Introduction

Since December 2019, the 2019 novel coronavirus (2019-nCoV) pneumonia pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has threatened millions of people worldwide [1]. By March 2020, the coronavirus disease 2019 (COVID-19) epidemic had been effectively controlled in Wuhan city, China. Infected patients were hospitalized and discharged from the hospital after recovering. Currently, almost all COVID-19 patients in China are in the recovery stage following infection and therapy [2].

Presently, due to the lack of effective antiviral drugs [3] and vaccines [4], convalescent plasma therapy [5] and specific human monoclonal antibodies [6], the treatment of COVID-19 remains the greatest challenge for medical staff and scientific researchers [7]. As a novel coronavirus, the dynamic immunity to and pathogenesis of this disease in the human body are unclear [8,9,10].

In this study, we focused on COVID-19 patients in the recovery stage after having been discharged 6 months and aimed to evaluate the dynamic changes of the IgM and IgG antibodies, the changes in the plasma levels of hypersensitive C-reactive protein (hs-CRP) and interleukin-6 (IL-6), the alterations in plasma lymphocyte subsets, and potential correlations between the dynamic changes in serum IgM and IgG levels and patient immunity.

Materials and methods

Patients

From July 1, 2020 to August 31, 2020, consecutive COVID-19 patients in the recovery stage after 6 month hospital discharge who were admitted to the Department of Novel Coronavirus Pneumonia Rehabilitation Clinic at Hubei University of Chinese Medicine Affiliated Hubei Hospital of Chinese Medicine were recruited.

All included patients who were admitted to our department met the criteria of the Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (Trial Version 7) proposed by the National Health Commission of China, which were diagnosed based on the most common symptoms of fever, generalized weakness and dry cough, the positive of nucleic acid detection in the nasal and throat swab sampling or other respiratory tract samplings by real-time polymerase chain reaction (PCR), and the multifocal ground glass changes on chest CT scan [11].

The severity in the acute stage was classified as mild, moderate, severe and critical according to the criteria of the National Health Commission of China. Mild was mild clinical symptoms and no pneumonia manifestations on imaging. Moderate was fever, respiratory symptoms and pneumonia manifestations on imaging. Severe was met one of the following: shortness of breath and respiratory rate ≥ 30 time/minute; oxygen saturation of finger ≤ 93% in resting state; arterial partial pressure of oxygen (PaO2)/fraction of inspiration oxygen (FiO2) ≤ 30 mmHg (1 mmHg = 0.133 kPa); significant progression of the lesion > 50% within 24–48 h on pulmonary imaging. Critical was met one of the following: respiratory failure with required mechanical ventilation; shock; combining with other organ failure and required Intensive Care Unit (ICU) monitoring treatment [11].

Inclusion and exclusion criteria

We performed a 4 week 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 from the patients (or their families).

The exclusion criteria were as follows: (1) patients discharged less than 6 months prior; (2) patients whose SARS-CoV-2 nucleic acid turned positive again; (3) patients presenting with infection after discharge; (4) patients who underwent surgery during discharge; (5) pregnant or lactating females; (6) patients with cancer; (7) patients with organ dysfunction during discharge; (8) patients with immune system diseases; and (9) patients in other studies.

Treatment

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

Ethics

Ethical approval was approved by the Research Ethical Committee of Hubei Hospital of Chinese Medicine on April 23, 2020 (grant no. HBZY2020-C26-01).

Data collection

The following data were collected from a consecutive series of 111 patients in the 4-week observation stage: (i) demographic data, including sex, age, severity of hospitalization, and histories of chronic disorder (chronic cardiovascular disease, chronic respiratory disease, chronic cerebrovascular illness, and diabetes); and (ii) serum biochemical data, including 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].

Peripheral venous blood samples were obtained using strict aseptic techniques from patients at weeks 0, 2 and 4. IgM and IgG were detected by the colloidal gold method using a novel coronavirus (2019-nCoV) IgM/IgG antibody detection kit (Livzon Reagent Co., Ltd., Zhuhai, China). Hs-CRP was determined by a Modular PPI automatic biochemical analyser (Roche Diagnostics (Shanghai) Co., Ltd., Shanghai, China). IL-6 was determined by a fully automatic electrochemiluminescence immunoassay system (Cobas e411 analyser series, Roche Diagnostics (Shanghai) Co., Ltd., Shanghai, China). Lymphocyte subsets were detected and counted by a BD FACSCalibur flow cytometer (BD, Bioscience, CA, USA). The following antibodies (BD, Bioscience, CA, USA) were used: FITC anti-human CD14, FITC-conjugated anti-CD4, ECD-conjugated anti-CD3, PC5-conjugated anti-CD8, PE-conjugated anti-CD19, and PC7-conjugated anti-CD16CD45.

Statistical analysis

The software package SPSS 11 (Chicago, IL, USA) was used for all data management and analyses. The descriptive data of continuous variables and the numbers (%) of categorical variables are presented as the mean ± standard deviation (mean ± SD). Multiple groups were compared with one-way analysis of variance. The χ2 test with Yates correction was used to compare categorical variables between the two groups. Correlations were evaluated with the Spearman rank test, and P < 0.05 was considered statistically significant.

Results

Patients

This study included 111 patients (51 males and 60 females) with COVID-19 in the recovery stage after having been discharged for 6 months. The median age of the patients was 50.3 years (range, 24–70 years). Chronic disorder histories in this population included chronic cardiovascular disease (21 person times), chronic respiratory disease (12 person times), chronic cerebrovascular illness (5 person times), and diabetes (10 person times).

The values of hs-CRP and IL-6 for all patients were within normal ranges (0–3 mg/L and < 7 pg/mL, respectively) at weeks 0, 2 and 4, and the comparisons of groups were meaningless.

A 54-year-old male patent with IgM(+) and IgG(−) was symptomless, had negative nucleic acid, 2019-nCoV open reading frame gene and 2019-nCoV nucleoprotein gene tests, had no infection on chest CT manifestations, and had no chronic respiratory disease. Three antibody tests for IgM and IgG revealed a status of IgM(+) and IgG(−). The lymphocyte subsets of the patient are given in Table 1.

Table 1 Lymphocyte subsets of the IgM(+) and IgG(−) patient
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Comparison of patients with different IgM and IgG antibodies

There were 110 patents included in the analysis. Thirty-two (29.1%) of the 110 patients were IgM(+) and IgG(+),38 (34.5%) were IgM(−) and IgG(+), and 40 (36.4%) were IgM(−) and IgG(−).

There were no significant differences in age, sex, clinical classification of hospitalization, or chronic disorders among the IgM(+) and IgG(+), IgM(−) and IgG(+), IgM(−) and IgG(−) groups (Table 2).

Table 2 Characteristics of patients with different IgM and IgG antibodies
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The number of patients with all lymphocyte subset testing items within normal ranges was 12/110 (10.9%) at week 0, 15/110 (13.6%) at week 2 and 18/110 (16.4%) at week 4. Specifically, 0 patients (0/32, 0.0%) in the IgM(+) and IgG(+) group, 4 (4/38, 10.5%) in the IgM(−) and IgG(+) group, and 8 (8/40, 20.0%) in the IgM(−) and IgG(−) group at week 0; 1 patient (1/32, 3.1%) in the IgM(+) and IgG(+) group, 5 (5/38, 13.2%) in the IgM(−) and IgG(+) group, and 9 (9/40, 22.5%) in the IgM(−) and IgG(−) group at week 2; and 3 patients (3/32, 9.4%) in the IgM(+) and IgG(+) group, 6 (6/38, 15.8%) in the IgM(−) and IgG(+) group, and 9 (9/40, 22.5%) in the IgM(−) and IgG(−) group at weeks 4 showed values for these items in the normal range (Table 3).

Table 3 Characteristics of total lymphocyte subsets in patients with differences in IgM and IgG antibodies
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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 (P < 0.05, Table 4). The comparisons of the three groups for the percentages of CD8 + at three time points, the IgM (+) and IgG (+) group was highest, IgM (-) and IgG (-) group was the lowest (P < 0.05, Table 4). The comparisons of the three groups for the percentages of NK cells and B lymphocytes at three time points, the IgM(−) and IgG(−) group was highest, IgM (+) and IgG (+) group was the lowest (P < 0.05, Table 4).

Table 4 Comparison of the dynamic percentage changes in lymphocyte subsets in patients with differences in IgM and IgG antibodies
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The percentages of CD8 + cells, NK cells and B lymphocytes in the IgM(+) and IgG(+) group were quite different from those in the IgM(−) and IgG(+) group and the IgM(−) and IgG(−) group, with much higher percentages of CD8 + cells and much lower percentages of NK cells and B lymphocytes at weeks 0, 2 and 4 (P < 0.05, Table 4). The comparisons of the three groups for the percentages of CD8 + cells at the three time points showed that the IgM(+) and IgG(+)group had the highest percentages, and the IgM(−) and IgG(−) group had the lowest percentages (P < 0.05, Table 4). The comparisons of the three groups for the percentages of NK cells and B lymphocytes at the three time points showed that the IgM (-) and IgG (-) group had the highest percentages, and the IgM (+) and IgG (+) group had the lowest percentages (P < 0.05, Table 4).

Based on the above results, we selected the candidate markers for correlation analysis, which was statistically significant in the analysis. The comparisons of the IgM(+) and IgG(+) group (r = 0.1711) versus the IgM(−) and IgG(+) group (r = 0.1579) (U = 0.0536, P = 0.9572), the IgM(+) and IgG(+) group (r = 0.1711) versus the IgM(−) and IgG(−) group (r = 0.1541) (U = 0.0700, P = 0.9442), and the IgM(−) and IgG(+) group (r = 0.1579) versus the IgM(−) and IgG(−) group (r = 0.1541) (U = 0.0165, P = 0.9868) were relevant, respectively (Table 5).

Table 5 Correlation analysis of lymphocyte subsets in patients with differences inIgM and IgG antibodies
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Comparison of patients within the IgM(+) and IgG(+) antibodies group

During the 4 week observation period, 12 of 32 patients converted from IgM(+) to IgM(−). The 32 patients were divided into the IgM(+) group and the IgM(−) group. There were no significant differences in age, sex, clinical classification of hospitalization or chronic disorders between the two groups (Table 6).

Table 6 Characteristics of patients in the IgM(+) and IgG(+) antibodies group
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The dynamic percentage changes in CD3 + , CD8 + and CD4 + cells for patients in the IgM(+) group were identical to those in the IgM(−) group (P > 0.05, Table 7). Compared with those of the IgM(+) group, the percentages of NK cells and B lymphocytes were significantly increased in the IgM(−) group at week 4 (P < 0.05, Table 7). In the IgM(−) group, the percentage of B lymphocytes was significantly greater at week 4 than at week 0 (P < 0.05, Table 7). We selected NK cells and B lymphocytes for correlation analysis, which was statistically significant. The comparisons of patients with IgM(+) (r = 0.2113) or IgM(−) (r = 0.2595) in the IgM(+) and IgG(+) antibodies group were relevant (U = 0.1238, P = 0.9015, Table 8).

Table 7 Comparison of the dynamic percentages changes in lymphocyte subsets in patients with IgM(+) or IgM(−) in the IgM(+) and IgG(+) antibodies group
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Table 8 Correlation analysis of lymphocyte subsets in patients with IgM(+) or IgM(−) in the IgM(+) and IgG(+) antibodies group
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Discussion

Mortality from COVID-19, which is high in Wuhan city, China, mainly occurs during hospitalization in the acute stage, during which the patient is at risk for lung and/or systemic complications [12]. SARS-CoV-2 is a very strange virus that can lead to cellular immune deficiency and an excessive immune response in the acute stage [13, 14].

The production of antibodies is the host’s immune response to viral infection; in the case of COVID-19, serum 2019-nCoV IgM/IgG are detectable as early as 4 days and reached a peak in the second week after symptom onset [15, 16]. Serological antibody testing may be helpful for the identification of suspected patients who show negative results from nucleic acid tests and in the diagnosis of asymptomatic infections [17].

The importance of antibody detection in convalescent patients seems to be completely different from that in infected patients [18, 19]. In this report, we enrolled 111 COVID-19 patients in the recovery stage after having been discharged for six months: 1 IgM(+) and IgG(−), 32 IgM(+) and IgG(+), 38 IgM(−) and IgG(+), and 40 IgM(−) and IgG(−) patients.

The important pro-inflammatory cytokines of hs-CRP and IL-6 cause cascade and amplify cytokine storm [20]. Hs-CRP is a nonspecific inflammatory marker that is widely used in the prediction of COVID-19 pneumonia [21] and is not affected by radiotherapy, chemotherapy, or corticosteroids. IL-6 is the key proinflammatory cytokine in the excessive immune response to SAP and is a potential, reliable and easy-to-use predictor of COVID-19 prognosis [22]. However, our results indicate that the values of hs-CRP and IL-6 for all patients were within the normal ranges 6 months after discharge.

The lymphocyte subsets in peripheral blood play an important role in preserving immune function, in which the immune cells restrict and regulate each other. Previous studies revealed that T cell subset counts were significantly decreased for COVID-19 patients during hospitalization [23,24,25]. Our findings suggest that serum lymphocyte subset counts are correlated with dynamic changes in serum IgM and IgG and are not related to inflammatory cytokines or the severity of the disease during hospitalization. The number of patients with all lymphocyte subset testing items within normal ranges was 12/110 (10.9%) at week 0, 15/110 (13.6%) at week 2 and 18/110 (16.4%) at week 4. The percentages of CD8 + , NK cells and B lymphocytes in the IgM(+) and IgG(+) group were quite different from those in the IgM(−) and IgG(+) group and the IgM(−) and IgG(−) group, with much higher percentages of CD8 + cells and much lower percentages of NK cells and B lymphocytes at weeks 0, 2 and 4. The correlation analysis among the three groups for the percentages for CD8 + , NK cells and B lymphocytes were relevant.

Recent studies have reported that plasma levels of lymphocyte subsets and inflammatory cytokines are associated with the severity of COVID-19 [26, 27]. Liu et al. [26] found that the degrees of lymphopenia and the levels of pro-inflammatory cytokines in severe COVID-19 patients were higher than those in mild cases and were associated with the severity of disease. Jiang et al. [27] reported that the counts of CD8 + and CD4 + cells in COVID-19 patients could be used as predictors of disease severity. Our results indicate that the immune status of patients (lymphocyte subsets and inflammatory cytokines) in the recovery stage is completely different from that in the acute stage.

We found that on conversion from IgM(+) to IgM(−), the patient’s immunity gradually improved, mainly manifested by the compensation in NK cells and B lymphocytes. In our study, we analyzed 12 patients with IgM(+) who converted to IgM(−) in the IgM(+) and IgG(+) group. Compared with those in the IgM(+) group, the percentages of NK cells and B lymphocytes were significantly increased in the IgM(−) group at week 4. The correlation analysis of NK cells and B lymphocytes for patients with IgM(+) or IgM(−) in the IgM(+) and IgG(+) antibodies group was relevant.

In conclusion, the dynamic changes in serum IgM and IgG are closely related to immunity for patients in the recovery stage, which is dominated by CD8 + for IgM(+) patients and gradually improves by the compensation of NK cells and B lymphocytes when IgM(+) converts to IgM(−). However, immunity does not recover when the patients test negative for SARS-CoV-2 antibodies. Future large-scale studies are required to clarify the dynamic changes in antibodies and immunity throughout the course of COVID-19.