key: cord-0723705-viq3o8cw authors: Zhou, Wei; Xu, Xiaomao; Chang, Zhigang; Wang, He; Zhong, Xuefeng; Tong, Xunliang; Liu, Taotao; Li, Yanming title: The dynamic changes of serum IgM and IgG against SARS‐CoV‐2 in patients with COVID‐19 date: 2020-07-24 journal: J Med Virol DOI: 10.1002/jmv.26353 sha: 3e7cf935ad3536657fb01ff5d1460f6a94c34790 doc_id: 723705 cord_uid: viq3o8cw Coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has become a worldwide pandemic since it emerged in December, 2019. Previous studies have reported rapid antibody response to SARS‐CoV‐2 with the first 2‐3 weeks after symptom onset. Here, we retrospectively described the dynamic changes of serum IgM and IgG specifically against SARS‐CoV‐2 in later weeks (mainly 4‐10 weeks) in 97 hospitalized patients with COVID‐19. We observed that serum IgM and IgG, especially in patients with moderate‐to‐high levels, declined significantly between week 4‐10 after illness onset. Notably, IgG levels in high percentage of patients (77.5%, 31/40) rapidly declined by half, from 212.5 (range, 163.7‐420.3) to 96.3 (range, 75.0‐133.4) AU/ml, within 1‐2 weeks in the second month and then sustained around 100 AU/ml until discharge from hospital. Significant reduction of IgM was also observed as SARS‐CoV‐2 nucleic acid converted to negative (p=0.002). In the recovery stage, serum IgG declined significantly (early vs. late recovery stage, n=16, p=0.003) with a median reduction of 50.0% (range, 3.7‐77.0%). Our results suggested the decline of IgM may be an indicator of virus clearance and recovery patients may have robust immunity against reinfection within at least 3 months after illness onset. Yet, the rapid reduction of IgG by half arises serious concerns on the robustness and sustainability of humoral immune response in the future period after discharge, which is crucial for immunity strategy and developing vaccine. This article is protected by copyright. All rights reserved. Coronavirus disease 2019 (COVID- 19) , an acute respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in Wuhan, China on December 12, 2019 and became a worldwide pandemic in 2020 1, 2 . Accumulatively, nearly 10 million confirmed COVID-19 cases and 500 thousand deaths have been reported around the world by June 27, 2020 according to World Health Organization (WHO). Most of the patients had typically clinical symptoms including fever, cough, fatigue and dyspnea which appeared 2-14 days after exposure 2, 3 . About 20% patients may progress to a severe or critical disease with a higher mortality rate than mild cases 3 . patients also have antibody response to virus infection [4] [5] [6] . Serum IgM and IgG against SARS-CoV-2 were detectable within the first several weeks after symptom onset 4, [7] [8] [9] . They were detectable as early as 4 days and reached a peak in the second week after onset 7, 8 . Almost all the infected cases had seropositive antibodies in the first 3 weeks post illness onset 4 . Thus, the serological testing for specific antibodies against SARS-CoV-2 in early stage may be helpful for the diagnosis of suspected cases in the context of high false-negative risk of RT-PCR assay in detecting virus nucleic acid 8, 10 . Serologic testing in conjunction with RT-PCR have been demonstrated to enhance the sensitivity of detecting symptomatic or asymptomatic individuals infected with SARS-CoV-2 11 . However, most of previous publications have only focused on the acute response phase within 2~3 weeks after onset in COVID-19. Our study aims to describe the dynamic changes of serum antibody against SARS-CoV-2 in the later weeks after onset, especially when the virus nucleic acid converts to negative and when patients are recovering from the disease. This article is protected by copyright. All rights reserved. Recovery was defined as SARS-CoV-2 nucleic acid conversion negative. To avoid false negative, only the patients with negative results in all of the subsequent nucleic acid tests after conversion were confirmed to be truly recovered. We defined early recovery stage (ERS) as the first 7 days and late recovery stage (LRS) as more than 14 days after the first recovery date 12 . The serum antibodies against SARS-CoV-2 (IgM and IgG) was detected by enzyme linked immunosorbent assay (iFlash 2019-nCoV IgM and IgG kits from YHLO Biotech Co., Ltd, Shenzhen, China) using iFlash3000 Chemiluminescence Immunoassay Analyzer (YHLO Biotech Co., Ltd, Shenzhen, China). Continuous variables were present as median and interquartile range (IQR). Independent continuous variables were compared using Mann-Whitney U test and paired variables with Wilcoxon signed-rank sum test. Categorical variables were present as number and percentage, and compared using χ 2 test or Fisher's exact test as appropriate. P value < 0.05 was considered statistically significant. All statistical analyses and scientific graphics were made by using GraphPad Prism 8.0 (GraphPad Software, Inc., CA. USA). After excluding 10 patients who only received antibody tests prior to 3 weeks after illness onset, a total of 97 COVID-19 patients (62 severe and 35 non-severe) were included in our study ( Table 1 ). The median age was 65 years (IQR, 53-72), and severe patients were older than non-severe patients (p=0.007). Male and current smokers were at higher risk of being severely ill (p=0.022 and 0.003, respectively). Cough, fatigue, dyspnea and fever were the most common Accepted Article symptoms, which were reported in 86.6, 71.1, 68.0 and 59.8% of all patients. Fever, dyspnea, myalgia and fatigue were more frequently manifested in severe patients (all p<0.01). No significant differences of comorbidities except malignancy were observed between severe and non-severe patients. All patients were discharged from hospital before April 8, 2020, except one critical patient who died from acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome (MODS) 33 days after symptom onset. Severe patients had shorter duration from symptom onset to admission to hospital (median 9.0 vs. 14.0 days, p=0.001) but longer duration of hospitalization (median 45.0 vs. 32.0 days, p<0.001). The median time for SARS-CoV-2 nucleic acid transferring negative was 34.0 days with no significant difference between severe and non-severe groups. We illustrated the overall profile of serum IgM and IgG against SARS-CoV-2 from week 4 to 10 after illness onset in Figure Median serum IgG was detected as high as more than 150 AU/ml in week 4-6 but markedly reduced to nearly 100 AU/ml which plateaued during week 7-10 ( Figure 1b ). When stratifying patients according to severity, we found the antibody profile of serum IgM and IgG in severe and non-severe patients were similar to that of the total sample. In addition, the difference of antibody levels between This article is protected by copyright. All rights reserved. severe and non-severe groups was not statistically significant, although non-severe patients had slightly lower median antibody levels in some weeks (Table 2) , which may be due to small sample size. No difference was observed between male and female. Since the immune response may differ greatly among individuals, the change pattern of serum antibodies revealed by analyzing unpaired date may be biased. To investigate the change pattern more precisely, we extracted paired data from patients who had antibody tests in at least two consecutive weeks and compared the serum antibody levels between consecutive weeks using Wilcoxon AU/ml (p<0.001) while IgG was not obviously altered. In the other week pairs, the antibody levels had no obvious change. Dynamic of serum antibody levels of 40 patients who received antibody tests in at least 3 weeks among week 4-9 after illness onset were shown in Figure 3 . In addition, the changes of antibody levels of each patients were shown in Figure 4 . We stratified patients according to antibody level of the first antibody test. Patients in low-IgM group (<20 AU/ml, n=12) had persistently low IgM levels in week 4-9 without obvious changes (Figure 3a, 4a) . Meanwhile, serum IgM of patients in moderate-IgM group (20~100 AU/ml, n=19) gradually declined (Figure 3b, 4b) and IgM of patients in high-IgM group (>100 AU/ml, n=9) had significant reduction of more than 50AU/ml in week 4-9 (Figure 3c, 4c) . Serum IgG of most patients in low-IgG group (<100 AU/ml, n=7) also sustained at low level (Figure 3d, 4d) . In moderate-IgG group (100~200 AU/ml, n=15), serum IgG of 7 patients increased firstly to a peak and then rapidly declined, while the others declined directly (Figure 3e, 4e) . In high-IgG group (>200 AU/ml, n=18), most patients had great reduction of >100 AU/ml in IgG (Figure 3f, 4f) . However, serum IgG in moderate and high groups sustained around 100 AU/ml after significant reduction until discharge from hospital. Notably, serum IgG in 60.0% (24/40) of patients rapidly declined within 1 week and 17.5% (7/40) within 2 weeks (no antibody tests in the intermediate week) and then sustained until discharge. The median levels of serum IgG before and after rapid decline were 212.5 (range, 163.7-420.3) and 96.3 (range, 75.0-133.4) AU/ml, respectively, and the median reduction was 52.7% (range, 39.8-75.2%). Therefore, it was reasonable to infer that most patients had a sudden reduction of IgG by half within 1 week in the second month after illness onset. In the rest of the patients, we found 4 patients with low IgG levels (<100AU/ml) also had sharp reduction of IgG by half (range, 45.5-60.4%) within 1-2 weeks. We analyzed paired data from 18 patients who had antibody tests in acute stage (AS, the period when the SARS-CoV-2 nucleic acid can be found in respiratory specimen) and ERS to investigate the changes of serum antibodies This article is protected by copyright. All rights reserved. with the nucleic acid conversion negative (i.e., recovery). The median days of transferring negative after illness onset in these patients were 46.0 (IQR, 36.8-50.8). If a patient had multiple antibody data in AS, only the last one was used. IgM level significantly decreased (AS: 28.2 (IQR, 8.7-230); ERS: 16.0 (IQR, 6.5-32.6); p=0.002) as the viral RNA turned negative (Figure 5a ). IgG level decreased from 133.0 (IQR, 81.9-209.9) to 96.4 (IQR, 87.1-118.4) AU/ml but the difference did not reach statistical significance (p=0.108, Figure 5b ). The current study described the dynamic changes of serum IgM and IgG against SARS-CoV-2 after 3 weeks following illness onset in hospitalized patients with COVID-19. In overall, IgM and IgG declined in the second month after illness onset, especially in patients with moderate-to-high levels of serum antibodies that represented a stronger antibody response to SARS-CoV-2. Antibody response is critical for virus clearance and preventing reinfection. Previous studies have reported rapid production of specific IgM and IgG to SARS-CoV-2 within the first week and peak level of antibodies by 2-3 weeks after disease onset 4, 10, 13 . The peak of antibodies may not persist for long-time duration. We found the decline of serum IgM after peak mainly occurred in the This article is protected by copyright. All rights reserved. second month (week 5-8) after onset. This was parallel to virological assessment results that SARS-CoV-2 nucleic acid of most patients turned negative in week 4-7 (median 34 days, IQR 23.8-44.2) since illness onset. Subsequent paired-data analysis revealed that IgM level significantly declined as virus was completely cleared. Thus, it seems to be conclusive that IgM titer increases rapidly within the first 2 weeks, persists in the following 1-2 weeks as virus are being cleared, and declines since 4 th week after illness onset as virus nucleic acid turns negative 14 . Therefore, the decline of IgM may be an indicator of virus clearance, which may help determine the true nucleic acid conversion negative in conjunction with RT-PCR testing. It could be served as criterion for discharge and ending quarantine, especially for mildly symptomatic or asymptomatic cases. Sustained IgG level is crucial for shaping memory immune to prevent reinfection. Previous studies revealed detectable antibody levels persisted for more than 2 years in SARS-CoV-and MERS-CoV-infected patients 15, 16 . It is unclear how long specific IgG against SARS-CoV-2 will last. The present study showed a significant reduction of IgG in week 5-7 that only continued for 1-2 weeks but a sustained IgG level since 7 th week after illness onset until discharge. The level of IgG decreased from over 150 AU/ml and then plateaued around 100 AU/ml. Serum IgG was still detectable in two patients as long as 11 weeks since illness onset. Analysis using paired data showed that IgG may occur in the process of recovery. Our findings were consistent with previous studies 17, 18 IgG 8 weeks after they were discharged from hospital 17 . Notably, the decline of IgG mainly occurred in patients with moderate-to-high IgG levels, indicating that stronger antibody response did not always necessarily convert to more robust long term humoral immunity against reinfection. Additionally, dynamic analysis in current study showed that IgG levels in most patients rapidly declined by half within 1-2 weeks in the second month after illness onset. Several patients with IgG levels <100AU/ml also had a rapid reduction by half. This may arise serious concerns that IgG would have logarithmically declined at certain time intervals in the future and recovery patients may become vulnerable to SARS-CoV-2 again. More serological studies investigating the dynamics of antibodies in recovery patients for a longer time period (3 months or more after discharge) are urgent need to determine the duration of humoral immunity, which is pivotal for immunity strategy and developing vaccine. The immune response to SARS-CoV-2 may differ by disease severity. Immunological survey demonstrated that critical ill cases had dramatically higher levels of IL-2R, IL-6, IL-10 and TNF-α but lower counts of T lymphocytes, CD4 + T cells and CD8 + T cells as compared with moderate cases 19 , so that IL-2R/lymphocyte may be a useful marker for monitoring the progression and predicting the progression 20 . Serological assays also revealed higher titers of IgG 4 and total antibody 10 in severe patients than non-severe patients, which was 10 . Our study revealed that serum IgM and IgG levels in 4 or more weeks after symptom onset were comparable between severe and non-severe patients. Notably, non-severe patients had lower median IgM levels than severe patients in week 4 and 5, but the difference did not reach statistical significance (p=0.850 and 0.052, respectively), which may be due to small sample size. Therefore, more studies are needed to clarify the precise association between antibody-mediated immune response and disease severity. Some limitations in our study should be noted. Firstly, this is a retrospective study and serological testing has not become regular until late February, 2020. Thus, most patients had incomplete weekly data of antibody testing from 4 th week to discharge, and only a small proportion of patients were included in paired analysis. Secondly, we did not discriminate binding antibodies specific to SARS-CoV-2 N and S protein (i.e. N-IgM, N-IgG, S-IgM, S-IgG). Thirdly, the sample size is relatively small with inherently reduced statistical power. Furthermore subgroups analysis stratified by age and comorbidities were not available in our study. In conclusion, our study described the dynamic changes of serum antibodies of patients with COVID-19 and provided deep insight into the clinical course and humoral immune response to SARS-CoV-2 during month 2-3 after illness onset. This study was supported by Special Foundation for National Science and This article is protected by copyright. All rights reserved. Coronary heart disease 15 A Novel Coronavirus from Patients with Pneumonia in China A pneumonia outbreak associated with a new coronavirus of probable bat origin Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. 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Emerg Infect Dis Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections Neutralizing Antibodies Responses to SARS-CoV-2 in COVID-19 Inpatients and Convalescent Patients Clinical and immunological features of severe and moderate coronavirus disease 2019 Using IL-2R/lymphocytes for predicting the clinical progression of patients with COVID-19 The authors declare that there are no conflict of interests. YL conceptualized the study. WZ, XX, ZC, HW, XZ, XT and TL collected and analyzed the data. YL and WZ interpreted the data. WZ drafted the manuscript.All authors critically revised the manuscript and approved the final version for publication. The data that support the findings of this study are available from the corresponding author upon reasonable request.