key: cord-0966685-uic18wv8
authors: Lei, Q.; Li, Y.; Hou, H.; Wang, F.; Zhang, Y.; Lai, D.; Jo-Lewis, B. N.; Xu, Z.; Zhang, B.; Chen, H.; Ouyang, Z.; Xue, J.; Lin, X.; Zheng, Y.; Yao, Z.; Wang, X.; Yu, C.; Jiang, J.; Zhang, H.; Qi, H.; Guo, S.; Huang, S.; Sun, Z.; Tao, S.-c.; Fan, X.
title: Antibody dynamics to SARS-CoV-2 in Asymptomatic and Mild COVID-19 patients
date: 2020-07-11
journal: nan
DOI: 10.1101/2020.07.09.20149633
sha: 82d1bf8d52894bca8f3ef1ced7f7f0c918cbc6f6
doc_id: 966685
cord_uid: uic18wv8

Humoral immunity in asymptomatic infections with SARS-CoV-2 has not been well established. 63 healthy contacts, 63 asymptomatic individuals, and 51 mild patients were enrolled in this study and screened using nucleic acid testing (NAT) and commercial kits of serum IgM and IgG antibodies against recombinant nucleoprotein (N) and spike (S) proteins of SARS-CoV-2. Asymptomatic and mild patients were classified into at least four types based on NAT and serological tests, especially 81% and 25.4% negative NAT but positive IgM/IgG responses, respectively. Antibody dynamics were further demonstrated by IgM and IgG profile responses to SARS-CoV-2 proteome. IgM antibody responses against S1 were elicited in asymptomatic individuals as early to the seventh day after exposure and peaked on days from 17d to 25d, which might be used as early diagnostic biomarkers. Moreover, asymptomatic individuals evoked weaker S1 specific IgM and neutralizing antibody responses than mild patients. Most importantly, S1 specific IgM/IgG responses and the titers of neutralizing antibody in asymptomatic individuals gradually vanished in two months. Our findings might have important implications for serological survey, public health and immunization strategy.

respectively. Antibody dynamics were further demonstrated by IgM and IgG profile responses to SARS-CoV-2 proteome. IgM antibody responses against S1 were elicited in asymptomatic individuals as early to the seventh day after exposure and peaked on days from 17d to 25d, which might be used as early diagnostic biomarkers. Moreover, asymptomatic individuals evoked weaker S1 specific IgM and neutralizing antibody responses than mild patients. Most importantly, S1 specific IgM/IgG responses and the titers of neutralizing antibody in asymptomatic individuals gradually vanished in two months. Our findings might have important implications for serological survey, public health and immunization strategy.

SARS-CoV-2 is an emerging coronavirus, which was first recognized as the causal agent of COVID-19 in December 2019 1 . COVID-19 has rapidly spread around the world, and on 11 March 2020, the WHO has declared COVID-19 a global pandemic 2 . As of 28 June 2020, there have been 9,843,073 confirmed cases and 495,760 deaths, reported in 215 countries and territories worldwide 3 .

The sharp rise in the number of global COVID-19 cases brings the fear of having viral transmission from asymptomatic individuals. Asymptomatic COVID-19 infection has been defined as a person infected with SARS-CoV-2 who has no clinical symptoms (such as fever, cough, or sore throat), yet nucleic acid testing positive for SARS-CoV-2 4 . As of February 11, 2020, there were 72,314 COVID-19 cases reported in China, and 889 cases (1.2%) belonged to asymptomatic infections 5 . As of April 14, 2020, a total of 6,764 asymptomatic infections reported in China, which accounts for about 5.9% of all cases. Importantly, 1,297 asymptomatic infections subsequently developed to the confirmed cases 6 . Moreover, an important role in the transmission of asymptomatic individuals has been documented in different countries [7] [8] [9] . Therefore, asymptomatic cases pose a significant infection control challenge.

To understand the humoral immunity against SARS-CoV-2 in asymptomatic infections, we first screened by RT-PCR from 11,776 personnel returning to work, and close contacts with the confirmed cases in different communities of Wuhan by investigation of clusters and tracing infectious sources. Only 12 asymptomatic individuals with a positive NAT result were found out.

We further conducted a serological survey with the serum samples collected from all participants, using a validated assay for IgM and IgG antibodies against the recombinant antigens containing the nucleoprotein and the spike protein of SARS-CoV-2. Another 51 asymptomatic infections were further discovered because they had positive results for IgG alone, or both IgG and IgM. 63 healthy contacts with both negative results for NAT and antibodies were selected as negative controls. 51 mild patients without any preexisting conditions were also screened from 1056 patients during hospitalization in Tongji Hospital. A total of 177 participants were enrolled in this study and serial serum samples (n=213) were collected. Clear exposure history or days after symptoms onset were obtained from 48 healthy contacts, 36 asymptomatic infections and 51 mild patients. The research was conducted between 17 February 2020 and 28 April 2020. Serum IgM and IgG profiles of 177 participants were further probed using a SARS-CoV-2 proteome microarray. Neutralizing antibody responses in different population were detected by a pseudotyped virus neutralization assay system. The dynamics of IgM and IgG antibodies and neutralizing antibodies were analyzed with exposure time or symptoms onset.

Firstly, we found that 63 asymptomatic individuals were classified into four categories, based on the results of both NAT and ELISA tests. 81% (51/63) asymptomatic individuals had negative NAT but IgG alone (28/63), or both IgG and IgM (23/63) positive. Interestingly, 6.3 % (4/63) were only NAT positive and 12.7% (8/63) were positive for both NAT and IgG (Table 1) . Mild patients were further defined as five types. Of mild patients, 25.4% (13/51) were NAT negative but positive IgG alone (4/51), or both positive for IgG and IgM (9/51). 60.3% reported positive results for NAT and IgG alone (10/51), or positive NAT, IgG and IgM (22/51). Importantly, 11.3% (6/51) mild patients were only NAT positive (Table 1) .

To better understand the role of humoral immunity against infection and disease progression, SARS-CoV-2 specific IgM (red) and IgG (green) antibodies were further detected in parallel using a SARS-CoV-2 proteome microarray ( Figure 1 ). Overall visualizations of IgM and IgG profiles were respectively shown in Figure 2 and 3 . Surprisingly, healthy contacts also elicited IgM responses to several structural proteins such as N-Cter, S2, and E, and most of non-structural proteins such as ORF-7b, ORF-8, NSP1, NSP4, NSP5, NSP7, NSP8, NSP9, NSP16, NSP14, NSP15, and RdRp of SARS-CoV-2 ( Figure 2 ). Low levels of IgG responses against N-Cter, and NSP1, NSP4, NSP7, NSP8, NSP9, NSP10, and RdRp were only induced in partial healthy contacts ( Figure 3 ). It is noteworthy that both asymptomatic individuals and mild patients elicited higher levels of antibody responses (IgM or IgG) against S1, N, N−Nter, N-Cter, S2 and ORF-7b ( Figure 4A and Figure 5A ) than healthy contacts. Except NAT alone positive asymptomatic individuals, other asymptomatic individuals and different subgroups of mild patients induced higher levels of S1 and N specific IgM or IgG responses than healthy contacts ( Figure 4B and 5B), although these antibody responses could not differentiate the same subgroup between asymptomatic and mild cases( Figure 4C and Figure 5C ). Interestingly, 3 healthy contacts were clustered as asymptomatic infections, and 15 asymptomatic infections and 5 mild patients were recognized as healthy contacts by IgM profile response to SARS-CoV-2 proteome ( Figure 2 ).

We further compared the dynamic change of S1, N, N−Nter, and N-Cter specific IgM and IgG antibodies in 48 healthy contacts and 36 asymptomatic individuals with different exposure time, and in 51 mild patients with days after symptoms onset ( Figure 6 and Figure 7 ). Early to the seventh day after exposure, S1 and N specific IgM and IgG responses were induced in asymptomatic individuals and reached a peak on days from 17d to 25d. Then all of these antibodies began to decline. Except N specific IgG response, other antibodies could not be detectable 2 months after exposure. Interestingly, mild patients had a very distinct dynamic change of antibodies. Early to 1d, S1 specific IgM responses were induced in mild patients and persistently increased until 29 after symptoms onset. Moreover, mild patients elicited higher levels of N specific IgM and IgG responses, which maintained for at least 65 days.

Finally, we analyzed the dynamic change of the neutralizing antibody responses using a pseudotyped virus based neutralization assay ( Figure 8 ). Interestingly, we found that 36.5% (23/63) asymptomatic individuals, mainly NAT positive (8/12), did not produce neutralizing antibody, 63.5% (40/63) asymptomatic infections and 19% (12/63) healthy contacts only induced low titers of neutralizing antibody, with the mean NT50 1:24 and 1:13, respectively ( Figure 8A ). Among three groups, mild patients stimulated the highest levels of neutralizing antibody with the mean NT50 1:269. Only 11.8% (6/51) mild patients, mainly NAT alone positive (4/6), did not elicit neutralizing antibody ( Figure 8B ). More importantly, the neutralizing antibody response in asymptomatic individuals was produced on 7d after exposure and peaked on days from 10d to 25d, then declined with exposure time. However, mild patients produced the neutralizing antibody early to 1d after symptom onset, and the titer rose persistently until 22 days and maintain for at least 65 days ( Figure 8C ).

Our study first demonstrates that asymptomatic infection should be defined as a person has positive NAT or/and antibodies without clinical symptoms. In particular, about 81% asymptomatic infections had negative NAT, which indicates that the proportion of asymptomatic infections might be much higher than the current rate reported in China. NAT should be used in conjunction with serological testing to early identify asymptomatic infections and to benefit accurate estimation of the asymptomatic proportion. However, the role in the transmission of different subgroups of asymptomatic individuals remains to be investigated.

Although our findings highlight the value of serological testing, current commercial kits have several significant limitations. IgM antibody responses against S1 protein might be meaningful to assist NAT for earlier identification of asymptomatic individuals and diagnosis of COVID-19 patients, because of rapid emergence and disappearance. N specific IgG responses persist in the infected host for a longer time and should be suitable for serological survey. Therefore, serological testing should be performed based on S or N specific antibody response, not by simultaneously testing responses to S and N antigens of SARS-CoV-2. Moreover, IgM and IgG antibodies against both antigens cannot differentiate the same subgroup of asymptomatic individuals and mild patients, which indicates the infectiousness of asymptomatic infections. To achieve this end, further investigation to define more accurate serological markers, such as profiling B cells epitopes from S antigen should be encouraged.

Most importantly, the persisting antibodies in asymptomatic individuals only reacted to N, not to S1 antigen. Correspondingly, the titers of neutralizing antibody in asymptomatic individuals were very low and decreased rapidly, 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". Together, our findings might indicate the risks of 'shield immunity' and asymptomatic individuals still need immunization with vaccines. Complying with strict public health measures remains the most important strategy to control the epidemic.

In order to identify and report SARS-CocV-2 infection case in time, on Jan 28, 2020, the National Health Commission of China updated the COVID-19 Prevention and Control Plan (3th edition) and first emphasized the identification and quarantine of mild cases and asymptomatic infections.

Close contacts with confirmed cases, persons with close social distance during extensive investigation of clusters and tracing infectious sources were considered as potential case sources.

RT-PCR screening was carried out to find positive cases with or without clinical symptoms. On April 8, 2020, the lockdown had been lifted in Wuhan. Personnel returning to work were also required to screen by RT-PCR. According to the COVID-19 Prevention and Control Plan (3th edition), all positive RT-PCR individuals were asked to provide detail information, including demography, preexisting conditions, exposure history, and symptoms, as well as screening records for the preceding 14 d. The negative persons also complied with home quarantine for 14d. An asymptomatic case was defined as an individual with a positive nucleic acid test result but without any relevant clinical symptoms during quarantine. A mild COVID-19 patient was defined as an individual with nasopharyngeal swabs that were positive for SARS-CoV-2, with symptoms such as fever, cough or sore throat, yet without lung image change. A close contact was defined as (1) anyone who had been within approximately 6 feet (2 meters) of a person infected with SARS-CoV-2 for longer than 10 min and (2) The half-maximal inhibitory concentration for plasma (NT50) of each serum sample was determined as the highest dilution ratio of serum with 50% neutralization rate by using log2 value of serum dilution of the same sample as the abscissa and different neutralization rate as the ordinate to draw the linear relationship line and calculated using nonlinear regression (SPSS).

All statistical analyses were carried out using SPSS or R software. Proteins were described as the medians and interquartile ranges (IQRs), and Mann-Whitney U test was conducted to test difference between two groups. The Log2NT50 was presented with mean (SD) and t test was used to test difference between two groups. The half-maximal inhibitory concentration for plasma (NT50) was determined using nonlinear regression (SPSS). Cluster analysis was performed with pheatmap package of R. Statistical significance was determined as a value of p< 0.05.

The study was approved by the Ethical Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China (IRB ID:TJ-C20200128). Written informed consent was obtained from all participants enrolled in this study. Comparison of IgM and IgG responses to S1 and N proteins in different subgroups of asymptomatic individuals and mild patients with that of healthy contacts. (C) Comparison of IgM and IgG response to S1 and N proteins in the same subgroup of asymptomatic individuals and mild patients. Medians and interquartile range value for each group are indicated. Differences between groups were analysed using the Mann-Whitney U test. *** indicated p <0.001, ** indicated p <0.01, * p indicated <0.05, and n.s.

indicated not significant. respectively. The blue line shows the dynamic change of neutralizing ability in mild patients at different times after symptom onset. Samples with neutralizing titers below 1:10 were plotted at the dotted line (log2NT50=1). Statistical significance in A and B was determined using unpaired t-test and horizontal bars indicate mean and SD. NT50: Half-maximal inhibitory plasma neutralizing.

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