key: cord-279550-7u2hksxm
authors: Wang, Kai; Long, Quan-Xin; Deng, Hai-Jun; Hu, Jie; Gao, Qing-Zhu; Zhang, Gui-Ji; He, Chang-Long; Huang, Lu-Yi; Hu, Jie-Li; Chen, Juan; Tang, Ni; Huang, Ai-Long
title: Longitudinal dynamics of the neutralizing antibody response to SARS-CoV-2 infection
date: 2020-08-03
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa1143
sha: 
doc_id: 279550
cord_uid: 7u2hksxm

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 symptom 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 28, 2020, over 16 million people worldwide have been reportedly infected and more than 650,800 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), 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 angiotensin-converting 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 mainly act against the receptor-binding domain (RBD) of the SARS-CoV-2 S protein, [7] [8] [9] 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. 10, 11 The serological antibody response continues for at least 3 weeks and, in some cases, substantially longer. 12, 13 However, the dynamics and roles of SARS-CoV-2-specific NAbs and their correlation with antibody responses have not been explored in COVID-19 patients more than two months after symptom onset.

A c c e p t e d M a n u s c r i p t 6 Previous studies of SARS-CoV and MERS-CoV demonstrated that the most immunogenic antigens are the S-and N-proteins, and development of serological tests (such as enzymelinked immunosorbent assay and magnetic chemiluminescence enzyme immunoassay) for SARS-CoV-2 IgG or IgM antibodies have focused on these viral proteins. However, it is still unknown whether serological antibodies predict neutralizing activities or protection against viral re-infection. 14 A c c e p t e d M a n u s c r i p t 7

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. A confirmed case of COVID-19 was defined as an individual with nasopharyngeal swabs that were positive for laboratory-based PCR testing. COVID-19 patients who meet following criteria can be discharged: with two consecutive negative RT-PCR results on respiratory tract samples, body temperature is back to normal for more than 3 days, respiratory symptoms improve obviously, pulmonary imaging shows obvious absorption of inflammation, can be discharged. On April 2nd and May 8th, 2020 (follow-up point 1 and follow-up point 2, respectively) two follow-up visits were conducted. Sequential serum samples were collected from patients in the acute phase (3 or 4 samples per patient) and the convalescent phase 

The SARS-CoV and SARS-CoV-2 pseudoviruses were generated as previously described, with some modifications. 18 Briefly, HEK293T cells (5 × 10 6 ) were co-transfected with pNL4- Relative luminescence units of Luc activity were determined using the Luciferase Assay Kit (Promega). The titers of NAbs were calculated as 50% inhibitory dose (ID 50 ), expressed as the highest dilution of plasma which resulted in a 50% reduction of luciferase luminescence compared with virus control, using a cut-off titer 1:20.

All serum samples were inactivated at 56 °C for 30 min and stored at −20 °C before testing. 

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. Two-tailed Pearson correlation test was used to calculate the correlation coefficient of NAb to IgG levels or cytokines.

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) , who meeting any of the following criteria: 1) respiratory distress (≥30 times/minutes), 2) the oxygen saturation≤93% at rest, 3) the arterial partial pressure of oxygen (PaO 2 ) / the fraction of inspired oxygen (FiO 2 ) ≤ 300 mmHg. The median length of the hospital stay was 22 d (IQR 15-26). patients during hospitalization ( Figure 2B ). The peak NAb levels varied among the patients; 6·7%, 73·3%, and 20% patients showed low (ID 50 < 500), medium-low (ID 50 500-999), and medium-high (ID 50 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 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 kinetic levels of NAbs and virus-specific IgG over time in COVID-19 patients are still unknown. To address this, we first determined the relationship between the NAb levels and virus-specific IgG levels in individual patients ( Figure 4A and Supplementary Figure 1) ; similar dynamic changes were observed for the NAbs and virus-specific IgG levels in some patients. Furthermore, to determine if there was a statistical correlation between NAb levels and virus-specific IgG levels in COVID-19 patients, serum samples were grouped by time (weeks) after symptom onset. A statistically significant positive correlation was only observed in samples obtained 3 weeks after symptom onset (p = 0·027, r = 0·410) ( Figure   4B ).

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 colonystimulating factor (M-CSF) (r = 0·454, p = 0·017) levels ( Figure 5 ).

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. 20 Herein, we described the dynamics of SARS-CoV-2-specific NAbs generated during both the acute and convalescent phases of A c c e p t e d M a n u s c r i p t 13 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 3-month 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-229E. 21 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. 25 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 A c c e p t e d M a n u s c r i p t 14 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. 26, 27 Although the role of memory T cells in the protective immune 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 13,28 . 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 one of the major antigens of the SARS-CoV-2. 29 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. 30 Several preliminary clinical trials have proven its effectiveness in treating SARS-CoV-2. 5, 6 The efficacy of CP transfusion is attributed to the neutralizing effect of antibodies; thus, the NAb titer is the major 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-2-specific 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 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. 

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Convalescent plasma as a potential therapy for COVID-19

We would like to thank Prof. Cheguo Cai (Wuhan University, Wuhan, China) for providing the pNL4-3.Luc.R-E-plasmid. 

The authors declare no competing interests.

A c c e p t e d M a n u s c r i p t 17 A c c e p t e d M a n u s c r i p t 18