key: cord-0894124-h0j6xmrd
authors: Lei, Qing; Li, Yang; Hou, Hong‐yan; Wang, Feng; Ouyang, Zhu‐qing; Zhang, Yandi; Lai, Dan‐yun; Banga Ndzouboukou, Jo‐Lewis; Xu, Zhao‐wei; Zhang, Bo; Chen, Hong; Xue, Jun‐biao; Lin, Xiao‐song; Zheng, Yun‐xiao; Yao, Zong‐jie; Wang, Xue‐ning; Yu, Cai‐zheng; Jiang, He‐wei; Zhang, Hai‐nan; Qi, Huan; Guo, Shu‐juan; Huang, Sheng‐hai; Sun, Zi‐yong; Tao, Sheng‐ce; Fan, Xiong‐lin
title: Antibody dynamics to SARS‐CoV‐2 in asymptomatic COVID‐19 infections
date: 2020-10-26
journal: Allergy
DOI: 10.1111/all.14622
sha: a36f3afbc5b897f2b28bad2d9e4aba24678838f1
doc_id: 894124
cord_uid: h0j6xmrd

BACKGROUND: The missing asymptomatic COVID‐19 infections have been overlooked because of the imperfect sensitivity of the nucleic acid testing (NAT). Globally understanding the humoral immunity in asymptomatic carriers will provide scientific knowledge for developing serological tests, improving early identification, and implementing more rational control strategies against the pandemic. MEASURE: Utilizing both NAT and commercial kits for serum IgM and IgG antibodies, we extensively screened 11 766 epidemiologically suspected individuals on enrollment and 63 asymptomatic individuals were detected and recruited. Sixty‐three healthy individuals and 51 mild patients without any preexisting conditions were set as controls. Serum IgM and IgG profiles were further probed using a SARS‐CoV‐2 proteome microarray, and neutralizing antibody was detected by a pseudotyped virus neutralization assay system. The dynamics of antibodies were analyzed with exposure time or symptoms onset. RESULTS: A combination test of NAT and serological testing for IgM antibody discovered 55.5% of the total of 63 asymptomatic infections, which significantly raises the detection sensitivity when compared with the NAT alone (19%). Serum proteome microarray analysis demonstrated that asymptomatics mainly produced IgM and IgG antibodies against S1 and N proteins out of 20 proteins of SARS‐CoV‐2. Different from strong and persistent N‐specific antibodies, S1‐specific IgM responses, which evolved in asymptomatic individuals as early as the seventh day after exposure, peaked on days from 17 days to 25 days, and then disappeared in two months, might be used as an early diagnostic biomarker. 11.8% (6/51) mild patients and 38.1% (24/63) asymptomatic individuals did not produce neutralizing antibody. In particular, neutralizing antibody in asymptomatics gradually vanished in two months. CONCLUSION: Our findings might have important implications for the definition of asymptomatic COVID‐19 infections, diagnosis, serological survey, public health, and immunization strategies.

SARS-CoV-2 is an emerging coronavirus, which was first recognized as the causative agent of COVID-19 in December 2019, 1 and has rapidly spread around the world. On March 11, 2020 , the WHO has declared COVID-19 a global pandemic. 2 

The combination of NAT and serological testing for IgM antibody significantly improves the detection sensitivity of asymptomatic COVID-19 infections, compared with NAT alone. S1-specific IgM antibody response with rapid emergence and disappearance might be helpful to assist NAT for early identification of infectious individuals. A majority of asymptomatics induce very low levels of neutralizing antibody that disappear in two months. to asymptomatic infections. 5 As of April 14, 2020, a total of 6764 asymptomatic infections reported in China, which accounts for about 5.9% of all registered cases. Among these, 1297 asymptomatic individuals, in fact presymptomatics and subsequently, developed to the confirmed cases with different severities of illness, while the others remained asymptomatic. 6 Beyond doubt, presymptomatics are really infectious. [7] [8] [9] Interestingly, several studies reported that asymptomatic COVID-19 infections also play important roles in the transmission. 10, 11 Therefore, both types of asymptomatics contribute significantly to disease transmission. To better control the pandemic of COVID-19, actively discovering, as well as early identifying and quarantining asymptomatics are urgently needed.

Until now, detection of asymptomatic infections has been relied on extensive NAT screening of quarantined individuals. The test sensitivity of NAT highly depends on the course and the type of clinical COVID-19 syndromes, the collection site, the transportation, and storage of specimens. About 30% false-negative rates of NAT have been reported in COVID-19 patients. 12 In particular, recent seroprevalence investigations strongly suggested that COVID-19 cases, especially asymptomatics are greatly underestimated in different countries and regions. SARS-CoV-2-specific IgG response in blood donors reached 3.08% during lockdown of Wuhan city, 13 consistent with the other report of the seropositivity in healthcare workers in Wuhan ranging from 3.2% to 3.8%. 14 Both studies indicate that the number of actual infections is at least five times higher than that of the reported cases in Wuhan. In Spain, there were 5% serological positive individuals of national population and 1/3 of them did not report symptoms. 15 Similar situation occurred in different regions of the United States. 16 Therefore, these missing asymptomatic cases that are infectious in the community have been substantially overlooked because of the limited sensitivity of NAT and passive approaches to discover them. 

In order to identify and report SARS-CoV-2 infected cases in time, the NHCC updated the COVID-19 Prevention and Control Plan (3rd edition) on January 28, 2020, which first emphasized the identification and quarantine of asymptomatic infections. 4 Specifically, close contacts with confirmed cases and persons with close social distance during extensive investigation of clusters and tracing infectious sources were required to screen by Real-time PCR (RT-qPCR) testing SARS-CoV-2 genes in nasopharyngeal swabs. On April 8, 2020, the lockdown had been lifted in Wuhan. Personnel returning to work

were also required to screen by RT-qPCR. All NAT-positive individuals were asked to provide detail information, including demography, preexisting conditions, exposure history, symptoms, as well as screening records, and accepted centralized isolation for the preced- 

Between February 17, 2020, and April 28, 2020, 1056 confirmed COVID-19 patients with different severities of illness were hospitalized in Tongji Hospital, Wuhan, China. Only 51 mild COVID-19 patients who had serial serum samples yet not any preexisting conditions were selected and collected for a total of 87 samples. After extensively screening, 11 766 epidemiologically suspected individuals, 63 asymptomatic infections, and 63 healthy controls were informed in the study. Among these, 48 healthy controls and 36 asymptomatics had clear exposure history, respectively. All participants were traced consecutively for 65 days. Serum specimens were collected from each individuals and were stored at −80°C until use.

Nasopharyngeal swabs of all participants on enrollment were collected and maintained in viral transport medium. Before detection, all specimens were thermally inactivated in 56°C for 30 minutes before detection. SARS-CoV-2 infection was confirmed using TaqMan One-Step RT-qPCR Kits (DAAN Gene) which detected ORF1ab and N genes and approved by the China Food and Drug Administration (CFDA). The RT-qPCR assay kits were performed according to manufacturers' instructions, and the cutoff Ct value was 40 for both genes. The Ct values of both genes were less than 40 and were defined as positive.

The IgM and IgG antibodies against recombinant both nucleoprotein (N) and spike (S) proteins of SARS-CoV-2 in serum specimens were detected by a chemiluminescence method according to the manufacturer's instructions (YHLO Biotech), respectively. The antibody levels ≥10 AU/mL are reactive (positive), and the results <10 AU/mL are negative.

The SARS-CoV-2 proteome microarray was prepared as our previous study with minor modifications. 20 Three more proteins, that is, ORF3a, ORF3b, and ORF7b, were expressed by our laboratory, and another protein RdRp was provided by H. Eric Xu's Lab. 21 The proteins printed on the microarray were listed in Table S1 . The proteins with indicated concentrations, along with the negative (GST, Biotin-control, and eGFP) and positive controls (Human IgG, Human IgM, and ACE2-Fc), were printed in triplicate on PATH substrate slide (Grace Bio-Labs, Oregon, USA) to generate identical arrays in a 2 × 7 subarray format using Super Marathon printer (Arrayjet). Protein microarrays were stored at −80°C until use.

The prepared SARS-CoV-2 proteome microarray was conducted to probe all serum samples, as previously described. 20 Briefly, a 14-chamber rubber gasket was mounted onto each slide to create individual chambers for the 14 identical subarrays. The arrays stored at −80°C were warmed to room temperature and then incubated in Overall visualization of IgG and IgM profiles was built by clustering analysis to generate heatmaps. The neutralization rate (%) for different dilutions was calculated as following:

The titer of neutralization antibody for each serum sample was expressed as the half-maximal neutralizing titer (NT50). NT50 of each serum sample was determined as the highest dilution ratio of serum with 50% neutralization rate.

All diagram and statistical analyses were carried out using Prism 8, SPSS, or R software when applicable used. Cluster analysis of IgM and IgG profiles was performed with pheatmap package of R, respectively. NT50 was determined using nonlinear regression by SPSS, and a loess 489 regression model was used to establish the

kinetics of neutralizing antibody. The analysis of variance (ANOVA), post hoc test (SNK), Student's t test, and Mann-Whitney U test were used to compare difference among different groups when required.

A statistical significant was considered when P < .05.

To understand the humoral immunity against SARS-CoV-2 in asymp- 

Currently, NAT is still the gold standard to diagnose COVID-19 patients. To explore the role of serological testing for early identifying asymptomatics, we first analyzed the characteristics of study population based on the results of both NAT and commercial serological test ( 

To better understand humoral immune responses against SARS-CoV-2, IgM (red) or IgG (green) antibody responses to 20 out of 28 predicted proteins of SARS-CoV-2 were further detected in parallel using a proteome microarray ( Figure S1A ). Three representative microarray pictures probed with sera collected from a healthy contact;

an asymptomatic infection and a mild patient were shown as Figure   S1B . Serum IgM (red) or IgG (green) antibodies against different proteins were captured and then detected by secondary antibodies la- were built separately. Overall visualizations of IgM ( Figure S2 ) or IgG ( Figure S3 ) profiles in 177 participants were performed by clustering analysis to generate heatmaps. Overall, the serum samples for all three groups, that is, healthy controls, asymptomatic infections, and mild cases, were clustered together, especially for IgG antibodies, although some samples were not correctly grouped which labeled with spots in different colors ( Figure S2 and Figure S3 ). Notably, clinically diagnosed healthy controls, asymptomatics, and mild cases were not divided into three complete independent groups, especially based on microarray-constructed IgM profiles ( Figure S2) , which indicate that we cannot distinguish these groups based on antibody detection alone and also confirm the presence of serological testing negative asymptomatic infections and mild cases. In addition, three healthy controls were not correctly clustered as asymptomatic infections based on the IgM profiles ( Figure S2 ), which highlight the significance of keeping social distance and repeated tests.

Based on the analysis of quantitative data, both asymptomatics and mild patients induced stronger IgM ( Figure 2A) and IgG responses ( Figure 2B ), especially against S1, N, N-Nter, and N-Cter out of 20 proteins than healthy controls, respectively. Although asymptomatic infections and mild cases had similar IgM profiles against 20 proteins of SARS-CoV-2 by clustering analysis (Figure S2 ), the levels of IgM responses to S1, N, N-Nter, N-Cter, and ORF7b were significantly higher in mild patients than asymptomatics (Figure 2A ).

Mild patients also tended to induce stronger IgG responses against S1, N, N-Nter, and N-Cter proteins than asymptomatic infections ( Figure 2B ). And then, we compared the levels of antibodies in different subgroups of asymptomatics and mild patients with that of healthy controls. Except NAT alone positive asymptomatic individuals, other subgroups of asymptomatics and mild patients elicited higher levels of S1-, N-, N-Nter-, and N-Cter-specific IgM or IgG antibodies than healthy controls ( Figure S4A ), although these antibody responses could not differentiate the same subgroup between asymptomatics and mild cases ( Figure S4B ). Taken together, our results demonstrated that IgM and IgG responses to only S1 and N from the 20 proteins of SARS-CoV-2 might differentiate both asymptomatics and mild patients from healthy controls.

To help establish serological tests, we further compared the dynamic changes of S1-, N-, N − Nter-, and N-Cter-specific IgM and IgG antibodies in 48 healthy controls, 36 asymptomatic individuals, and 51 mild patients, who had either clear exposure history or serial samples after symptoms onset (Figure 3 and Figure S5 ). Early to the seventh day after exposure, S1-and N-specific IgM and IgG responses were induced in asymptomatic individuals and peaked on days from 17 days to 25 days and then began to decline. Except N-specific IgG response, other antibodies in asymptomatics could not be detectable 2 months after exposure (Figure 3 and Figure S5 ). Compared to asymptomatics, mild patients had distinct dynamic changes of these antibodies. Early to 1 day after symptoms onset, IgM antibody against the N protein rapidly evolved and persisted at a high level, while S1-specific IgM responses were induced in mild patients and persistently increased until 29 days after symptoms onset. In addition, S1-or N-specific IgM and IgG responses in mild patients maintained for at least 65 days (Figure 3 ).

Antibody responses to different proteins of SARS-CoV-2. Serum proteome microarray was used to probe IgM or IgG antibody against 20 proteins of SARS-CoV-2 in all samples collected from 63 healthy controls, 63 asymptomatic individuals, and 51 mild patients. The results were expressed as mean {log 2 (Fluorescence intensity)} ± SD in different groups. A, Comparison of IgM responses to five proteins among three groups. B, Comparison of IgG responses to five proteins among three groups. Both analysis of variance (ANOVA) and post hoc test (SNK) were conducted to test difference in means among healthy controls, asymptomatics, and mild patients. ***P < .001, **P < .01, *P < .05, and ns indicating no significance F I G U R E 3 Dynamic changes of S1and N-specific IgM and IgG responses. Serum proteome microarray was used to probe antibody responses in the samples collected from 48 healthy individuals, 36 asymptomatic individuals, and 51 mild patients. The result of each serum sample was expressed as log 2 (fluorescence intensity). 48 healthy controls and 36 asymptomatic infections having clear exposure history were plotted in sections according to the exposure time. 51 mild COVID-19 patients with serial sera samples (n = 87) were segmented according to days after symptoms onset. The yellow, green and blue line showed the mean level of antibody responses in healthy controls, asymptomatic infections and mild patients, respectively. A, Dynamic changes of S1-and N-specific IgM responses. B, Dynamic changes of S1and N-specific IgG responses

To better understand the role of humoral immunity against infection, we analyzed the dynamic changes of neutralizing antibody responses using pseudotyped virus-based neutralization assay platforms ( Figure 4) . Interestingly, we found that 38.1% (24/63) asymptomatic individuals, mainly NAT-positive (8/12), did not produce neutralizing antibody. 61.9% (39/63) of asymptomatic infections and 19% of (12/63) healthy controls only produced low titers of neutralizing antibody, with the geometric mean NT50 of 1:24 and 1:13, respectively ( Figure 4A ). Among three groups, mild patients stimulated the highest levels of neutralizing antibody with the geometric mean NT50 of 1:269, whereas 11.8% (6/51) mild patients, mainly NAT alone positive (4/6), did not elicit neutralizing antibody ( Figure 4B ). In order to investigate the duration of neutralizing antibody, the dynamics of neutralizing antibody response were also established for three groups. As early as 7 days after exposure, neutralizing antibody rapidly evolved in asymptomatics individuals, peaked on days from 10 days to 25 days, then decayed rapidly within our observed period. ( Figure 4C ) As early as 1 day post symptom onset, mild patients also produced low level of neutralizing antibody, and then, the titer rose persistently until 22 days and maintain high levels for at least 65 days ( Figure 4D ).

It remains very difficult to early and actively find out all asymptomatic COVID-19 infections from healthy population, based on the current control strategy. In this study, five epidemiological strategies were adopted as recommended by the NHCC to discover these 63 asymptomatics from more than 10 000 epidemiologically suspected individuals, which provide us opportunities to establish and verify laboratory tests for early and actively screening asymptomatic infec-

tions. Firstly, we demonstrated that the current used NAT alone has a very low sensitivity (only 19%) to screen asymptomatic infections, which was supported by the following reports. For instance, the strategy of nucleic acid screening for all citizen had been performed in Wuhan after lift of the lockdown, and there were only another 300 NAT-positive asymptomatics out of about 10 million citizens. 22 The latest seroprevalence studies also revealed that the proportion of asymptomatic infections might be much higher than the incidence rate reported in China 5, 6, 13, 14 Combining with the results of seroprevalence investigations in different countries, 15 based on both NAT and serological testing should be performed to find out as many infectious SARS-CoV-2 infected individuals as possible. In our study, NAT in conjunction with serological testing for IgM discovered 55.5% of the total asymptomatic infections, except an additional 17.6% IgM-positive mild patients. Therefore, the joint screening strategy will significantly attribute to early identifying and actively discovering infectious sources, and repeated tests may further improve the detection sensitivity. 25 Under these context, we provide a new perspective that asymptomatic COVID-19 infection should be defined as a person has positive NAT or/and IgM antibody response, yet without clinical symptoms and radiological changes of the lung. This definition will give an additional 36.5% IgM seropositivity of asymptomatics, which is more reasonable and practical than the current used.

Furthermore, both humoral immune responses to the antigens of SARS-CoV-2 and antibody dynamics in asymptomatics determined by our serum proteome microarray analysis provide scientific foundation for the development and application of serological tests. Our study found that asymptomatics mainly evolved IgM and IgG antibodies against S1 and N proteins out of 20 proteins of SARS-CoV-2, supported by the facts that both S and N proteins also are the major targets in commercial or homemade serological tests. 26, 27 Although asymptomatic individuals have a long duration of viral shedding, 18 the timing of serological tests is also very significant for assisting diagnosis. 28, 29 As demonstrated in this study, S1-specific IgM antibody responses were induced in asymptomatics as early as 1 week after exposure and disappeared within two months, which coincides within the duration of viral shedding of SARS-CoV-2. Because of rapid emergence and disappearance, S1-specific IgM antibody re- pandemic. 32 We found that 63.5% of asymptomatic individuals only elicited low levels of neutralizing, and the rest did not produce neutralizing antibodies at all. In addition, 11.8% mild patients also did not produce neutralizing antibody, in line with other reports. 18, 33, 34 In particular, we demonstrated that neutralizing antibody in asymptomatic individuals decreased rapidly and disappeared in a short time, which indicate that the effectiveness of antibody-mediated immunity could not be used to guarantee the accuracy of an "immunity passport" or "risk-free certificate." Our findings might suggest the risks of "shield immunity" and notably, that asymptomatic individuals might still need immunization with vaccines. Strict public health strategies including lockdown of city, tracing infectious sources and quarantine, keeping social distance, isolate protection of healthy individuals such as wearing mask and washing hands were performed during the phase of high prevalence in Wuhan, so these undetected asymptomatics might not play important roles in disease transmission. Therefore, complying with strict public health measures remains the most important strategy to control the pandemic of COVID. 

The authors declare no conflicts of interest. Dr Lei has nothing to disclose. Dr Li has nothing to disclose. Dr Hou has nothing to disclose. Dr Wang has nothing to disclose. Dr Ouyang has nothing to disclose. Dr Zhang has nothing to disclose. Dr Lai has nothing to disclose. Dr Banga Ndzouboukou has nothing to disclose. Dr Xu has nothing to disclose. Dr Zhang has nothing to disclose. Dr Chen has nothing to disclose. Dr Xue has nothing to disclose. Dr Lin has nothing to disclose. Dr Zheng has nothing to disclose. Dr Yao has nothing to disclose. Dr Wang has nothing to disclose. Dr Yu has nothing to disclose. Dr Jiang has nothing to disclose. Dr Zhang has nothing to disclose. Dr Qi has nothing to disclose. Dr Guo has nothing to disclose. Dr Huang has nothing to disclose. Dr Sun has nothing to disclose. Dr Tao has nothing to disclose. Dr Fan has nothing to disclose. Zi-yong Sun https://orcid.org/0000-0002-6443-9755

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