key: cord-0856557-p7dgvpxh authors: Gallais, F.; Gantner, P.; Bruel, T.; Velay, A.; Planas, D.; Wendling, M.-J.; Bayer, S.; Solis, M.; Laugel, E.; Reix, N.; Schneider, A.; Glady, L.; Panaget, B.; Collongues, N.; Partisani, M.; Lessinger, J.-M.; Fontanet, A.; Rey, D.; Hansmann, Y.; Kling-Pillitteri, L.; Schwartz, O.; De Seze, J.; Meyer, N.; Gonzalez, M.; Schmidt-Mutter, C.; Fafi-Kremer, S. title: Evolution of human antibody responses up to one year after SARS-CoV-2 infection date: 2021-05-14 journal: nan DOI: 10.1101/2021.05.07.21256823 sha: bd2b412b8020808e258cd6080d1858ccc6f9a77a doc_id: 856557 cord_uid: p7dgvpxh Assessment of the kinetics of SARS-CoV-2 antibodies is essential to predict protection against reinfection and durability of vaccine protection. Here, we longitudinally measured Spike (S) and Nucleocapsid (N)-specific antibodies in 1,309 healthcare workers (HCW) including 393 convalescent COVID-19 and 916 COVID-19 negative HCW up to 405 days. From M1 to M7-9 after infection, SARS-CoV-2 antibodies decreased moderately in convalescent HCW in a biphasic model, with men showing a slower decay of anti-N (p=0.02), and a faster decay of anti-S (p=0.0008) than women. At M11-13, anti-N antibodies dramatically decreased (half-life: 210 days) while anti-S stabilized (half-life: 630 days) at a median of 2.41 log Arbitrary Units (AU)/mL (Interquartile Range (IQR): 2.11 -2.75). One case of reinfection was recorded in convalescent HCW (0.47 per 100 person-years) versus 50 in COVID-19 negative HCW (10.11 per 100 person-years). Correlation with live-virus neutralization assay revealed that variants D614G and B.1.1.7, but not B.1.351, were sensitive to anti-S antibodies at 2.3 log AU/mL, while IgG [≥] 3 log AU/mL neutralized all three variants. After SARS-CoV-2 vaccination, anti-S levels reached 4 logs regardless of pre-vaccination IgG levels, type of vaccine, and number of doses. Our study demonstrates a long-term persistence of anti-S IgG antibodies that may protect against reinfection. By significantly increasing cross-neutralizing antibody titers, a single-dose vaccination strengthens protection against escape mutants. Since the beginning of the pandemic, hypothesis of waning humoral immunity in COVID-19 convalescent patients has raised many concerns about reliability of population-based seroprevalence studies and more critically about long-term antibody protection against reinfection and by extension the durability of vaccine protection. COVID-19 leads to the development of protective neutralizing antibodies in the vast majority of cases [1] [2] [3] [4] . Several reports suggested a rapid decline of SARS-CoV-2 antibodies as early as 3 months after infection 3, 5 , while others reported persistence of antibody responses up to five months 4, 6 . A recent rigorous study investigating T and B cell responses in convalescent COVID-19 reported that substantial immune memory is generated after COVID-19, and 95% of subjects retained immune memory at ≈ 6 months after infection 7 . Furthermore, the presence of SARS-CoV-2 anti-spike (S) and anti-nucleocapsid (N) IgG antibodies were associated with a reduced risk of SARS-CoV-2 reinfection within 6 months after initial infection 8 Here, using validated serological assays 13-15 on a large cohort of healthcare workers (HCW) recovered from a mild COVID-19, we first described the dynamics of SARS-CoV-2 humoral response up to one year after COVID-19 and analyzed the incidence of reinfection within this period. Second, we used the S-Fuse live-virus neutralization assay 16 , to assess the sensitivity of infectious SARS-CoV-2 variants to anti-S antibodies before and after vaccination several months after primary infection. . 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. against SARS-CoV-2 before M11-13 sampling. Only one asymptomatic reinfection was reported after nine months in this cohort. Conversely, among the 916 COVID-19 negative HCW, 50 (5.5%) reported a symptomatic SARS-CoV-2 infection (8 before M3-6, 29 before M7-9 and 13 before M11-13) confirmed by a positive RT-PCR test and by seroconversion in 56% and 100% of them, respectively. We first sought to analyze the dynamics of SARS-CoV-2 humoral responses and its determinants in the aftermath of COVID-19 (e.g. natural history after primary infection in the absence of vaccination). Seropositivity rates differ widely depending on: (1) isotypes (IgM or IgG), (2) antibody targets (N or S), Approximately half of individuals (51.0% and 58.1%, respectively) had still detectable antibodies at . 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) anti-S IgG titers at M7-9 and on decay speed between M3-6 and M7-9 by univariate (not shown) and multivariate analyses. No significant difference of anti-S IgG titers was found in univariate analysis according to gender. However, antibody titers declined faster in men in univariate analysis from M3-6 to M7-9. By multivariate analysis ( Figure 2D ), anti-S IgG titers also decayed faster in men than in women with an acceleration of antibody titers decrease of -0.033 log AU/mL per month (95% confidence interval (95%CI, -0.053 to -0.014; p=0.0008) in the first condition. Another factor significantly associated with faster decay was the rhesus-negative (Rh-) status, impacting the decay by a factor of -0.021 log AU/mL per month (95%CI, -0.002 to -0.040; p=0.0008). Notably, no significant effect of age, BMI, blood group, DSO or initial Ct values on anti-S titer slope was observed ( Figure 2D ). Regarding ELISA anti-N IgG, a significant decay of ratios was observed between the four study visits ( Figure 2E ). Interestingly, a triphasic kinetic dynamics of anti-N IgG index over time was observed with a first steep decay between M1 and M3-6 (median, -0.26 optical density (OD) ratio Sample/Cut-Off . 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 May 14, 2021. ; https://doi.org/10.1101/2021.05.07.21256823 doi: medRxiv preprint (S/CO) per month), then a slower decay up to M7-9 (-0.02) before a second drop to M11-13 (-0.07; all p<0.0001) ( Figure 2F ). Thus, the t1/2 of each phase was of 60, 690 and 210 days, respectively. This pattern differed from those of anti-S IgG titers. Univariate and multivariate analyses were conducted for anti-N index similarly to anti-S titers in order to identify potential predictor factors of anti-N IgG index dynamics. Higher antibody index were found at M7-9 in men in univariate analysis compared to women. Moreover, a slower decay from M3-6 to M7-9 was revealed by multivariate analysis in men (0.046 OD ratio S/CO per month; 95%CI 0.007-0.087; p=0.02) and in older participants (0.017 per 10year age; 95%CI 0.002-0.032; p=0.03) ( Figure 2G ). Thus, male participants displayed a faster decay of anti-S antibodies and conversely a slower decay of anti-N antibodies. . 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. To assess whether SARS-CoV-2 variants are sensitive to anti-S antibodies that persist at M11-13 with or without prior vaccination, sera collected at M11-13 from 28 convalescent COVID-19 (13 vaccinated and 15 unvaccinated) were analyzed with the S-Fuse live-virus neutralization assay 16 ( Figure 5A ). The 13 vaccinated HCW received a single dose, including 8 with ChAdOx1 nCoV-19 vaccine (AstraZeneca), . 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. Figure 5D ). Altogether, our findings suggest that former COVID-19 positive individuals benefit from a single-dose vaccine and efficiently neutralize current SARS-CoV-2 variants. The duration and effectiveness of adaptive immunity directed against SARS-CoV-2 after primary infection are key questions to understand the coronavirus disease 2019 (COVID-19) pandemic. The present study involving a large cohort of HCW followed prospectively over one year provides, for the first time, crucial information on persistence of circulating SARS-CoV-2 antibodies after mild COVID-19. We demonstrate that: i) anti-SARS-CoV-2 antibody titers evolve differently in men and women; ii) anti-S IgG stabilize at a median titer of 2.41 log AU/mL (IQR: 2.11 -2.75) one year after symptom onset . 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. Unlike anti-S antibody titers which stabilize over time, we observed a steep decay of anti-N IgG titers after seven to nine months post-infection, with only less than 20% seropositive after one year. Previous studies with a shorter monitoring period after infection found discrepant results regarding anti-N IgG persistence, depending on commercial assays used. One study described a sustained humoral response up to ten months after infection 18 whereas an other in reported a significant decrease early after infection in line with our findings 19 . These differences could be explained by increased avidity that compensates antibody loss or by changes in recognized epitopes over time 18 . Overall, our study show that serological assays targeting nucleocapsid should not be preferred for seroprevalence studies, even if they have the advantage to differentiate between natural infection and post-vaccine immunity. We evaluated several host factors as potential predictors of antibody titers and of their kinetics up to seven to nine months after primary infection. While no differences in SARS-CoV-2 IgG titers were observed, their kinetic was influenced by gender and rhesus. Notably, men displayed a significantly faster decay of anti-S IgG and conversely a significantly slower decrease of anti-N IgG titers between . 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 May 14, 2021. ; https://doi.org/10.1101/2021.05.07.21256823 doi: medRxiv preprint M3-6 and M7-9 after infection, independently of age and of titers measured at M3-6. Sex difference in SARS-CoV-2 immune response was previously described early after infection. Takahashi and colleagues reported that female patients had more robust T cell activation than male patients in the early phase of SARS-CoV-2 infection 20 . Other studies reported a higher peak of anti-S antibody titers in men early after infection followed by a steeper decay compared to females 7,21-23 . The early greater humoral response in convalescent men have been linked to the higher risk of severe disease in this population and to prolonged virus shedding 24,25 . However, this sex difference was also observed independently of age, severity of symptoms, or duration of symptoms 7, 21 . We showed that differences in antibody kinetics depending on gender were still observed later than six months, independently of case severity since only mild and few moderate and asymptomatic cases were monitored in our study. The sex differences in immune responses may be multifactorial, notably based on sex steroids concentrations, on transcriptional factors and on incomplete inactivation of immunoregulatory genes on the second X chromosome in females 26, 27 . Previous studies reported a relationship between the ABO and rhesus blood groups and the COVID-19 susceptibility, suggesting that type O blood and rhesus-negative status may protect against severe COVID-19 28,29 . In our study, Rh-status was associated with faster decay of anti-S IgG titers over time, while no association was observed with ABO blood groups. Although our study provides crucial data on the natural history of mild COVID-19, it is important to note that there are some limitations. Neutralization experiments were performed on a small subset of the cohort due to insufficient volume of remaining sera. However, the strong correlation between CMIA IgG levels and neutralizing titers observed in this study and reported by the manufacturer and by other studies 13, 30, 31 , allows an extrapolation of the results to the entire cohort. Assessment of reinfection was based on participant reports during visits, as no RT-PCR surveillance was planned in the study. Therefore, it cannot be excluded that the COVID-19 positive participants had unnoticed asymptomatic reinfection during follow-up. However, no COVID-19 positive HCW except the case of reinfection had a significant increase of both anti-S and anti-N levels during follow-up. Another . 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 May 14, 2021. ; https://doi.org/10.1101/2021.05.07.21256823 doi: medRxiv preprint limitation is the unbalanced sex distribution with predominance of women. This sex ratio reflects the sex distribution of the healthcare workers in our hospital. Nevertheless, the sex difference in immune response was observed by using univariate and multivariate analysis. Furthermore, we were not able to investigate the kinetic of memory B cells because of the lack of adequate samples. Finally, our results were obtained in participants with a median age of 39 years (IQR 30-51), hence we cannot exclude that older individuals may experience different evolution of their humoral response overtime. Altogether, our data demonstrate a long-term persistence of anti-S IgG titers that may protect convalescent COVID-19 against reinfection by variants D614G and B.1.1.7. By increasing the levels of cross-neutralizing antibodies, SARS-CoV-2 vaccine may strengthen their protection especially against variants harboring antibody escape mutations like B1.351. Future work will help determining whether vaccine-induced antibodies evolve in the same manner and whether their kinetic is different between men and women. We characterized SARS-CoV-2 antibody persistence in COVID-19 healthcare workers (HCW) from Strasbourg University Hospitals, France up to 13 months after infection. Participants were recruited as follows ( Figure 1 ): among 1,496 HCW initially screened by SARS-CoV-2 serology between 6 th April and 7 th May 2020 in our institution, all participants with a COVID-19 history proven either by serology at screening or by a previous RT-PCR were recruited and followed at M1, M3-6, M7-9 and M11-13. In parallel, participants displaying negative serology without a history of positive RT-PCR for SARS-CoV-2 were recruited to evaluate the incidence of infection. Participants completed a questionnaire at each visit in reference to sociodemographic characteristics, COVID-19 exposure, symptoms, virological findings and eventually vaccination. . 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. Confirmation assay. All M1 sera associated with at least one positive result using the above-mentioned assays or with a history of positive SARS-CoV-2 RT-PCR were retrospectively analyzed with the Abbott Architect SARS-CoV-2 IgG Quant II assay (Abbott, Sligo, Ireland) to confirm the serological positive status and to measure the anti-S IgG titer, if allowed by remaining serum volume. Sera collected during follow-up were also analyzed with this commercial assay for the entire selected cohort of COVID-19 HCW to define the serological status at each timepoint. This assay is an automated chemiluminescence microparticle immunoassay (CMIA) that quantify anti-RBD IgG with 50 AU/mL as a positive cut-off and a maximal threshold of quantification of 40,000.0 AU/mL (80,000 AU/mL with 1:2 dilution). According . 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 May 14, 2021. ; https://doi.org/10.1101/2021.05.07.21256823 doi: medRxiv preprint to the manufacturer, this CMIA displays clinical sensitivity and specificity of 98.81% and 99.55% after 15 DSO, respectively. Antibody titers measured with this assay are announced by the manufacturer to correlate, with a high probability (>95%), to neutralizing antibody titers assessed by plaque reduction assay on SARS-CoV-2 reference strain. This correlation was confirmed by previous studies 13, 30 . Chi-squared test, Kruskal-Wallis rank sum test and Fisher's exact test were conducted to identify any significant changes in categorical variables over time and between groups. Non parametric Wilcoxon paired tests and Mann-Whiney tests were conducted to compare quantitative data over time or between groups, respectively. All tests were two-sided with an α level of 0.05. To model anti-S (logtransformed) and anti-N IgG titers over time a triphasic decay was used, and the half-life (t1/2) of each decay phase was calculated. To assess characteristics of patients with a faster/slower decay in anti-S and anti-N IgG titers, non- . 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 May 14, 2021. ; https://doi.org/10.1101/2021.05.07.21256823 doi: medRxiv preprint This analysis was conducted on data from an on-going prospective, interventional, monocentric, longitudinal, cohort study enrolling healthcare workers from the Strasbourg University Hospitals (ClinicalTrials.gov Identifier: NCT04441684). The protocol was approved by the institutional review board of CPP Sud Méditerranée III. All participants provided a written informed consent. We are grateful to all the study participants who donated blood, The team of Cellule d'Appel , Pôle des Ressources Humaines who scheduled the planning of participants, the DRCI team including Eric . 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 May 14, 2021. ; https://doi.org/10.1101/2021.05.07.21256823 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. The copyright holder for this preprint this version posted May 14, 2021 . 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 cumulative probabilities of remaining free of SARS-CoV-2 infection among COVID-19 negative (COVID-19-) participants (red curve) and reinfection among former COVID-19 positive (COVID-19+) participants (blue curve) are shown on one year of follow-up (with 95% confidence interval, dotted lines). Exposition starts since the first negative serology for the COVID-19-group and two months after initial SARS-CoV-2 infection for the COVID-19+ group, as described previously 8 . SARS-CoV-2 infection/re-infection was assessed either by RT-qPCR or/and serology. Vaccinated individuals were censored at the time of the first vaccine dose. The number of exposed participants is defined under the x axis. Comparison of survival curves was performed using log-rank test. Comparison and p value were computed using the Graphpad Prism version 9.0.0 software. . 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) for all participants at M11-13 of according to the viral strain and the vaccination status. The number of participants is included at the center of the pie. *p value < 0.05; **p value < 0.01; ***p value < 0.001, ****p value <0.0001; calculated with non-parametric Wilcoxon paired tests or Spearman correlation. P values and correlation coefficients computed using the Graphpad Prism version 9.0.0 software. . 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 May 14, 2021. ; https://doi.org/10.1101/2021.05.07.21256823 doi: medRxiv preprint Figure 1 . 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) The copyright holder for this preprint this version posted May 14, 2021. ; https://doi.org/10.1101/2021.05.07.21256823 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. . 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 May 14, 2021. ; https://doi.org/10.1101/2021.05.07.21256823 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 May 14, 2021. ; https://doi.org/10.1101/2021.05.07.21256823 doi: medRxiv preprint Distinct Early Serological Signatures Track with SARS-CoV-2 Survival. Immunity Evolution of antibody immunity to SARS-CoV-2. Nature Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients Persistence and decay of human antibody responses to the receptor binding domain of SARS-CoV-2 spike protein in COVID-19 patients Rapid Decay of Anti-SARS-CoV-2 Antibodies in Persons with Mild Covid-19 Robust neutralizing antibodies to SARS-CoV-2 infection persist for months Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection Antibody Status and Incidence of SARS-CoV-2 Infection in Health Care Workers. The New England journal of medicine Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England Emerging SARS-CoV-2 variants reduce neutralization sensitivity to convalescent sera and monoclonal antibodies Resurgence of COVID-19 in Manaus, Brazil, despite high seroprevalence Detection of a SARS-CoV-2 variant of concern in South Africa Effect of previous SARS-CoV-2 infection on humoral and T-cell responses to single-dose BNT162b2 vaccine Evaluation of the performance of SARS-CoV-2 serological tools and their positioning in COVID-19 diagnostic strategies. Diagnostic microbiology and infectious disease Serologic responses to SARS-CoV-2 infection among hospital staff with mild disease in eastern France