Detection of IgM and IgG Antibodies to Human Parvovirus B19 in Sera of Patients with Thymoma-Associated Myasthenia Gravis

Abstract

Much uncertainty still exists about the viral etiology of myasthenia gravis (MG). To address this, we explored the relationship between human parvovirus B19 (PVB19) infection and MG by investigating the presence of PVB19-specific antibodies in serum. A total of 131 patients with MG (including 47 with thymoma-associated MG, 14 with hyperplasia-associated MG, and 70 with unknown thymic lesions) and 172 healthy volunteers were enrolled in this study. Enzyme linked immunosorbent assay was conducted to detect virus-specific antibodies in cell-free serum. The data were analyzed using Pearson chi-square (χ ²) and Fisher's exact tests. In the 131 patients with MG, there was no significant difference between male (53.41 ± 14.65 years) and female (50.19 ± 15.28 years) groups regarding mean age (p > 0.05). Among all MG subgroups, the largest age group comprised participants aged 30–60 years. We found that the frequency of detecting immunoglobulin G (IgG) antibodies against PVB19 VP1 and VP2 was significantly higher among patients with MG (68.70%) than in healthy controls (41.86%) (p < 0.001). In particular, the positive rate for anti-PVB19 IgG in patients with thymoma-associated MG (35/47, 74.47%) was significantly higher than that in healthy participants (72/172, 41.86%; p < 0.001). The findings of this study indicate that PVB19 infection may play a role in the etiopathogenesis of MG, particularly in patients with thymoma-associated MG. The study protocol was registered at ClinicalTrials.gov with the identifier ChiCTR-1900023338.

Introduction

Myasthenia gravis (MG) is an autoimmune neuromuscular disease that is mainly mediated by acetylcholine receptor autoantibodies, muscle-specific kinase autoantibodies, and lipoprotein-related protein 4 autoantibodies, resulting in injury to the postsynaptic muscle membrane (4,13,19). MG is typically characterized by fluctuating muscle weakness and fatigability and can be primarily classified into ocular myasthenia gravis (OMG) and generalized myasthenia gravis (GMG), based on clinical peculiarities (8 –10). Thymic abnormalities, including thymic hyperplasia and thymoma, commonly occur together with MG (3,20,28) and are related to the occurrence of muscle weakness in patients (16). The past decade has seen increasingly rapid advances in understanding regarding the etiology of MG. However, to date, the etiology of MG remains unclear. Studies have shown that MG can be provoked by environmental factors such as hormones, vitamin D, and diet (6,10). Moreover, the disease can likely be triggered by infection, particularly viral infections (6,10). The interplay between genetic factors, environmental infectious agents, and autoimmunity responses jointly contributes to the etiology of MG (10). Exposure to infectious factors, namely, hepatitis E virus (HEV), West Nile virus, and Zika virus, has been shown to be related to the occurrence of MG (12,22,30). In general, however, the results are obviously inconsistent or negative, or only a few cases are studied. Therefore, other viruses should be considered.

Parvovirus B19 (PVB19) is a small single-stranded DNA virus; its genome encodes three proteins that have known functions (17). PVB19 belongs to the family Parvoviridae, subfamily Parvovirinae, and genus Erythroparvovirus. PVB19 is universally distributed worldwide and infects human beings. Although it was named as erythrovirus (26), extensive research has shown that PVB19 can also infect nonerythroid blood cells, which is crucial for the induction of multiple autoimmune diseases (17,23). More recently, studies have emerged reporting findings regarding the role of PVB19 in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis, (RA), and Hashimoto's thyroiditis (HT) (2,7,15,24,29). These findings encouraged us to study the role of PVB19 in MG etiopathogenesis.

There are several manners in which to investigate the role of viruses in the etiology of autoimmune diseases, including detection of viral DNA and protein, and/or virus-specific antibodies in serum and tissue samples (7,12,30). However, some evidence suggests that the serostatus of PVB19 DNA is irrelevant to PVB19 infection in humans (2). Furthermore, simply detecting viral DNA does not provide valid evidence of virus replication (1). On the contrary, detection of virus-specific antibodies to PVB19 indicates the presence of human PVB19 antigen and its replication (25). Therefore, we focused our investigation on the detection of virus-specific antibodies to PVB19. Our recent study (11) demonstrated that PVB19 infection is closely associated with thymic hyperplasia-associated MG, based on several cellular and molecular methods. And very interestingly, we found that PVB19 infection existed in the two cases of thymoma (11). As that study was retrospective, we could not detect PVB19 immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies in patient serum. Therefore, in this study, we aimed to detect PVB19 IgG and IgM antibodies in the serum of patients with MG as well as healthy individuals using serological methods. For the first time, we present serological evidence of PVB19 infection in the patients with MG. This study provides new insights that will be useful in further research into the etiology and possible pathogenesis of MG.

Materials and Methods

Specimen collection

As a prospective study, the researchers were blinded to the pathological diagnosis results regarding thymic abnormities of the included patients and whether they were infected with PVB19. We collected 131 blood samples from inpatients (including 47 with thymoma-associated MG, 14 with hyperplasia-associated MG, and 70 without thymic lesions) who met our inclusion criteria in the department of thoracic surgery and department of neurology of Tangdu Hospital in 2019. All patients provided their signed informed consent before participating in our study. Blood samples were tested for PVB19 infection using protein detection with enzyme linked immunosorbent assay (ELISA). The control group contained a large number of 172 healthy blood donors. Table 1 summarizes the demographic characteristics of the study population.

Table 1.

Baseline Characteristics of Patients with MG and Healthy Controls

Groups	Gender		Age group, year			Mean age, year (SD)
Groups	Male	Female	<30	31–60	>60	Mean age, year (SD)
Experimental group
Total patients with MG^a (n = 131)	63	68	15	72	44	51.74 (16.09)
Ocular MG (n = 45)	25	20	3	26	16	53.58 (16.17)
Generalized MG (n = 80)	34	46	12	40	28	50.08 (16.46)
Thymic hyperplasia-associated MG (n = 14)	6	8	2	10	2	48.29 (14.98)
Thymoma-associated MG (n = 47)	23	24	4	28	15	50.85 (14.11)
Control group (n = 172)	NA

All patients with MG, with or without thymic disorders.

MG, myasthenia gravis; NA, not available (According to the requirements of ethics, the age and gender of blood donors cannot be revealed.); SD, standard deviation.

All patients were followed up at Tangdu Hospital and were diagnosed based on the ITMIG Consensus Statement on the Use of World Health Organization Histological Classification of Thymoma and Thymic Carcinoma (21) and Association of British Neurologists' Management Guidelines for MG (27). All patients with MG and control participants were of Chinese origin. Participants were enrolled according to the following inclusion criteria: patients with a diagnosis of MG with thymic abnormalities such as thymoma and thymic hyperplasia, aged 18 to 85 years, and able to provide their informed consent. Patients were excluded according to the following criteria: history of autoimmune diseases such as HT, RA, SLE, or systemic sclerosis; pregnancy; severe organ dysfunction; and serum lipemia.

Detection of IgM and IgG antibodies in serum samples

All tests were evaluated using a microplate reader (iMark; BioRad, Hercules, CA). Blood samples (2–5 mL) were collected from all participants. After centrifugation (8/2 × g for 10 min at 25°C; Heraeus Multifuge ×3, Thermo Fisher, US), the serum was collected and stored at −70°C. Before assaying, all samples were diluted in sample diluent at 1:100 (10 μL serum in 1.0 mL phosphate buffer), and absorbance was measured at 450 nm. The presence of IgM and IgG antibodies against PVB19 VP1 and VP2 structural proteins was investigated using serum samples from all 303 participants with ELISA (PVB19 IgG/IgM ELISA; IBL International GmbH, Hamburg, Germany). Serum samples were tested to detect IgM and IgG antibodies, reflecting a current or past PVB19 infection. ELISAs were carried out according to the manufacturer's instructions.

The measurement unit was international units (U). Test results were evaluated as follows: 9 U was negative, ≥9 to <11 U was equivocal, and ≥11 U was positive. Repeated tests in 2 to 4 weeks using a fresh blood sample was recommended if the results were equivocal; the sample was judged to be negative if the results of a repeated test were also equivocal. The detection limit of the test was 0.1 U, the intra-assay coefficient of variation (CV) was 1.44–4.48%, and the interassay CV was 10.61–13.96%. The specificity was 99.28% and the sensitivity was 97.42%.

Statistical analysis

Data from the experimental results were analyzed using Pearson chi-square (χ ²) test and Fisher's exact test with IBM SPSS software version 23.0 (IBM Corp., Armonk, NY). A two-sided p-value of <0.05 was considered to indicate a statistically significant difference.

Ethical standards

All work was conducted with the formal approval of the Hospital Ethics Committee (IEC) of the Institution for National Drug Clinical Trials, Tangdu Hospital, Fourth Military Medical University, China (protocol #201907-03), and the trial was registered as per the applicable legislation. Written informed consent was obtained from each participant before the study. The study was implemented in line with the ethical standards set out in the 1964 Helsinki Declaration and its subsequent amendments.

Results

Basic clinical characteristics of patients with MG

As given in Table 1, the mean age of the 131 patients with MG (68 women and 63 men) was 51.74 ± 16.09 years, with no significant difference between male (53.41 ± 14.65 years) and female (50.19 ± 15.28 years) groups regarding mean age (p > 0.05). However, there were obvious differences in the distribution of patients with MG among the different age groups. The age group of 30–60 years contained the largest number of patients among all MG subgroups. The second largest age group comprised participants >60 years old; patients <30 years old formed the smallest age group.

Antibodies to PVB19 VP1 and VP2 in the serum of patients with MG and healthy controls

As given in Table 2, the study results demonstrated that among the 131 patients with MG, 90 (68.70%) were positive for anti-PVB19 IgG antibodies. Specifically, this frequency among patients with thymic hyperplasia-associated MG was 57.14% (8/14), and that among patients with thymoma-associated MG was 74.47% (35/47). The results showed that the positive rate of anti-PVB19 IgG antibody in patients with MG was significantly higher than that in the healthy control group (p < 0.001). MG can be classified as OMG and GMG based on the clinical characteristics. We found no difference between these two subgroups with respect to the positive rate of anti-PVB19 IgG and anti-PVB19 IgM antibodies. However, the frequency of anti-PVB19 IgG in both the OMG (30/45, 66.67%) and GMG (56/80, 70.00%) subgroups was significantly higher than that in the healthy control group (72/172, 41.86%; p = 0.003). In another sense, patients with MG can be divided into those with hyperplasia-associated MG and thymoma-associated MG groups. As shown in the experimental findings, the positive rate of anti-PVB19 IgG antibody among patients with thymoma-associated MG (74.47%) was significantly higher than that in the control group (41.86%; p < 0.001). Surprisingly, there was not a large difference between patients with hyperplasia-associated MG (57.14%) and controls (41.86%; p = 0.267). Finally, there was no difference in the positive rate of anti-PVB19 IgM antibody between all experimental groups (10/175, 5.71%) and the control group (9/172, 5.23%; p > 0.05).

Table 2.

Results of Serological Testing for Parvovirus B19 Antibodies Among Patients with Myasthenia Gravis and Healthy Controls

Groups	Serum-positive, n (%)		p₂ ^b
Groups	PVB19 IgG antibodies	PVB19 IgM antibodies	p₂ ^b
Experimental group
Total patients with MG	90 (68.70)	9 (6.87)	<0.001
Patients with MG, without thymic disorders	35 (63.64)	5 (9.09)	0.005
Ocular MG	30 (66.67)	3 (8.57)	0.003
Generalized MG	56 (70.00)	6 (7.50)	<0.001
Thymic hyperplasia-associated MG	8 (57.14)	0 (0.00)	NS
Thymoma-associated MG	35 (74.47)	3 (6.38)	<0.001
Control group	72 (41.86)	9 (5.23)
p ₁ ^a	<0.001	NS

p ₁ refers to the comparison of different examination items between the entire experimental group and the healthy control group.

p ₂ refers to the comparison of PVB19 IgG antibodies between each experimental subgroup and the healthy control group.

IgG, immunoglobulin G; IgM, immunoglobulin M; NS, not significant; PVB19, parvovirus B19.

Discussion

Our recent study revealed the existence of both PVB19 DNA and proteins in thymic tissues of the patients with thymic hyperplasia-associated MG. Meanwhile, we found that PVB19 DNA and proteins were also detected, respectively, in two cases and one case of thymoma group (11). It indicates that viral infection was closely and mainly related to thymic hyperplasia-associated MG. Hence, the finding prompted us to investigate whether viral infection was also related to thymoma-related MG.

In this study, we found that the frequency of IgG antibodies against PVB19 VP1 and VP2 was significantly higher among patients with MG than in healthy controls. In addition, the positive rate for anti-PVB19 IgG in patients with thymoma-associated MG was also significantly higher than that in healthy participants. To the best of our knowledge, this is the first report of the presence of PVB19 antibody in thymoma-associated MG.

The results of our study also showed that the largest number of patients with OMG and other subgroups of MG was in the age group of 30–60 years, which is in accordance with the findings of Fortin et al. (9). The age of patients with MG, thymoma, or thymic hyperplasia was evenly distributed according to gender. These results show that the included patients were basically consistent with respect to the overall disease distribution among those with MG, thymoma, or thymic hyperplasia. Therefore, our research findings based on these cases can be considered reliable and representative.

Evidence shows that clearance of PVB19 peripheral viremia after acute infection is slower than has been previously thought (18), supporting the hypothesis that PVB19-infected individuals have chronic infection status for a longer period and thus, there is greater opportunity to cause immune disruption. During this infectious period, the humoral immune system plays an important role in clearing viremia by producing specific antibodies against VP1 and VP2, two structural viral proteins of PVB19. Viremia decreases with the emergence of IgM antibody, which is gradually replaced by more persistent IgG antibodies (23). IgM antibodies are detectable in late stages of viremia. However, this stage can last for several weeks or months after acute infection, suggesting a current or very recent infection (18). IgG antibodies are detectable ∼15 days postinfection, with a peak at days 35–40. IgG remains high for several months and persists over a long period, indicating resolution of infection and past or chronic infection (5,23). Moreover, anti-VP1u IgG antibody can provide lifelong immunity in the body and prevent reinfection (14).

In our study, IgG antibodies were detected in the serum of many patients with MG, indicating a past or chronic infection with PVB19. The results of this study demonstrate, for the first time, an association between PVB19 infection and MG, using serological methods in a large cohort study of 131 patients and 172 healthy individuals. The data showed that the positive rate of anti-PVB19 IgG antibody in patients with MG was significantly higher than that in healthy controls. This suggests that PVB19 may be an environmental infectious factor in MG. Moreover, our findings further confirm PVB19 infection in patients with thymoma-associated MG. As shown in the experimental results, the positive rate of anti-PVB19 IgG antibody among patients with thymoma-associated MG was significantly higher than that in the control group. One possible explanation might be that PVB19 is involved in the maintenance of a persistent inflammatory response in the thymus of patients with thymoma, which eventually triggers autoimmunity. Surprisingly, however, we found no correlation between PVB19 infection and thymic hyperplasia-associated MG (p = 0.267), which was possibly due to the small number of included patients with thymic hyperplasia-associated MG (14 patients with MG and thymic hyperplasia).

It is generally accepted that the two subtypes of MG (OMG and GMG) may have different pathogeneses, including different pathogenic antibodies and mean ages of onset. Therefore, it is important to determine whether there are differences between the two subsets of MG. We observed significant differences in PVB19 IgG antibodies between the OMG/GMG and control groups (p = 0.003); however, no significant results were found between the two MG subgroups in our study (p = 0.699). This suggested that PVB19 infection was involved in the thymic pathogenesis of MG but was not relevant to the different clinical subtypes according to clinical manifestations.

In conclusion, for the first time, we detected anti-PVB19 IgG antibodies in the serum of patients with thymoma-associated MG, supporting its association with PVB19 infection. Considerably more research is needed to determine the role of PVB19 in MG etiopathogenesis.

Footnotes

Author Disclosure Statement

The article has been reviewed by all authors, and all of the authors have approved its submission for publication. The authors declare that there is no potential conflict of interest regarding the materials presented in this article.

Funding Information

We thank the National Program on Key Basic Research Project (973 Program) under grant number 2015cb553703 for funding this study.

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