The dynamic changes of serum IgM and IgG against SARS‐CoV‐2 in patients with COVID‐19

Abstract 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 in the first 2 to 3 weeks after symptom onset. Here, we retrospectively described the dynamic changes of serum immunoglobulin M (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 to 10 after illness onset. Notably, IgG levels in high percentage of patients (77.5%, 31 of 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 to 2 weeks in the second month and then sustained at around 100 AU/mL until discharge from hospital. Significant reduction of IgM was also observed as SARS‐CoV‐2 nucleic acid turned negative (P = .002). In the recovery stage, serum IgG declined significantly (early vs late recovery stage, n = 16, P = .003) with a median reduction of 50.0% (range, 3.7%‐77.0%). Our results suggested that the decline of IgM may be an indicator of virus clearance and recovered patients may have a robust immunity against reinfection within at least 3 months after illness onset. Yet, the rapid reduction of IgG by half rises serious concerns on the robustness and sustainability of the humoral immune response in the period after discharge, which is crucial for immunity strategy and developing a vaccine.

clinical symptoms including fever, cough, fatigue, and dyspnea, which appeared 2 to 14 days after exposure. 2,3 About 20% of patients may progress to a severe or critical disease with a higher mortality rate than mild cases. 3 Similar to patients infected by SARS-CoV or MERS-CoV, COVID-19 patients also have antibody response to virus infection. [4][5][6] Serum immunoglobulin M (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 the early stage may be helpful for the diagnosis of suspected cases in the context of high false-negative risk of reversetranscription polymerase chain reaction (RT-PCR) assay in detecting virus nucleic acid. 8,10 Serologic testing in conjunction with RT-PCR have demonstrated enhanced sensitivity of detecting symptomatic or asymptomatic individuals infected with SARS-CoV-2. 11 However, most of the previous publications have only focused on the acute response phase within 2 to 3 weeks after onset of 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 turns negative and when patients are recovering from the disease.

| Levels of serum antibodies at different weeks
We illustrate the overall profile of serum IgM and IgG against SARS-CoV-2 from week 4 to 10 after illness onset in Figure 1. 3) AU/mL, respectively, which were significantly lower than those in week 4 to 6 (all P < .05). Median serum IgG was detected as high as 150 AU/mL or more in week 4 to 6 but markedly reduced to nearly 100 AU/mL, which plateaued during week 7 to 10 ( Figure 1B).
When stratifying patients according to severity, we found the antibody profile of serum IgM and IgG in severe and nonsevere patients were similar to that of the total sample. In addition, the difference of antibody levels between severe and nonsevere groups was not statistically significant, although nonsevere patients had slightly lower median antibody levels in some weeks (Table 2), which may be due to the small sample size. No difference was observed between male and female.

| Changes of serum antibodies using paired data
As 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 the Wilcoxon signed-rank sum test (Figure 2).
In week 5 to 6 (n = 21), the IgM level reduced from 32.9 (IQR, In the other week pairs, the antibody levels had no obvious change.  The serum IgG of most patients in low-IgG group (<100 AU/ mL, n = 7) also sustained at a low level ( Figures 3D and 4D). In the moderate-IgG group (100-200 AU/mL, n = 15), serum IgG of seven patients increased first to a peak and then rapidly declined, while the others declined directly (Figures 3E and 4E). In the high-IgG group (>200 AU/mL, n = 18), most patients had great reduction of >100 AU/mL in IgG (Figures 3F and 4F). However, serum IgG in moderate and high groups sustained at around 100 AU/mL after significant reduction until discharge from hospital.

| Rapid reduction of serum IgG
Notably, serum IgG in 60.0% (24 of 40) of patients rapidly de- In the rest of the patients, we found 4 patients with low IgG levels (<100 AU/mL) also had sharp reduction of IgG by half (range, 45.5%-60.4%) within 1 to 2 weeks.

| Antibody changes as the nucleic acid turns negative
We analyzed paired data from 18 patients who had antibody tests in the acute stage (AS, the period when the SARS-CoV-2 nucleic acid can be found in the respiratory specimen) and ERS to investigate the changes of serum antibodies as the nucleic acid turns negative (ie, recovery). The median days from illness onset to recovery in these  Figure 5B).

| 929
Thus, it seems to be conclusive that IgM titer increases rapidly within the first 2 weeks, persists in the following 1 to 2 weeks as the virus is being cleared, and declines by the 4th week after illness onset as the 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 serve as a criterion for discharge and in ending quarantine, especially for mildly symptomatic or asymptomatic cases.
Sustained IgG level is crucial for shaping memory immune to prevent reinfection. Previous studies revealed that 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 happened within 1-2 weeks, which sustained since 7th 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 Changes of serum immunoglobulin M (IgM) and IgG in 4 to 9 weeks after symptom onset stratified by antibody level in the first antibody test in 40 patients who received antibody tests in at least 3 weeks. A-C, Low, moderate, and high IgM group (<20 AU/mL, n = 12; 20-100 AU/mL, n = 19; >100 AU/mL, n = 9, respectively). D-F, Low, moderate, and high IgG group (<100 AU/mL, n = 7; 100-200 AU/mL, n = 15; >200 AU/mL, n = 18, respectively) necessarily convert to more robust long-term humoral immunity against reinfection.
Additionally, dynamic analysis in the current study showed that the IgG levels in most patients rapidly declined by half within 1 to 2 weeks in the second month after illness onset. Several patients with IgG levels <100 AU/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 recovered 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 urgently need to determine the duration of humoral immunity, which is pivotal for immunity strategy and developing a 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 interleukin-2R (IL-2R), IL-6, IL-10, and tumor necrosis factor-α but lower counts of T lymphocytes, CD4 + T cells, and CD8 + T cells as compared with moderate cases, 19 so that the 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 in nonsevere patients, which was associated with clinical outcomes. This may suggest an effect of antibody-dependent enhancement (ADE). ADE, characterized by enhanced virus entry mediated by antibodies and induction of severe inflammatory activation, may contribute to lung injury. 21 This was previously observed in SARS-CoV-1-infected rhesus macaques. 22  F I G U R E 5 Dynamic changes of antibody against SARS-CoV-2 as the nucleic acid turns negative. A, Serum IgM levels. B, Serum IgG levels. AS, acute stage; ERS, early recovery stage; IgM, immunoglobulin M; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. The antibody levels are compared by Wilcoxon signed-rank sum test. *P < .05; **P < .01; ***P < .001 data of antibody testing from the 4th week to discharge, and only a small proportion of patients were included in paired analysis. Second, we did not discriminate binding antibodies specific to SARS-CoV-2 N and S protein (ie N-IgM, N-IgG, S-IgM, S-IgG). Third, the sample size was relatively small with inherently reduced statistical power. Furthermore, subgroup 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 to 3 after illness onset. The antibody levels are compared by Wilcoxon signed-rank sum test. *P < .05; **P < .01; *** P < .001