Assessment of the humoral immune status of varicella-zoster virus in patients with diffuse connective tissue diseases

Background Diffuse connective tissue diseases (DCTDs) require long-term immunosuppressive treatment, increasing the risk of varicella-zoster virus (VZV) infection. This study aims to evaluate the humoral immune status against VZV in DCTD patients and explore factors that may influence their immune levels. Methods This is a retrospective cohort study that collected data from adult DCTD patients (≥18 years) attending our outpatient clinic. The geometric mean concentration (GMC) of VZV-specific IgG antibodies in the patients’ sera was measured using the enzyme-linked immunosorbent assay (ELISA). Results A total of 280 RA patients, 272 SLE + MCTD patients and 280 healthy controls were included. SLE + MCTD patients had significantly higher VZV IgG antibody levels than RA patients (p < 0.05) but showed no significant difference compared to healthy controls (p > 0.05). Notable differences were observed particularly among female patients and those aged 30–49 years, (p < 0.05). SLE + MCTD patients in an active disease state had significantly higher VZV IgG antibody titers than RA patients (p < 0.05). Additionally, patients with a history of herpes zoster, regardless of being in the SLE + MCTD, RA, or control group, exhibited higher VZV IgG titers (p < 0.05). Conclusion Although DCTD patients, particularly those with SLE and MCTD, exhibit higher VZV IgG antibody levels, they still face a higher risk of developing herpes zoster (HZ), which may be related to their underlying disease and immunosuppressive treatment. The presence of antibodies alone may not provide complete protection, necessitating consideration of cellular immune mechanisms. It is recommended to enhance monitoring of VZV antibody levels in high-risk patients and consider herpes zoster vaccination to reduce HZ-related complications.

Recent research has shed light on the humoral immune status of VZV in DCTD patients.Krasselt et al. (10) found that patients with SLE and RA had altered humoral immunity to VZV compared to healthy controls.Their study revealed lower VZV-specific IgG levels in RA patients, while SLE patients showed no significant difference from controls.However, the implications of these findings for HZ risk and the impact of various treatment regimens remained unclear.In particular, there is a lack of in-depth understanding of several key issues: whether there is a difference in VZV-specific IgG antibody levels between DCTD patients and healthy individuals, whether significant differences exist in VZV immune status among patients with different types of DCTDs [RA (11), SLE (10) and MCTD], how factors such as disease duration, disease activity, and immunosuppressive treatment (12)(13)(14)(15) affect VZV immune levels in DCTD patients, and whether the VZV immune status of DCTD patients is related to their risk of developing herpes zoster.Understanding the VZV immune status of this special population is of great significance for formulating prevention strategies and optimizing treatment plans.Therefore, this study aims to evaluate the humoral immune status of DCTD patients against VZV through a retrospective cohort study and explore factors that may influence their immune levels.We will also compare the VZV antibody levels of these patients with those of a healthy control group to determine whether DCTD patients have a higher risk of herpes zoster.Through this study, we hope to provide a scientific basis for the prevention, diagnosis, and treatment of DCTD patients, thereby improving their quality of life and prognosis.

Study design and participants
Over an eight-month period, all adult DCTD patients (≥18 years) attending routine consultations at our outpatient clinic were invited to participate in this retrospective cohort study.There were no further restrictions on gender, age, or specific treatments to ensure a representative sample of outpatients.Diagnoses were based on the clinical judgment of rheumatologists, typically following the classification criteria established by the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) (16) in 2010.DCTD activity, mainly according to the last disease activity score 28, Systemic Lupus Erythematosus disease activity index and EULAR primary Sjögren's syndrome disease activity index score, or rheumatologists criteria.The condition is classified as stable with no activity and mild activity, while other patients are classified as active.The treatment drug regimen for enrolled patients has not undergone significant adjustments in the past year.Clinical data and herpes zoster history were taken during the visit or was available from the existing medical records.Herpes zoster infection occurred within the past year.Vaccination data was sourced from the Jiangsu Provincial Integrated Service Management Information System for Vaccination.Healthy controls were randomly selected from routine hospital diagnoses, matched by age and gender to the case group.

Serum collection and serological testing
Approximately 5 mL of venous blood was drawn from each participant and stored at −70°C until analysis.VZV-specific IgG antibodies were measured using ELISA at the central laboratory of the Affiliated Suqian First People's Hospital of Nanjing Medical University.The quantification of varicella immunoglobulin G (IgG) antibodies' geometric mean concentrations (GMC) is conducted through glycoprotein-based enzyme-linked immunosorbent assay (gpELISA).The ELISA kit used (from Institut Virion/Serion GmbH) measures specific IgG class antibodies against the viral envelope glycoprotein of VZV bound to microtitration wells.A positive result was defined as a varicella virus IgG antibody concentration ≥50 mIU/mL.

Statistical analysis
Statistical analysis was performed using SPSS v22.0.Continuous data were described using means (M) and standard deviations (SD), while categorical data were described using absolute or relative frequencies.Fisher's exact test was employed to compare categorical variable frequencies.For continuous data comparisons, normality tests were followed by student's t-test or Mann-Whitney U test.A p-value <0.05 was considered statistically significant.

Results
A total of 280 RA patients (mean age 50.0 ± 13.3 years) and 272 SLE + MCTD patients (with SLE accounting for approximately 50%, mean age 49.5 ± 14.3 years) were included.Additionally, 280 healthy controls (mean age 50.1 ± 10.2 years) were randomly selected from routine diagnoses.None of the participants had received the herpes zoster vaccine.Detailed information about the included patients and control group is presented in Table 1, and the medication details for the patient group are shown in Table 2.There were no significant differences in age and gender between the patient and healthy control groups.
The VZV IgG antibody titers in SLE + MCTD patients were slightly lower than those in the control group (p > 0.05) but significantly higher than in RA patients (p = 0.017).Additionally, the VZV IgG antibody positivity rate in the control group was higher than in both RA patients and SLE + MCTD patients (p = 0.057).Stratified analysis (excluding those with a history of herpes zoster) showed that among the two patient groups (SLE + MCTD and RA), the VZV IgG concentration in SLE + MCTD patients treated with a combination of glucocorticoids and conventional + biological DMARDs (GC/csDMARDs + bDMARDs) was significantly higher than that in RA patients (p < 0.05).Age stratification revealed that ODCTDs patients aged 30-49 had higher VZV IgG antibody titers compared to RA patients (p < 0.05), while no statistically significant differences were observed in other age groups (p > 0.05).Furthermore, when the disease was active, the VZV IgG antibody titers in the SLE + MCTD patient group were 3670.9 (2255.8-5086.0)mIU/mL, significantly higher than in the RA patient group, which had titers of 2092.9 (1513.1-2672.6)mIU/ mL (p < 0.05) (Figure 1).
In our cohort, the average age at which SLE + MCTD patients developed herpes zoster (HZ) was 46.4 ± 13.3 years, compared to 42.3 ± 11.2 years for RA patients.The incidence rates of HZ in the SLE + MCTD patient group, the RA patient group, and the control group were 8.1, 3.2, and 2.9%, respectively (p < 0.05).The average time from the onset of rheumatic disease to the development of HZ was shorter in SLE + MCTD patients compared to RA patients (3.3 ± 1.9 vs. 6.4 ± 2.9 years, p < 0.05, Table 1).When comparing the VZV IgG levels between SLE + MCTD patients with positive HZ history and any other group (including SLE + MCTD patients without HZ history, but also RA and controls groups), SLE + MCTD patients show higher titers after at least one episode of HZ within 2 years (Supplementary Table S1).In the SLE + MCTD patient group, the use of glucocorticoids, regardless of dosage, was associated with a higher risk of developing HZ (p < 0.05).

TABLE 1
Characteristics of the studied patients and controls.