key: cord-278491-cnqxsno8 authors: Wang, K.; Long, Q.-X.; Deng, H.-J.; Hu, J.; Gao, Q.-Z.; Zhang, G.-J.; He, C.-L.; Huang, L.-Y.; Hu, J.-L.; Chen, J.; Tang, N.; Huang, A.-L. title: Longitudinal dynamics of the neutralizing antibody response to SARS-CoV-2 infection date: 2020-07-17 journal: nan DOI: 10.1101/2020.07.14.20151159 sha: doc_id: 278491 cord_uid: cnqxsno8 Background Coronavirus disease 2019 (COVID-19) is a global pandemic with no licensed vaccine or specific antiviral agents for therapy. Little is known about the longitudinal dynamics of SARS-CoV-2-specific neutralizing antibodies (NAbs) in COVID-19 patients. Methods Blood samples (n=173) were collected from 30 COVID-19 patients over a 3-month period after symptom onset and analyzed for SARS-CoV-2-specific NAbs, using the lentiviral pseudotype assay, coincident with the levels of IgG and proinflammatory cytokines. Results SARS-CoV-2-specific NAb titers were low for the first 7-10 d after symtom onset and increased after 2-3 weeks. The median peak time for NAbs was 33 d (IQR 24-59 d) after symptom onset. NAb titers in 93.3% (28/30) of the patients declined gradually over the 3-month study period, with a median decrease of 34.8% (IQR 19.6-42.4%). NAb titers increased over time in parallel with the rise in IgG antibody levels, correlating well at week 3 (r = 0.41, p < 0.05). The NAb titers also demonstrated a significant positive correlation with levels of plasma proinflammatory cytokines, including SCF, TRAIL, and M-CSF. Conclusions These data provide useful information regarding dynamic changes in NAbs in COVID-19 patients during the acute and convalescent phases. Coronavirus disease 2019 (COVID-2019) is a novel respiratory disease that is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the outbreak of SARS-CoV-2 last year, it has spread rapidly and caused a global pandemic. 1 As of July 3, 2020, over 10 million people worldwide have been reportedly infected and more than 512,000 individuals have died of COVID- 19. 2 Currently, considerable progress is being made to understand SARS-CoV-2 pathogenesis, epidemiology, antiviral drug development, and vaccine design. However, no licensed specific antiviral drugs or prophylactic vaccines are available. Developing effective viral inhibitors and antibody-based therapeutics to prevent or treat COVID-19 infection is a high global priority. The SARS-CoV-2 RNA genome encodes 29 structural and non-structural proteins, including spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins, and the ORF1a/b polyprotein. 3 The S glycoprotein is responsible for SARS-CoV-2 attachment and entry into target host cells via its binding to the angiotensinconverting enzyme 2 (ACE-2) receptor. 4 Virus-specific neutralizing antibodies (NAbs) play a key role in reducing viral replication and increasing viral clearance. 5, 6 NAbs act against the receptor-binding domain (RBD) of the SARS-CoV-2 S protein, effectively blocking viral entry. Thus, serological testing, especially to detect NAbs, is essential in determining the onset of the serological immune response, evaluating the potential capacity of the host body for viral clearance, and identifying donors for passive antibody therapy trials. In COVID-19 patients, NAbs can be detected within 2 weeks of symptom onset. 7, 8 The serological antibody response continues for at least 3 weeks and, in some cases, substantially longer. 9, 10 However, the dynamics and roles of SARS-CoV-2specific NAbs and their correlation with antibody responses have not been explored in COVID-19 patients more than two months after symptom onset. In this study, we first analyzed the 3-month longitudinal dynamics of in vitro NAb titers in 30 recovered COVID-19 patients. Second, we evaluated the correlation between the dynamics of NAb titers and serological IgG levels, as well as inflammatory cytokine levels. Our study may provide useful information regarding dynamic changes in NAbs in COVID-19 patients during the acute and convalescent phases and aid in the development of vaccines against SARS-CoV-2. A total of 30 COVID-19 patients who had recovered and were discharged from the Yongchuan Hospital of Chongqing Medical University were included in our cohort. . 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 July 17, 2020. The codon-optimized gene encoding the SARS-CoV S protein (AAP13567.1) and SARS-CoV-2 S protein (QHD43416) with the 19 C-terminal amino acids deleted were synthesized by Sino Biological Inc (Beijing, China) and cloned into the the pCMV3 vector, respectively. The HIV-1 NL4-3 ΔEnv Vpr luciferase reporter vector (pNL4-3.Luc.R-E-), constructed by N. Landau, 11 was provided by Cheguo Cai, Wuhan University (Wuhan, China). The vesicular stomatitis virus G (VSV-G)expressing plasmid pMD2.G was provided by Prof. Ding Xue, Tsinghua University (Beijing, China). HEK293T cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in Dulbecco's modified 5 . 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 July 17, 2020. . https://doi.org/10.1101/2020.07.14.20151159 doi: medRxiv preprint Eagle's medium (DMEM; Hyclone, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Rockville, MD, USA), 100 mg/mL streptomycin, and 100 U/mL of penicillin at 37 °C in 5% CO2. HEK293T cells transfected with human ACE2 (293T-ACE2) were cultured under the same conditions, with the addition of G418 (0·5 mg/mL) to the medium. The SARS-CoV and SARS-CoV-2 pseudoviruses were generated as previously described, with some modifications. 12 Briefly, HEK293T cells (5 × 10 6 ) were co- 6 . 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 July 17, 2020. . https://doi.org/10.1101/2020.07.14.20151159 doi: medRxiv preprint The 293T-ACE2 cells (2 × 10 4 cells/well) were seeded in 96-well plates. For the neutralization assay, 50 μL of pseudovirus (3·8 × 10 4 copies) was incubated with serial dilutions of serum samples from patients and human control serum as a negative control for 1 h at 37 °C and then added to the 96-well 293T-ACE2 plates. Titers of NAbs were calculated as the 50% inhibitory dose (ID50). Continuous variables were expressed as median (inter-quartile range, IQR) and categorical variables were expressed as number (percentage, %). Comparisons between two groups were performed using the Mann-Whitney U test or Fisher's exact test. A two-sided α of <0·05 was considered statistically significant. Statistical analyses were performed using R software, v3.6.0. Twotailed Pearson correlation test was used to calculate the correlation coefficient of NAb to IgG levels or cytokines. 7 . 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 July 17, 2020. . Of the total 30 patients in the cohort, 60·0% (18/30) were female, and 10·0% (3/30) were categorized as severe based on the COVID-19 Treatment guidelines (National Health Commission of the People's Republic of China) ( Table 1 ). The median length of the hospital stay was 22 d (IQR 15-26). 8 . 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 July 17, 2020. . https://doi.org/10.1101/2020.07.14.20151159 doi: medRxiv preprint We analyzed the longitudinal dynamics of virus-specific IgG and NAb levels in 30 patients who were positive for SARS-CoV-2 using real-time RT-qPCR. Sequential patients during hospitalization ( Figure 2B ). The peak NAb levels varied among the patients; 6·7%, 73·3%, and 20% patients showed low (ID50 < 500), medium-low (ID50 500-999), and medium-high (ID50 1000-2500) NAb titers, respectively ( Figure 2C ). There was no statistical difference among peak NAb titers that occurred during hospitalization and convalescence ( Figure 2D ). The duration and maintenance of peak of NAb levels in COVID-19 patients is of great concern. Thus, we compared NAb levels between the peak time point and the final follow-up time point. A decline in NAb levels was observed in 93·3% (28/30) of SARS-CoV-2 infected patients, with a median decrease of 34·8% (IQR 19.6-42.4%) ( Figure 3A ). Patients were also grouped according to their rate of 9 . 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 July 17, 2020. . decrease in NAb levels; more than 20% of the patients showed a >70% decrease in NAb levels during this time period (21/30) ( Figure 3B ). The We analyzed the correlation between cytokine and chemokine levels and NAb levels in COVID-19 patients during the acute phase. Interestingly, we observed that NAb levels were positively correlated with stem cell factor (SCF) (r = 0·616, p = 0·001), TNF-related apoptosis-inducing ligand (TRAIL) (r = 0·514, p = 0·008), and macrophage colony-stimulating factor (M-CSF) (r = 0·454, p = 0·017) levels ( Figure 5 ). . 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 July 17, 2020. . https://doi.org/10.1101/2020.07.14.20151159 doi: medRxiv preprint Virus-specific NAbs have been considered an important determinant for viral clearance. The pseudovirus-based assay is suitable for the high-throughput screening of SARS-CoV-2 NAbs in plasma donors without the requirement of BSL-3 laboratories. The assay has been widely used for evaluating NAbs in highly pathogenic viruses, such as Ebola, SARS-CoV, MERS-CoV, and highly pathogenic influenza viruses. 14 Herein, we described the dynamics of SARS-CoV-2-specific NAbs generated during both the acute and convalescent phases of SARS-CoV-2 infection using a pseudovirus-based neutralization assay. We found that SARS-CoV-2-specific NAb titers were low before day 7-10, peaked at approximately day 33 after symptom onset, and then gradually declined over a 3month period. Meanwhile, SARS-CoV-2-specific NAbs were detected concurrently with and positively correlated with IgG antibodies in our cohort, indicating that the NAb response may play an important role in viral clearance. Our understanding of the duration and nature of protective immunity to SARS-CoV-2 is currently very limited. The kinetics of antibody-mediated immunity to SARS-CoV-2 infection and how long this immunity lasts are unknown. Our data suggest that NAb titers in patients were variable, and the protective humoral immune response to SARS-CoV-2 may abate over time, which is in accordance with findings in patients infected with other human coronaviruses, such as HCoV- . 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 July 17, 2020. . https://doi.org/10.1101/2020.07.14.20151159 doi: medRxiv preprint 229E. 15, 16 The short-term humoral immune response in COVID-19 patients is also highly consistent with that observed in patients infected with SARS-CoV and MERS-CoV, 17, 18 who show a rapid decrease in virus-specific antibody titers within 3-4 months. Among the 30 recovered patients in our study, two patients showed very low NAb titers during the acute phase and 3-month follow-up, indicating that other immune responses, involving T cells and inflammatory cytokines may have contributed to viral clearance. These data suggest that the antibody titers may diminish with time or some recovered patients may not produce a high-titer response during SARS- Recently, in a rhesus macaques model, SARS-CoV-2 infection evoked a robust protective immune response when the animals were re-exposed to SARS-CoV-2 one month after the initial viral infection. 19 However, natural infection and volunteer challenge studies hint that coronavirus infections, including those with HCoV-229E and HCoV-OE43, cannot induce stable protective immunity; thus, reinfection occurs frequently. Moreover, a SARS-CoV antigen-specific memory B cell response was not detectable in recovered SARS patients at 6-years after disease onset, whereas SARS-CoV-specific memory T cells persisted in recovered SARS patients. 20, 21 Although the role of memory T cells in the protective immune 12 . 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 July 17, 2020. . https://doi.org/10.1101/2020.07.14.20151159 doi: medRxiv preprint response to SARS-CoV-2 needs further evaluation, a robust T cell response is required for viral clearance. We also described here, the dynamic correlation between SARS-CoV-2-specific NAbs and serological total IgG levels. NAb titers appeared concomitantly and correlated moderately with IgG levels at week 3 after symptom onset, which is consistent with other reports regarding COVID-19 recovered patients 10, 22 . The antigen epitope used for IgG detection in our study contained the nucleoprotein peptide, as well as the RBD domain of the spike protein, which partially explains the discrepancy in NAb titers and IgG levels at weeks 4, 9, and 14 after symptom onset. The nucleoprotein is the most abundant protein in the SARS-CoV-2 viral particle and possesses the strongest immunogenicity. The binding antibodies detected by the total IgG assay may also be involved in viral clearance through antibody-dependent cytotoxicity, Therefore, the roles of binding antibodies and NAbs in disease progression need further evaluation. Currently, adaptive immunotherapy using convalescent plasma (CP) from recovered COVID-19 patients is being employed as a potential therapeutic approach to confer antiviral protection. 23 Several preliminary clinical trials have proven its effectiveness in treating SARS-CoV-2. 6, 24 The efficacy of CP transfusion is attributed to the neutralizing effect of antibodies; thus, the NAb titer is the major 13 . 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 July 17, 2020. . https://doi.org/10.1101/2020.07.14.20151159 doi: medRxiv preprint determinant for CP therapy. Monitoring NAb levels and their duration will provide valuable data for evaluating the effectiveness of CP therapy. In our study, the levels of NAbs declined gradually over the 3-month follow-up period, with a median decrease of 34.8%. Thus, CP samples with high titers of NAbs from patients in the early stage of convalescence will be more suitable for clinical use. There are some limitations to this study, which should be addressed. Due to the small sample size, we could not find any correlation between the dynamics of NAb titers and clinical characteristics contributing to different clinical outcomes. Serological blood samples were collected up to 3 months after symptom onset; data collected over longer follow-up times should be obtained to demonstrate the duration of humoral immunity after SARS-CoV-2 infection. The lack of data to determine an anamnestic immune response, such as tests for SARS-CoV-2specific memory B cells, memory T cells, and specific cytokine-dependent memory cells, hampered the evaluation of the immune response, especially protective immunity against viral reinfection. These are major issues that should be investigated in future studies. In summary, we determined the dynamics of NAb titers within 3 months after symptom onset in 30 SARS-CoV-2-infected patients and found a positive correlation between NAb titers and IgG antibodies. Our work provides valuable 14 . 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 July 17, 2020. . https://doi.org/10.1101/2020.07.14.20151159 doi: medRxiv preprint insight into the humoral immunity against SARS-CoV-2 infection. We also described a pseudotype system for measuring NAb titers, which could be expanded to antiviral drug screening and vaccine development. . 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 July 17, 2020. . https://doi.org/10.1101/2020.07.14.20151159 doi: medRxiv preprint Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. 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 July 17, 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 July 17, 2020. . Patients (n = 30) A pneumonia outbreak associated with a new coronavirus of probable bat origin Coronavirus Disease (COVID-19) Situation Reports COVID-19: Epidemiology, Evolution, and Cross-Disciplinary Perspectives The potential danger of suboptimal antibody responses in COVID-19 Effectiveness of convalescent plasma therapy in severe COVID-19 patients Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma Antibody responses to SARS-CoV-2 in patients with COVID-19 Neutralizing antibody response in mild COVID-19 Convalescent plasma therapy for the treatment of patients with COVID-19: Assessment of methods available for antibody detection and their correlation with neutralising antibody levels SARS-CoV-2 seroprevalence and neutralizing activity in donor and patient blood from the San Francisco Bay Area Vpr Is Required for Efficient Replication of Human Immunodeficiency Virus Type-1 in Mononuclear Phagocytes Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV Comparison of lentiviral vector titration methods Pseudotyping Viral Vectors With Emerging Virus Envelope Proteins The time course of the immune response to experimental coronavirus infection of man Disappearance of Antibodies to SARS-Associated Coronavirus after Recovery A serological survey on neutralizing antibody titer of SARS convalescent sera Challenges of convalescent plasma infusion therapy in Middle East respiratory coronavirus infection: a single centre experience SARS-CoV-2 infection protects against rechallenge in rhesus macaques Lack of Peripheral Memory B Cell Responses in Recovered Patients with Severe Acute Respiratory Syndrome: A Six-Year Follow-Up Study Long-lived effector/central memory T-cell responses to severe acute respiratory syndrome coronavirus (SARS-CoV) S antigen in recovered SARS patients Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications We would like to thank Prof. Cheguo Cai (Wuhan