Comparison of Varicella-Zoster Virus Specific Immunity of Patients with Diabetes Mellitus and Healthy Individuals

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1 BRIEF REPORT Comparison of Varicella-Zoster Virus Specific Immunity of Patients with Diabetes Mellitus and Healthy Individuals Shigefumi Okamoto, 1,a Atsuko Hata, 2,a Kay Sadaoka, 1 Koichi Yamanishi, 1 and Yasuko Mori 1,3 1 Laboratory of Virology and Vaccinology, Division of Biomedical Research, National Institute of Biomedical Innovation, and 2 Department of Infectious Diseases, Kitano Hospital, The Tazuke Kofukai Medical Research Institute, Osaka, and 3 Division of Clinical Virology, Department of Microbiology and Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan Cell-mediated immunity (CMI) has been shown to be critical for the prevention and control of varicella-zoster virus (VZV) related diseases. Because a large population-based study has revealed that diabetes mellitus is a risk factor for herpes zoster, we studied VZV-specific immune responses of patients with diabetes mellitus and compared them with those of healthy individuals. In this study, we found that patients with diabetes mellitus had significantly lower CMI to VZV than did healthy individuals. These results suggest that the increased risk for herpes zoster among patients with diabetes mellitus may be related to decreased VZV-specific CMI. Varicella-zoster virus (VZV) causes chickenpox and becomes latent in sensory ganglia. Herpes zoster (HZ) is caused by the reactivation of latent VZV in sensory trigeminal and dorsal root ganglia. Reactivated VZV replicates in the skin and produces the characteristic HZ rash, which is accompanied by acute Received 9 May 2009; accepted 25 June 2009; electronically published 12 October Potential conflicts of interest: The varicella vaccine (Oka/Biken) used in this study was provided by Biken, which produces the vaccine. Financial support: The Ministry of Health, Labour and Welfare of Japan Research, Tokyo, Japan (Promotion of Emerging and Re-emerging Infectious Diseases grant H18-Shinko-013 to Y.M. and A. H.). a S.O. and A.H. contributed equally to this work. Presented in part: 34th International Herpesvirus Workshop, Ithaca NY, July 2009 (poster 5.05). Reprints or correspondence: Dr. Yasuko Mori, Laboratory of Virology and Vaccinology, Div. of Biomedical Research, National Institute of Biomedical Innovation, 7-6-8, Saito-Asagi, Ibaraki, Osaka , Japan (ymori@nibio.go.jp). The Journal of Infectious Diseases 2009; 200: by the Infectious Diseases Society of America. All rights reserved /2009/ $15.00 DOI: / pain and often by postherpetic neuralgia [1]. The reactivation probably occurs when VZV-specific T cell immunity has decreased below some threshold level [1]. In fact, an age-related waning of VZV-specific cell-mediated immunity (CMI) has been demonstrated, which suggests that CMI is important in the prevention and control of VZV infection [2]. The incidence of HZ is substantially greater among persons with certain disorders, including hematologic malignancies, solid tumors, human immunodeficiency virus (HIV) infection, hematopoietic stem cell transplantation, and systemic lupus erythematosis [3]. Furthermore, associations between HZ and diabetes mellitus have been investigated, albeit with different results [4, 5]. Recently, however, a large population-based study clearly showed that diabetes mellitus is an independent risk factor for HZ [6]. Diabetes mellitus is often accompanied by impaired CMI, and previous studies have shown that patients with diabetes mellitus contract infections more often than do individuals without diabetes mellitus [7]. However, no reports have shown whether the VZV-specific CMI is lower in persons with diabetes mellitus than it is in healthy individuals. Therefore, in this study, we performed an interferon (IFN) g enzyme-linked immunospot (ELISPOT) assay to measure the VZV-specific CMI in subjects with and subjects without diabetes mellitus. Patients and methods. Blood samples for the IFN-g ELI- SPOT assay and glycoprotein antigen based enzyme-linked immunosorbent assay (gpelisa) were collected during a single phlebotomy session. This study was approved by the ethical committees of each of the involved institutions. Written informed consent was obtained from all 63 patients with diabetes mellitus and all 67 healthy volunteers. The mean age of the patients with diabetes mellitus was 57.3 years (range, years) and that of the healthy volunteers was 56.2 years (range, years). The age discrepancy between the 2 groups was not significant, by the Mann-Whitney U test ( P p.499). Fortyfour of the patients with diabetes mellitus and 45 of the healthy volunteer were male. Stratified by decade, 14 (22.2%) of the patients with diabetes mellitus were aged years, 23 (36.5%) were aged years, 23 (36.5%) were aged years, and 3 (4.8%) were aged years. Of the healthy volunteers, 15 (22.4%) were aged years, 29 (43.2%) were aged years, 22 (32.8%) were aged years, and 1 (1.5%) was aged years. Among both groups, there was no history of herpes zoster, acquired immunodeficiency syndrome, leukemia, lymphoma, other malignancies, renal failure, bronchial 1606 JID 2009:200 (15 November) BRIEF REPORT

2 asthma, collagen diseases, or chronic infection. Healthy volunteers did not have symptoms of diabetes mellitus. Whole blood was collected from donors into Venoject heparin-containing tubes (Terumo), and peripheral blood mononuclear cells (PBMCs) were isolated and frozen as described previously [8]. The frozen PBMCs were thawed at 37 C and washed twice in complete medium (RPMI 1640 medium supplemented with 5% heat-inactivated fetal bovine serum, 1 mm L-glutamine, 20 mg/ml gentamycin, and 50 mm b-mercaptoethanol) with 40 U/mL of benzonase (Novagen). The cells were suspended in complete medium and counted. After another wash, the cells were resuspended in complete medium at a concentration of cells/ml and used for assays. We confirmed that freezing the PBMCs did not decrease their response to VZV. The IFN-g ELISPOT assay was performed as described elsewhere [8]. Varicella vaccine ( 3 10 pfu/well), which was pro- 4 vided by Biken, was treated with ultraviolet light (5000 J/m 2 ), and used as the VZV antigen. Phytohemagglutinin (PHA)-L 4 (Wako) at 2 mg/ml was included as a positive control. VZV glycoprotein (VZVgp) was purified as described elsewhere [8] with use of the VZV Oka parental strain and Medical Research Council (MRC) 5 cells. The values were calculated by subtracting the optical density from the wells containing MRC-5 glycoprotein from the optical density from the wells containing VZVgp, and the values of the test serum samples were referenced against a standard curve determined from 6 2-fold serial dilutions of the standard. IFN-g ELISPOT counts and gpelisa antibody titers were compared between patients with diabetes mellitus and healthy individuals with use of the Mann-Whitney U test. Spearman s correlation coefficient by rank test was used to analyze the correlation between IFN-g ELISPOT counts and the percentage of hemoglobin A1c (HbA1c) in samples from patients with diabetes mellitus. Results. We studied the CMI to VZV by IFN-g ELISPOT assay in the 63 patients with diabetes mellitus and 67 healthy volunteers. As shown in Figure 1A, the mean ELISPOT counts for the patients with diabetes mellitus were statistically significantly lower than the mean counts for the healthy volunteers. When compared across groups stratified by decade, the mean ELISPOT count was lower for patients with diabetes mellitus in their 40s, 50s, and 60s than it was for healthy volunteers in the same age groups (Figure 1B), but the differences were not statistically significant (for subjects in their 40s, P p.132; in their 50s, P p.257; in their 60s, P p.223). We also measured the ELISPOT count when the PBMCs were stimulated with PHA, an internal quality control for the integrity of the PBMC samples. Figure 1C shows that the ELISPOT values for patients with diabetes mellitus were similar to those for healthy volunteers. The VZV-specific humoral immunity in the 2 groups was then determined using a gpelisa assay. The anti-vzv antibody titer was not significantly different between 2 groups (Figure 1D), even with increasing age (Figure 1E). Among the patients with diabetes mellitus, there were 3 with symptoms of diabetes mellitus related chronic heart diseases and 19 with renal diseases other than renal failure. However, the differences of CMI and humoral immunity against VZV between patients with diabetes mellitus with and without these diseases were not statistically significant (data not shown). Fifty-six people among the patients with diabetes mellitus had HbA1c measured at the same time that IFN-g ELISPOT assay and the gpelisa were performed. Therefore, we next investigated whether there was a correlation between CMI to VZV and the severity of diabetes mellitus, as determined by the level of HbA1c; these findings are shown as a scatter plot (Figure 2A). The slope of the approximation curve indicated only a very small difference. Furthermore, Spearman s correlation coefficient by rank test showed only a weak correlation (correlation coefficient, 0.16). We also found that there was no statistically significant difference in CMI scores between individuals with 8% and 18% HbA1c (Figure 2B). Discussion. Symptomatic VZV reactivation generally results from a decrease in virus-specific CMI. Several reports have shown such a decrease in patients with leukemia, lymphoma, and hematopoietic stem cell transplants and in infants with HIV infection [3]. In contrast, although one study shows that diabetes mellitus is one of the risk factors for herpes zoster [6], the level of VZV-specific CMI in patients with diabetes mellitus has not been investigated. In the present study, we found that the VZV-specific CMI, but not the humoral immunity, was statistically significantly lower among patients with diabetes mellitus than it was among healthy volunteers. These results suggest that the decreased VZV-specific CMI in patients with diabetes mellitus may explain the diabetes mellitus associated increased risk for developing HZ, although socioeconomic factors were not considered in the present study. Patients with diabetes mellitus contract infections more often than do healthy individuals. In a review, Geerlings and Hoepelman [7] concluded that the innate immune responses (ie, chemotaxis, phagocytosis, and cell killing) by polymorphonuclear cells and monocytes/macrophages are lower in patients with diabetes mellitus than they are in healthy individuals. Furthermore, some microorganisms may adhere better to host tissues in a high-glucose environment and therefore become more virulent in patients with diabetes mellitus than in healthy individuals [7]. On the other hand, the serum antibody concentrations in patients with diabetes mellitus are normal, and patients with diabetes mellitus respond to being vaccinated with pneumococcal vaccine just like healthy individuals [9], although the CMI against antigens is lower in patients with diabetes mellitus than in healthy individuals [10, 11]. Further- BRIEF REPORT JID 2009:200 (15 November) 1607

3 Figure 1. Cell-mediated immunity and humoral immunity to varicella zoster virus (VZV) in patients with diabetes mellitus (DM) or healthy volunteers (HV). Enzyme-linked immunospot (ELISPOT) counts (VZV antigen stimulation) were compared between samples obtained from DM (black bar) and HV (white bar) in total (A) or stratified by age (40 49, 50 59, and years of age; B). ELISPOT counts (phytohemagglutinin [PHA] stimulation; C) were compared between DM and HV samples. Glycoprotein antigen based enzyme-linked immunosorbent assay (gpelisa) antibody in serum samples from DM and HV subjects as a single group (D) or stratified by age (40 49, 50 59, and years of age; E). Data are shown as the mean value standard error of the mean. PBMC, peripheral blood mononuclear cell; SFC, spot-forming cell. more, a recent report has shown that insulin degrading enzyme, which may be associated with the pathogenesis of type 2 diabetes, is a cellular receptor mediating VZV infection and cellto-cell spread [12]. This report and our observation suggest that decreased immune responses in patients with diabetes mellitus might underlie their decreased protection against microorganisms. Heymann et al [6] have reported that individuals with a diagnosis of diabetes mellitus were at significantly higher risk for HZ, regardless of age, among the patients they tested (odds ratio [OR] in subjects!45 years old, 1.61; OR in subjects years old, 1.5; OR in subjects 65 years of age, 1.5). In our study, the mean IFN-g ELISPOT count for anti-vzv CMI was lower in patients with diabetes mellitus than it was in healthy volunteers for subjects in their 40s, 50s, and 60s, although the differences were not statistically significant (Figure 1B). Presumably this is because the sample size was too small. Therefore, a large cohort study with age-matched control subjects is needed to determine the exact relationship between CMI to VZV and diabetes mellitus. Heymann et al [6] also studied the correlation between the incidence of HZ and severity of diabetes mellitus. Although individuals!45 years old with high levels of HbA1c (18%) had a statistically significantly increased risk of HZ, compared with patients whose HbA1c level was!5%, similar differences in risk were not observed among subjects 45 years of age. In the present study, we found no significant differences in CMI between individuals with 8% and those with 18% HbA1c (Figure 2B). However, because the sample size was small, a large cohort study will be necessary to provide an exact answer to the question of whether there is a correlation between the incidence of HZ and severity of diabetes mellitus. Humoral immunity is thought to be less important than CMI in host defense [1]. Our study also suggests that the mean of anti-vzv antibody titer in patients with diabetes mellitus is almost equal to that in healthy volunteers. VZV-specific anti JID 2009:200 (15 November) BRIEF REPORT

4 Figure 2. Correlation between cell-mediated immunity to varicella zoster virus (VZV) and the severity of diabetes mellitus. A, Enzyme-linked immunospot (ELISPOT) counts (VZV antigen stimulation) and hemoglobin A1c (HbA1c) levels, presented as a scatter plot. B, ELISPOT counts were compared among subjects with!7%, 7% 8%, and 18% HbA1c. Data are shown as the mean standard error of the mean. PBMC, peripheral blood mononuclear cell; SFC, spot-forming cell. body did not decrease with increasing age, whereas VZV-specific CMI did increase [13]. Because increasing age induces a significant increase in the development of HZ, humoral immunity may not be associated with protection from developing HZ. The herpes zoster vaccine that we used is licensed in the United States and Europe for use among the elderly population. Oxman et al [14] demonstrated that the vaccine reduces the risk of developing HZ by 51.3% in subjects 60 years old and that it has a 66.5% efficacy for preventing postherpetic neuralgia BRIEF REPORT JID 2009:200 (15 November) 1609

5 in this age group. This vaccine enhances both the humoral immune responses and the CMI to VZV [1, 2, 15]. In the present study, the patients with diabetes mellitus had a lower CMI response to VZV than did healthy volunteers, whereas the ELISPOT value with PHA stimulation was very similar for the patients with diabetes mellitus and healthy volunteers. Because the PHA response assays the integrity of PBMC samples [2], our results suggest that the PBMCs of patients with diabetes mellitus are immunologically similar to those of healthy volunteers. The result is raised a hypothesis that the HZ vaccine will boost VZV-specific immunity in patient with diabetes mellitus, which should be determined. Acknowledgments We thank Ms. Yuko Ueda (National Institute of Biomedical Innovation) and Ms. Mieko Matsuda and Ms. Fukue Inoue (Kitano Hospital, Tazuke Kofukai Medical Research Institute), for their technical assistance. Varicella vaccine and antigens for gpelisa were kindly provided by Dr. Yasuyuki Gomi and Mr. Toyokazu Ishikawa (Biken, Kanonji Institute, Research Foundation for Microbial Diseases of Osaka University). References 1. Arvin AM. Varicella-zoster virus. Clin Microbiol Rev 1996; 9: Levin MJ, Smith JG, Kaufhold RM, et al. Decline in varicella-zoster virus (VZV) specific cell-mediated immunity with increasing age and boosting with a high-dose VZV vaccine. J Infect Dis 2003; 188: Harpaz R, Ortega-Sanchez IR, Seward JF. Prevention of herpes zoster: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 2008; 57:1 30; quiz CE Brown GR. Herpes zoster: correlation of age, sex, distribution, neuralgia, and associated disorders. South Med J 1976; 69: Ragozzino MW, Melton LJ 3rd, Kurland LT, Chu CP, Perry HO. Population-based study of herpes zoster and its sequelae. Medicine (Baltimore) 1982; 61: Heymann AD, Chodick G, Karpati T, et al. Diabetes as a risk factor for herpes zoster infection: results of a population-based study in Israel. Infection 2008; 36: Geerlings SE, Hoepelman AI. Immune dysfunction in patients with diabetes mellitus (DM). FEMS Immunol Med Microbiol 1999; 26: Sadaoka K, Okamoto S, Gomi Y, et al. Measurement of varicella-zoster virus (VZV) specific cell-mediated immunity: comparison between VZV skin test and interferon-gamma enzyme-linked immunospot assay. J Infect Dis 2008; 198: Lederman MM, Schiffman G, Rodman HM. Pneumococcal immunization in adult diabetics. Diabetes 1981; 30: Casey JI, Heeter BJ, Klyshevich KA. Impaired response of lymphocytes of diabetic subjects to antigen of Staphylococcus aureus. J Infect Dis 1977; 136: Plouffe JF, Silva J Jr., Fekety R, Allen JL. Cell-mediated immunity in diabetes mellitus. Infect Immun 1978; 21: Li Q, Ali MA, Cohen JI. Insulin degrading enzyme is a cellular receptor mediating varicella-zoster virus infection and cell-to-cell spread. Cell 2006; 127: Takahashi M, Okada S, Miyagawa H, et al. Enhancement of immunity against VZV by giving live varicella vaccine to the elderly assessed by VZV skin test and IAHA, gpelisa antibody assay. Vaccine 2003; 21: Oxman MN, Levin MJ, Johnson GR, et al. A vaccine to prevent herpes zoster and postherpetic neuralgia in older adults. N Engl J Med 2005; 352: Levin MJ, Oxman MN, Zhang JH, et al. Varicella-zoster virus-specific immune responses in elderly recipients of a herpes zoster vaccine. J Infect Dis 2008; 197: JID 2009:200 (15 November) BRIEF REPORT