Relationship between the dynamic changes of serum 2019-nCoV IgM/IgG and patient immunity after discharged six months

Objectives To investigate the relationship between the dynamic changes of serum 2019-nCoV IgM/IgG and immunity alteration for patients after discharged six months. Methods 1 with IgM(+) and IgG(-), 32 with IgM(+) and IgG(+), 38 with IgM(-) and IgG(+), and 40 with IgM(-) and IgG(-) were included. Demographic data were collected. IgM and IgG antibodies, hypersensitive C-reactive protein (hs-CRP), interleukin-6 (IL-6) and lymphocyte subsets in serum were determined on weeks 0, 2 and 4. Results Hs-CRP and IL-6 for all patients were within the normal ranges. All testing items of the lymphocyte subsets were 12/110 (10.9%) of weeks 0, 15/110 (13.6%) of weeks 2 and 18/110 (16.4%) of weeks 4 within the normal ranges. The percentages of CD8+, NK cells and B lymphocytes in the IgM(+) and IgG(+) group were quite different from the IgM(-) and IgG(+) group and the IgM(-)and IgG(-) group, with much more the percentages of CD8+ and much less the percentages of NK cells and B lymphocytes on weeks 0, 2 and 4. 12 patients with IgM(+) had converted to IgM(-) in the IgM(+) and IgG(+) group, and the percentages of NK cells and B lymphocytes were signicantly increased on weeks 4. Conclusions The changes of serum IgM and IgG are closely related to immunity for patients in recovery stage. However, immunity isn't recovery with the turning negative of antibodies.


Introduction
Since December 2019, the pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caucused 2019 novel coronavirus (2019-nCoV) pneumonia has been threatened worldwide for millions people [1]. Till March 2020, the Coronavirus disease 2019 (COVID- 19) epidemic had been effectively controlled in Wuhan city, China. Infected patients were hospitalized and were discharged from hospital with recovery. Currently, almost all of COVID-19 patients in China have been in recovery stage after infection and therapy [2].
Presently, lack of effective antiviral drugs [3], effective vaccines [4], convalescent plasma therapy [5] and speci c human monoclonal antibody [6], the treatment of COVID-19 are still the greatest challenge for medical staff and scienti c research workers [7]. As a novel coronavirus, the dynamic immunity and pathogenesis of human body are unclear [8][9][10].
In this study, we focused on COVID-19 patients in recovery stage after discharged six months and aimed to evaluate dynamic changes of IgM and IgG antibodies, the change levels of hypersensitive C-reactive protein (hs-CRP), interleukin-6 (IL-6) and lymphocyte subsets alteration in plasma, and the potential correlation with the dynamic changes of serum IgM and IgG and patient immunity.

Inclusion and Exclusion Criteria
We did the four weeks clinical study for patients with COVID-19 at our department, which was registered in the Chinese Clinical Trial Registry (ChiCTR2000034794). Inclusion criteria were age between 18 and 70 years old, and signed informed consent by patients (or their families).

Treatment
The enrolled patients were treated with a standardized management, such as breathing training and oral Chinese medicine daily.

Ethics
The ethics was approved ethical approval by the Research Ethical Committee of Hubei Hospital of Chinese Medicine (granted No. HBZY2020-C26-01).

Data collection
The following data were collected from a consecutive series of 111 patients in four-week observation stage: i) demographic data including gender, age, severity of hospitalization, and chronic disorder histories (chronic cardiovascular disease, chronic respiratory disease, chronic cerebrovascular illness, and diabetes); ii) Serum biochemical data in included antibodies against SARS-CoV-2 (IgM and IgG), hs-CRP, IL-6 and lymphocyte subsets (including the percentage of CD3+/ CD8 +/ CD4+/natural killer (NK) cells/B lymphocytes).

Statistical Analysis
The software package SPSS 11 (Chicago, IL, USA) was used to all data management and analyses. Descriptive data of continuous variables and number (%) for categorical variables was presented as the mean ± the standard deviation (mean ± SD). Comparisons of multiple groups were analyzed with oneway analysis of variance. The χ2 test with Yates correction was used to compare the categorical variables between the two groups respectively. Correlations were evaluated with the Spearman rank test and P < 0.05 was statistically signi cant.

Patients
This study included 111 patients (51 males and 60 females) with COVID-19 in recovery stage after discharged six months. The median age of the patients was 50.3 years (ranges, 24-70 years). Chronic disorder histories were chronic cardiovascular disease (21 person times), chronic respiratory disease (12 person times), chronic cerebrovascular illness (5 person times), and diabetes (10 person times).
* For the B lymphocytes, the comparisons of the IgM(-) and IgG(+) group versus the IgM(-) and IgG(-) group on weeks 0 and 2 were signi cant difference respectively (P<0.05).
For the B lymphocytes, the comparison of the IgM(+) and IgG(+) group versus the IgM(-) and IgG(+) group on weeks 4 was signi cant difference (P<0.05).

Comparison of Patients within IgM(+) and IgG(+) Antibodies
During the 4-week observation period, 12 of 32 patients had IgM(+) converted to IgM(-).The 32 patients were divided into the IgM(+) group and the IgM(-) groups. There were no signi cant differences in age, gender, clinical classi cation of hospitalization and chronic disorders for the two groups (Table 5). The dynamic percentages changes of CD3+, CD8+ and CD4+ for patients in the IgM(+) group were unanimous with those outcomes in the IgM(-) group (P 0.05, Table 6). Compared with the IgM(+) group, the percentages of NK cells and B lymphocytes were signi cantly increased in the IgM(-) group on weeks

Discussion
Mortality of COVID-19 mainly occurs in the hospitalization in the acute stage, which carries a high mortality risk in Wuhan city of China, and the patient is at risk for lung and/or systemic complications [11]. SARS-CoV-2 is a very weird virus, which showed cellular immune de ciency and excessive immune response in the acute stage [12,13].
The production of antibodies is the host's immune response to viral infection, which serum 2019-nCoV IgM/IgG were detectable as early as 4 days and reached a peak in the second week after symptom onset [14,15]. Serological antibody testing may be helpful for the identi cation of suspected patients with negative of nucleic acid and for the diagnosis of asymptomatic infections [16].
The important pro-in ammatory cytokines of hs-CRP and IL-6 cause cascade and amplify cytokine storm [19]. Hs-CRP is a nonspeci c in ammatory marker widely used in the prediction of COVID-19 pneumonia [20], which is not affected by radiotherapy, chemotherapy, corticosteroids. IL-6 is the key proin ammatory cytokines in excessive immune response for SAP, which is a potential, reliable and easy-to-use predictor for COVID-19 prognosis [21]. However, our results suggested that the values of hs-CRP and IL-6 for all patients were within the normal ranges after discharged six months.
Lymphocyte subsets in peripheral blood play an important role in preserving immune function, which cellular immune regulate with each cell restricting and regulating one another. Previous researches revealed that T cell subset counts were signi cantly decreased for COVID-19 patients during hospitalization [22][23][24]. Our ndings suggest that serum lymphocyte subsets counts is correlated with the dynamic changes of serum IgM and IgG and not related to in ammatory cytokines and severity of hospitalization. All testing items of the lymphocyte subsets were 12/110 ( Recent studies had reported that lymphocyte subsets and in ammatory cytokines in plasma were associated with the severity of COVID-19 [25,26]. Liu et al [25] found that the degrees of lymphopenia and proin ammatory cytokines in severe COVID-19 patients were higher than in mild cases, and were associated with the severity of disease. Jiang et al [26] reported that the counts of CD8+ and CD4+ cells of COVID-19 patients were used as predictors of disease severity. Our results tell that the immune status of patients (lymphocyte subsets and in ammatory cytokines) in the recovery stage is completely different from that in the acute stage.
We found that with the conversion of IgM(+) to IgM(-), the patient's immunity gradually improved, mainly manifested by the compensation of NK cells and B lymphocytes. In our study, we analyzed 12 patients with IgM(+) had converted to IgM(-) in the IgM(+) and IgG(+) group. Compared with the IgM(+) group, the percentages of NK cells and B lymphocytes were signi cantly increased in the IgM(-) group on weeks 4 (P<0.05, Table 6).
In conclusion, the dynamic changes of serum IgM and IgG are closely related to patient immunity for patients in recovery stage, which is dominate with CD8+ for IgM(+) and gradually improve by the compensation of NK cells and B lymphocytes with the conversion of IgM(+) to IgM(-). However, the immunity is not recovery with the turning negative of antibodies of SARS-CoV-2. Future large-scale studies are required to clarify the dynamic changes of antibodies and immunity for the throughout course of COVID-19.