020. . We including infiltrates on chest radiograph and a PaO2/FiO2 ratio of less than 300, or pediatric criteria equivalent 30, 31 . MIS-C was defined using the Center for Disease Control definition; <21 years of age, fever >38°C for >24 hours, laboratory evidence of inflammation, hospital admission, multisystem involvement, no alternative plausible diagnosis, and positive SARS-Cov-2 serology 32 . Subjects less than 21 years of age recruited in the inpatient setting were assigned to COVID-ARDS vs MIS-C group following review of their charts by specialists in adult and pediatric critical care medicine. Sequential Organ Failure Assessment (SOFA) scores were calculated on all subjects using previously validated adult and pediatric score tools to provide additional clinical insight into subject disease severity 33-35 . This study was approved by the Institutional Review Board at Columbia University Irving Medical Center. Written consent was obtained from CPD subjects. Due to the limitations placed on direct contact with infected subjects and a need to conserve personal protective equipment, verbal informed consent was obtained from surrogates of critically ill COVID-ARDS subjects and verbal parental consent was obtained for MIS-C subjects. 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 14, 2020. . https://doi.org/10.1101/2020.07.12.20151068 doi: medRxiv preprint Blood samples were obtained at time of outpatient donation for CPD subjects, at time of admission for MIS-C subjects, and following diagnosis of ARDS for COVID-ARDS patients. Plasma was isolated from whole blood via centrifugation. Aliquots were frozen at -80˚C prior to analysis. The ectodomain of the SARS-CoV-2 spike trimer 37 was cloned into mammalian expression vector pCAGGS (Addgene, Watertown, MA), with a foldon tag followed by 6xHis tag and Strep tag II at the C-terminal. This expression vector was transiently transfected into HEK293F cells and the spike trimer secreted in the supernatant was purified 3-5 days post transfection by metal affinity chromatography using an Ni-NTA (Qiagen) column. SARS-CoV-2 nucleocapsid protein (N) was cloned into pET28a(+) vector (Millipore-Sigma, Burlington, MA) with an AAALE linker and 6xHis tag at the C-terminal. The NP construct was then used to transform into E. coli BL21 (DE3) pLysS cells and the target protein was 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 14, 2020. . produced and purified from the bacterial lysate by metal affinity chromatography using an Ni-NTA (Qiagen) column, followed by size-exclusion chromatography on a Superdex 200 10/300 GL column. SARS-CoV-2 spike trimer and N were coated on 96 well ELISA plates at 4℃ overnight, and unbound proteins were then removed washing with PBS, following by blocking with PBS/3% non-fat dry milk. Plasma samples were serially diluted in PBST (0.1% Tween-20 in PBS) + 10% bovine calf serum starting with 1:100, and five successive four-fold dilutions into each well of the coated plate which was incubated at 37℃ for 1hr, followed by washing 6 times with PBST. Peroxidase affiniPure goat antihuman IgG (H+L) antibody, anti-human IgM antibody (Jackson Immune Research, New Grove, PA), or anti-human IgA antibody (Thermofisher, Waltham, MA) was subsequently added into each well and incubated for 1hr at 37℃, washed and TMB substrate (Sigma, St. Louis) was added and the reaction was stopped using 1M sulfuric acid. Absorbance was measured at 450 nm and expressed as an optical density, or OD450 value. Identical serial dilutions were performed for all samples with no missing titrations. We adapted a pseudovirus-based neutralization strategy we previously developed to measure inhibition of infection by high biocontainment enveloped viruses in a large number of samples under low-level biocontainment 17, 18 . For this assay, SARS-CoV-2 S protein is pseudotyped onto recombinant vesicular stomatitis virus (VSV) that expresses red fluorescent protein (RFP) but does not express the VSV attachment protein, G (VSV-ΔG-RFP). Initially, VSV-ΔG-RFP pseudotyped with VSV G is used to infect 293T (human kidney epithelial) cells that were co-transfected with full length codon optimized SARS-CoV-2 S-protein (Epoch Life Science, Sugar Land, TX), the viral entry receptor ACE2 (Epoch Life Science, Sugar Land, TX) and green fluorescent protein (GFP). Infected HEK293T cells are then 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 14, 2020. . 12 mixed at a 2 to 1 ratio with Vero (African green monkey kidney) cells which have high endogenous expression of ACE2 38 . The cells are then combined with diluted serum or plasma in 96 well plates. During the assay, infected, S protein-expressing HEK293T cells generate VSV-ΔG-RFP viruses that bear S protein which subsequently infects and drives RFP expression in Vero cells and undergo multiple cycles of entry and budding in the HEK293T cells due to the co-expression of S protein with ACE2. The GFP and RFP signals are measured 24-48hrs after plating (Infinite M1000 PRO microplate reader, Tecan, Männedorf, Switzerland), resulting in robust amplification of the S protein pseudovirus-driven RFP signal between 24-48hr. Inhibition of RFP signal amplification indicates S protein neutralizing activity in patient plasma ( Figure S1 ). Identical, five-fold serial dilutions were performed for all samples and there were no missing titration data points for any of the samples. aliquoted, and stored at -80°C. Virus stock was quantified by limiting dilution plaque assay on Vero E6 cells as described 39, 40 . Two-fold dilutions of plasma in 50µL of Dulbecco`s Modified Eagle Media (DMEM) were incubated with 200 plaque forming unit (pfu) of SARS-CoV-2 in 50µL of DMEM for 30min at 4°C. 100µL of DMEM 4%FBS containing 4x10 4 Vero E6 cells were added on the top of the former mix in order to have final dilution of sera from 1:50 to 1:6400 (4 wells per dilution). Cells were then incubated for 3 days at 37°C, 5% CO2. Cytopathic effect was revealed by crystal violet staining, and scored by an 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 14, 2020. . https://doi.org/10.1101/2020.07.12.20151068 doi: medRxiv preprint 13 observer blinded to the study design and sample identity. Neutralization end point titers were expressed as the value of the last serum dilution that completely inhibited virus-induced cytopathic effect. For quantitation of neutralization titers in the pseudovirus assay, RFP signal driven by the pseudovirus normalized to the GFP signal derived from the SARS-Cov-2 S protein and ACE2 transfected cells was measured at 24 and 48hr; the ratio of normalized RFP at 48 hours (RFP48) to normalized RFP at 24 hours (RFP24) was calculated. This ratio provides a read-out of multicycle infection of the S protein/ACE2 transfected cell monolayer by S protein-bearing pseudoviruses. Heatmaps of this ratio at all titrations for all samples were generated using the Python data visualization library, seaborn 41 . Neutralizing activity for each sample was calculated by taking the sum of the reciprocal of the RFP48/RFP24 ratio at all 6 plasma dilutions for each sample as described 42 , and also by percent All statistical analysis was performed using Prism 8 software (GraphPad, San Diego, CA). Comparisons of clinical data between groups was performed using the Mann-Whitney U test. Comparisons of antibody levels and neutralization activity were performed using one-way Analysis of Variance (ANOVA) and Tukey's multiple comparisons test. Correlation analysis was performed using simple linear regression. 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 14, 2020. . https://doi.org/10.1101/2020.07.12.20151068 doi: medRxiv preprint We wish to express our gratitude to the Medical ICU nurse champions, Cora Garcellano, Tenzin Drukdak, Harriet Avila Raymundo, Lori Wagner, and Ricky Lee, who led the efforts to obtain patient samples for the adult ARDS patients, to Evelyn Hernandez and Lorena Gomez for their roles as clinical coordinators, and to the nurses and clinical staff in the pediatric intensive care unit of Morgan Stanley Children's hospital. We acknowledge the dedication, commitment, and sacrifice of the other nurses, providers, and personnel who helped care for these patients during the COVID-19 crisis. We acknowledge the suffering and loss of our COVID-19 patients and of their families and our community. This work was supported by NIH grant AIAI106697 and AI128949 awarded to D.L.F., NIH grants AI121349, NS091263, NS105699, and AI146980 awarded to M.P., and AI114736 awarded to A,M. S.W. is supported by NIH K08DK122130; T.C. is supported by NIH K23 AI141686. The funders/sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. 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 14, 2020. . Hartman, H., Wang, Y., Schroeder, H.W., Jr. & Cui, X. Absorbance summation: A novel approach for analyzing high-throughput ELISA data in the absence of a standard. PLoS One 13, e0198528 (2018). 43. Pessi 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 14, 2020. 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 14, 2020. . https://doi.org/10.1101/2020.07.12.20151068 doi: medRxiv preprint Figure 2 . Reduced SARS-CoV-2 neutralizing activity associated with MIS-C compared to severe and mild disease independent of age. a, b Neutralizing activity for SARS-CoV-2-specific antibodies was determined using a pseudovirus assay in which multi-cycle replication of SARS-CoV-2 S protein and red fluorescent protein (RFP)-expressing viruses was tested in the presence of 4 serial 1:5 dilutions of patient plasma. (see methods). For each sample, neutralizing activity is calculated based on the inhibition of RFP signal across dilutions. Shown is a heat map (a, left) of the normalized RFP signal across plasma titrations from patients with MIS-C (white bar, n=15); COVID ARDS (red bar, n=13), convalescent plasma donors (CPD, black bar, n=19), and control plasma from pre-pandemic donors (grey bar; Neg, n=8). The sum of the reciprocal values at the dilutions are shown (a, right) compiled from each of the patient and control groups. ±IQR. P value as calculated by one-way ANOVA with Tukey's multiple comparisons test. These values are also plotted against patient age (b) with the best fit line and P values calculated using simple linear regression. c, The neutralizing activity and anti-S IgG levels of MIS-C patients (n=10) during the acute phase of illness and at a follow-up visit 2-4 weeks after hospital discharge. P value as calculated by paired t-test. d, The anti-S IgG absorbance sums were plotted against SARS-CoV-2 neutralizing activity from patients with MIS-C (white circles, n=15, r 2 =0.5); COVID ARDS (red triangles, n=13, , r 2 =0.29) and CPD (black squares, n=19, r 2 =0.29). Best fit lines, r 2 values, P values and Y-intercepts as calculated by simple linear regression. e, The ratio of the neutralizing activity to anti-S IgG level for patients in each group is compiled. ±IQR. P value as calculated by one-way ANOVA with Tukey's multiple comparisons test. 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 14, 2020. Extended data figure 2. a, Plasma neutralizing activity in the pseudovirus assay was correlated with the end point titers in a live virus microneutralization assay based on inhibition of cytopathic effect (see methods). b, Using the results of the pseudovirus neutralization assay, the percent inhibition of S protein mediated multicycle replication was calculated for each patient plasma sample across all dilutions (see methods). Shown (b) are the percent inhibition values plotted against the plasma dilution factors in patients with MIS-C (white circles, n=15); patients with COVID ARDS (red triangles, n=14); convalescent plasma donors (CPD, black squares, n=19), and control plasma from pre-pandemic donors (grey triangles; Neg, n=8). 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 14, 2020. The pseudovirus-based assay was used to assess neutralizing activity against SARS-CoV-2 in sera that tested positive and negative for anti-S protein antibodies by ELISA. For each sample, neutralization activity is calculated based on inhibition of the pseudoviurs-driven RFP signal (see Figure 2 and methods). Shown is a heat map (left) of the normalized RFP signal across plasma titrations from de-identified patient samples that were sent to the clinical lab for ELISA-based SARS-CoV-2 antibody testing and were found to be positive for anti-S IgG by ELISA (black bar, n=18); and negative for anti-S IgG (white bar, n=30). The sum of the reciprocal values at the dilutions are shown (right) compiled from each of the groups. ±IQR. P value as calculated by Student's t-test. 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 14, 2020. . https://doi.org/10.1101/2020.07.12.20151068 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. 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