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Gender influence in EBV antibody response in multiple sclerosis patients from Kuwait
Journal of Neuroimmunology 285 (2015) 57–61
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Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Gender influence in EBV antibody response in multiple sclerosis patients from Kuwait Rabeah Al-Temaimi a,⁎, Raed Alroughani b,c, Sindhu Jacob d, Fahd Al-Mulla d a
Human Genetics Unit, Department of Pathology, Faculty of Medicine, Kuwait University, Kuwait Division of Neurology, Department of Medicine, Amiri Hospital, Kuwait, Kuwait Neurology Clinic, Department of Medicine, Dasman Diabetes Institute, Kuwait d Molecular Pathology Unit, Department of Pathology, Faculty of Medicine, Kuwait University, Kuwait b c
a r t i c l e
i n f o
Article history: Received 2 March 2015 Received in revised form 18 May 2015 Accepted 20 May 2015 Available online xxxx Keywords: Multiple sclerosis Kuwait Epstein–Barr virus EBNA1 VCA HLA-DRB1*1501
a b s t r a c t Background: Epstein–Barr virus (EBV) infection is implicated with multiple sclerosis (MS) risk, exacerbation, and progression. The HLA-DRB1*1501 haplotype is a strong MS risk factor consistently documented in MS populations. There are no studies of EBV infections and HLA-DRB1*1501 haplotype associating with MS from Kuwait where MS prevalence has increased significantly. Objectives: To determine the association of EBV infection with MS incidence, and to investigate HLA-DRB1*1501 as a potential genetic risk factor for MS in Kuwait. Methods: This is a case–control study involving 141 MS patients and 40 healthy controls. Antibody titers against EBV antigens' viral capsid antigen (VCA) and Epstein–Barr nuclear antigen 1 (EBNA1) were measured using enzyme-linked immunosorbent assays. HLA-DRB1*1501 haplotype assessment was done using rs3135005 TaqMan genotyping assay. Results: Antibody titers against EBV were significantly elevated in MS patients compared to healthy controls (anti-EBNA1, p = 0.008; anti-VCA, p = 0.028). MS males had higher antibody titers to EBNA1 than healthy male controls (p = 0.005) and female MS patients (p = 0.03). HLA-DRB1*1501 haplotype genotypes failed to generate a risk association with MS or EBV antibody titers (p = 0.6). Conclusion: An increased immune response to EBV infection is associated with MS incidence influenced by the type of antigen and sex. HLA-DRB1*1501 haplotype is not associated with MS risk in our Kuwaiti MS cohort. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is an inflammatory demyelinating disorder resulting from an autoimmune reaction against myelin and myelin associated antigens in the central nervous system (CNS), however the exact etiology remains unknown. Repeated demyelination events in the white matter result in subsequent neuro-axonal degeneration and oligodendrocyte cell death in the CNS manifesting primarily as lesions in the white matter of the brain and spinal cord causing perturbed central sensory and motor nerve conduction. MS incidence is 2:1 female:male ratio at a typical age of onset between 20–40 years of age (Liguori et al., 2000). MS is considered a multi-factorial complex disorder where genetic and environmental factors play a role in MS pathogenesis and relapse risk (Milo and Kahana, 2010). Kuwait has become a high-risk area for MS, its prevalence has increased from 4.4 in 1990 to 85 cases per 100,000 individuals in 2014 (Al-Din et al., 1990; Alroughani et al., 2014). One of the most consistently reported ⁎ Corresponding author at: Human Genetics Unit, Dept. of Pathology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait. E-mail address: [email protected] (R. Al-Temaimi).
http://dx.doi.org/10.1016/j.jneuroim.2015.05.021 0165-5728/© 2015 Elsevier B.V. All rights reserved.
association with MS risk is the association of viral infections, specifically Epstein–Barr virus (EBV) infection (Owens and Bennett, 2012; Lucas and Taylor, 2012). EBV is a human gamma-herpes virus that specifically infects nasopharyngeal epithelial cells and resting B-lymphocytes. EBV has the ability to activate or persist in a latent phase within the cells of infected individuals throughout their lives. EBV can mimic the stimuli of antigens and T-cell receptors in activating naïve B-lymphocytes into antigen specific memory B-cells. As infected memory B-cells differentiate into plasma cells EBV switches to lytic reproductive phase to produce new EBV particles (Laichalk and Thorley-Lawson, 2005). Human immune response against flourishing EBV production is to eliminate production houses; mainly EBV-infected plasma cells, effectively via the action of cytotoxic CD8+ T-cells (Hislop et al., 2007). EBV infection specificity to immune cells has resulted in its association with the incidence of several autoimmune disorders and malignancies in individuals predisposed to such disorders. Systemic lupus erythematosus, rheumatoid arthritis, Burkitt's lymphoma, and Hodgkin's lymphoma are examples of such disorders (Toussirot and Roudier, 2008; Saha and Robertson, 2011). In recent years, the role of EBV infections as an MS risk factor preluding MS incidence in susceptible individuals has become a field of
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interest in MS research (Owens and Bennett, 2012; Pender, 2011). Although EBV association with MS has become common knowledge in the field of MS research, no study has been reported from Kuwait investigating its association among Kuwaiti MS patients. Moreover, the most common known genetic risk factor associated with MS is the human leukocyte antigen (HLA) DRB1*1501 haplotype. HLA-DRB1*1501 has consistently proven its association in many case–control and familial genetic studies of MS risk and inheritance. (Hillert et al., 1994; Barcellos et al., 2006; Schmidt et al., 2007). However, the exact mechanism of how this haplotype increases susceptibility to MS is still unknown, but some evidence suggests its association in antigen detection, specifically EBV antigens. (Sundqvist et al., 2012; Kumar et al., 2014). Here we investigated the association of EBV antibody titers in Kuwaiti MS case–control study in relation to gender, antibody titers, HLA-DRB1*1501 genotype, infection stage, and other clinical variables pertinent to MS disease course. 2. Materials and methods 2.1. Patient and healthy control recruitment Blood samples from 141 Kuwaiti MS patients and 40 healthy control individuals were collected. Patients were recruited at the MS clinic in the Dasman Diabetes Institute (DDI). Information of procedures to be performed was fully explained to patients prior to procurement of their informed consent in agreement with the Joint Committee for the Protection of Human Subjects at Kuwait's Health Sciences Center (HSC), and in agreement with the Ethical Review Committee (ERC) of DDI. The inclusion criteria were as follows: 1) Patients aged 14–60 years, 2) patients with clinically definite multiple sclerosis (CDMS) with clear clinical course (RRMS, SPMS, PPMS, Benign), 3) availability of a detailed clinical history (demographics, age of onset, disease duration, Expanded Disability Status Scale (EDSS) score, and treatments received), 4) being born in Kuwait and have resided in Kuwait from birth to at least early adult life, and 5) willingness to provide a blood sample. Exclusion criteria included patients who have Clinically Isolated Syndrome (CIS), possible MS (not yet confirmed) or other demyelinating disorders such as acute disseminated encephalomyelitis/neuromyelitis optica, and patients with incomplete data or those who are unable to provide informed consents. Progression index (PI) was computed for each patient to determine the rate of disease progression by dividing EDSS score over disease duration since diagnosis. 2.2. EBV ELISA Blood samples were centrifuged at 2500 ×g for 5 min in a swinging bucket centrifuge at room temperature. Blood phases were isolated into plasma, buffy coat, and compact red blood cell pellet fractions. Plasma fractions were subjected to three ELISA assays for the presence of antibodies against specific viral antigens associated with EBV infection stage. Immunoglobulins against viral coat antigens (VCA), and Epstein–Barr nuclear antigen 1 (EBNA1) titers were assayed using GenWay's EBV VCA IgG, and EBNA1 IgG kits (GenWay Biotech Inc., CA, USA). The manufacturer's protocols were followed throughout the procedure. In brief, diluted plasma samples and ready-to-use standards were pipetted into the wells of microtiter plates pre-coated with respective EBV antigens. Plasma antibodies binding to immobilized EBV antigens were facilitated by incubation for 1 h at room temperature. The plate was rinsed with diluted wash solution to remove unbound material. The ready-to-use anti-human-IgG peroxidase conjugate was added and incubated at room temperature. After a second washing step, the substrate TMB solution was added and incubated at room temperature in the dark. The color development was terminated by the addition of a stop solution. The resulting dye was measured spectrophotometrically at 450 nm wavelength. The concentration of antibodies is directly proportional to the intensity of the color, and can be inferred in arbitrary
units (U/mL) from the standard curve plotted using ready-to-use standards. Positive EBV EBNA1 and VCA antibody titers were recorded as positive if titers were N20 U/mL, whereas ≤20 U/mL titers were recorded as negative. Segregation of the antibody titers into high (N50 U/mL) and low (50–20 U/mL) was performed to reach a grouped view of the non-normally distributed variation. Viral infection stage was determined according to De Paschale and Clerici, by categorization of EBV infection stage according to comparative assessment of antibody titers of both antigens into: acute infection, reactivation, past infection, and negative (De Paschale and Clerici, 2012). 2.3. HLA-DRB*1501 genotyping Blood samples were collected following patient and control consent. An additional 7 patients and 15 healthy controls provided frozen blood samples that were omitted from plasma antibody analyses. Approximately 3–4 mL of blood was collected from each individual and an aliquot of 200 μL of blood was retained for DNA extraction prior to blood fractionation. Genomic DNA was extracted using QIAamp DNA blood mini kit (Qiagen, CA, USA). The manufacturer's protocol was followed with minor modifications. In brief, whole blood samples were added to 25 μL of proteinase K and 200 μL of lysis buffer. Incubation at 56 °C was increased to 30 min to maximize lysis. DNA was precipitated in 200 μL absolute ethanol and bound to a filtered spin column. The column was washed twice in two different concentrations of wash buffer, followed by a dry spin at maximum speed for 1 min. DNA was eluted with nuclease free water. Genomic DNA yield and quality were ascertained using a NanoDrop spectrophotometer. HLA-DRB1*1501 genotyping was performed using tagSNP rs3135005 shown previously to correlate with the HLA-DRB1*1501 polymorphism (r2 = 0.93) and is in complete linkage disequilibrium with it (De Jager et al., 2008). TaqMan® rs3135005 genotyping assay (Life Technologies, CA, USA) was used for genotyping according to standard protocols and analyzed using the ABI 7500 Fast Real-time PCR system (Life technologies, CA, USA). 2.4. Statistical analysis All statistical analyses were performed using GraphPad prism 6.0 software. None of the ELISA results was normally distributed. Mann– Whitney rank sum test was used to compare groups with a two-tailed significance of p b 0.05. Hodges–Lehmann relative risk (RR) was used at 95% confidence interval (CI) for differences between median comparisons, and p b 0.05 was considered significant. In addition, Fisher exact test and Chi-square test with Yate's corrections and Student t-test were used when appropriate for the effect of HLA-DRB1*1501 locus on EBV antibody titers, infection stage, interferon treatment, and MS PI index. Positive HLA-DRB1*1501 genotype was assigned to homozygous/heterozygous allele A, while ancestral allele G homozygosity was assigned negative. 3. Results 3.1. EBV related antibody titers in MS patients A total of 141 MS patients were compared to 40 healthy controls' plasma EBV related antibody titers, patient and control demographics and clinical characteristics are depicted in Table 1. Most MS patients had RRMS course. Female to male ratio were at ~2:1 in both MS patients and healthy controls to account for gender difference in disease incidence. Five healthy controls and five MS patients were negative (no immunity) for antibodies against both VCA and EBNA1, whereas the remaining samples had detectable titers. Total anti-EBNA1 titers in MS patients differed significantly from healthy controls with higher positive response in MS patients than that of healthy individuals (p = 0.008, RR 22.59, 95% CI: 5.07–40.45). Grouping of the anti-EBNA1 titer into high
R. Al-Temaimi et al. / Journal of Neuroimmunology 285 (2015) 57–61 Table 1 Demographics and clinical characteristics of the studied cohort. Characteristic
MS cohort, n = 141
Healthy control cohort, n = 40
Nationality Sex [n (%)] Female Male Age [n (%)] ≤20 21–30 31–40 41≤ MS type (RR/SP/PP) Duration of disease (mean ± SD) EDSS [median (interquartile range)] Type of treatment [n (%)] IFNβ 1a (Avonex, Rebif) IFNβ 1b (Betaferon) Other treatments (Gilenya, Tysabri) Not on treatment EBNA1 IgG positive ≥ 50 U/mL [n (%)] EBNA1 IgG titers (median) VCA IgG positive ≥ 50 U/mL [n (%)] VCA IgG titers (median) EBV infection stage [n (%)] No immunity Acute infection Reactivation Past infection HLA-DRB1*1501 [n (%)] Positive Negative
Kuwaiti
Kuwaiti
93 (66) 48 (34)
27 (67.5) 13 (32.5)
11 (7.8) 58 (41.1) 50 (35.5) 22 (15.5) 138/2/1 4.83 ± 4.76 2 (1–3)
1 (2.5) 27 (67.5) 9 (22.5) 3 (7.5) NA NA NA NA
23 (16.3) 15 (10.6) 84 (59.6) 19 (13.5) 104 (73.7) 79.32 U/mL 104 (73.7) 80.58 U/mL
19 (47.5) 47.7 U/mL 23 (57.5) 55.4 U/mL
5 (3.5) 29 (20.6) 65 (46.1) 42 (29.8) N = 148 41 (27.7) 107 (72.3)
5 (12.5) 12 (30) 13 (32.5) 10 (25) N = 59 18 (30.5) 41 (69.5)
(N50 U/mL) and low (50–20 U/mL) resulted in a significant high response to EBNA1 antigen among MS patients when compared to healthy controls (p = 0.003). Similarly, anti-VCA titers among MS patients versus healthy controls were significantly different, albeit with non-significant risk association (p = 0.028, RR 20.42, 95% CI: 0.59– 39.66). Anti-VCA titer stratification into high and low was not significant. Categorization of EBV infection stage was performed according to comparative assessment of antibody titers of both antigens into: acute infection, reactivation, past infection, and negative (De Paschale and Clerici, 2012). This stratification allowed for EBV infection stage comparison among healthy controls and MS patients. There was a significant association between reactivation of EBV infection and MS when compared to healthy individuals (p = 0.02). Considering reactivated EBV infections' EBNA1 titers between MS and healthy controls, no significant association was revealed. However, when past infection titers of persistent immune surveillance was examined, anti-EBNA1 titers differed significantly (p = 0.046) indicative of a stronger immune surveillance in MS patients. The MS cohort was divided into four groups based on their treatment history: patients currently receiving any form of interferon beta treatment (Avonex, Betaferon, or Rebif) (26.9%), patients with previous history of interferon beta therapy in which the change of treatments was due to increased aggressiveness of disease or side effects (43.3%), patients on other treatments (16.3%), and patients without any treatment (13.5%). Anti-EBNA1 and anti-VCA titers did not differ significantly between patients currently on interferon beta treatments when compared to patients on other treatments or no treatment. 3.2. Gender differences in MS Our MS and healthy control cohorts were reflective of gender ratio associating with MS diagnoses (1.9:1 F:M). EDSS scores and progression index was computed for each MS patient. In total, 12 patients were excluded from EDSS and PI association analyses among male and female MS patients because they lacked an EDSS score (score of zero), or/and were recently diagnosed (disease duration = 0). There was no
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significant difference in the mean EDSS score between male and female MS patients since both genders had similar EDSS score distribution ranging from mild (0–2.5), intermediate (3–5.5) to severe (6–8). When comparing MS progression index classified as slow (PI ≤ 0.5) or fast (PI N 0.5), we found a significant difference between male and female patients (p = 0.006, RR 0.66, 95% CI: 0.52–0.86) (Fig. 1a). AntiEBNA1 titers between female MS patients and female healthy controls did not show any significant differences. However, when male MS patients are compared to male healthy controls, titers were significantly higher in MS males (p = 0.005, RR 41.2, 95% CI: 12.7–67) (Fig. 1b). Furthermore, male to female healthy controls' anti-EBNA1 titers did not reveal any significant differences, whereas male to female MS patients' titers showed a significant increase that was evident in MS males (p = 0.03) (Fig. 1c). Anti-VCA titers similarly showed a significant difference in male MS patients' VCA IgG titers compared to male healthy controls (p = 0.0003), whereas female MS patients' titers did not differ from female healthy controls (Fig. 1d). Female to male MS patients' VCA IgG titers did not differ significantly. There were no differences in EBV infection stage between MS male and female patients. 3.3. HLA-DRB*1501 and EBV immune response HLA-DRB*1501 haplotype was assayed in 141 MS patients, and 40 healthy controls, in addition to 15 healthy controls and 7 MS patients' DNA that were added to strengthen the power of our analysis; using the tagSNP rs3135005. Resultant rs3135005 genotypes are presented in Table 1. Of the 148 MS patients 11 were homozygous A and 30 were heterozygous AG and were assigned positive for HLA-DRB*1501 haplotype, whereas 2 healthy controls were homozygous A, and 16 were heterozygous AG. We found no association between positive HLA-DRB1*1501 genotype and MS risk. Stratifying MS patients into HLA-DRB1*1501 positive and negative did not result in any significant association with EBV antibody titers separately, or based on infection stage. In addition, no relationship was found between HLA-DRB1 positivity and EDSS, PI, or response to INF-beta treatment. 4. Discussion Viral infectious agents are the most convincing environmental factors to be associated with MS risk and incidence, primarily due to their ability to modulate the immune response. Studies in different populations reported the association of EBV infection with MS onset along with other viruses (Santiago et al., 2010; Virtanen and Jacobson, 2012). Different parameters of EBV infection have been analyzed to arrive at a more specific association, such as EBV infection stage, EBV cycle (lytic/lysogenic), EBV specific antibody titers, EBV viral load, EBV genotypes, and EBV infection in MS interacting with other environmental factors such as vitamin D deficiency and smoking (Kakalacheva et al., 2011; Lucas and Taylor, 2012; Pender and Burrows, 2014). Here we have shown that EBV related antibody response is altered in pharmaceutically managed MS patients when compared to healthy controls. More specifically, this alteration is determined by the type of EBV antigen and sex. Anti-EBNA1 had a stronger association with MS incidence which could be explained by the antigenic similarities between EBNA1 and myelin (Lunemann et al., 2008; Mameli et al., 2014). It is possible that EBNA1 would incite an amplified immune response to the one already in progress under the influence of MS neuro-inflammation. The resultant increase in cross-reacting antibody titers can be of diagnostic value for measuring neuro-inflammation if specific thresholds were to be identified. In addition, MS patients were shown to have increased B-cell transformation events albeit with similar kinetics to healthy individuals suggesting an increased propensity for having latent-EBV infected B-cells (Torring et al., 2014). This suggests higher EBV reactivation events in MS patients due to the presence of more latent EBV infected B-cells. We found such increased EBV reactivation to be significantly evident in MS patients. Moreover, past infections' anti-EBV titers were
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Fig. 1. Significant associations of gender influenced characteristics assayed in MS patients. (a) Progressive MS clinical course was significantly higher in MS males than females (p = 0.006); (b) Anti-EBNA1 titers were higher in MS males that healthy males (p = 0.005) and higher than MS females (p = 0.03) (c). (d) Similarly, anti-VCA titers were higher in MS males than healthy males (p = 0.0003). In (b–d); the line in the middle represents the median value, the box represents the interquartile range with whiskers representing the minimum and maximum values, and (*) represents the degree of p-value significance.
significantly higher in MS patients possibly due to increased immune surveillance driven by the autoimmune response to cross-reactive myelin antigens. The risk of developing MS is 2-times higher in females compared to males. However, very little is understood about the underlying mechanisms that contribute to this gender bias. This phenomenon is not restricted to MS, it is extended to other autoimmune disorders such as systemic lupus erythematosus (SLE) and rheumatoid arthritis. The reasons for gender bias in MS are unclear. The immune response in male MS patients to EBV antigens VCA and EBNA1 was significantly higher when compared to male healthy controls and female MS patients. Similar finding in a Lebanese study was reported for anti-EBNA1 IgG but not for anti-VCA IgG (Mouhieddine et al., 2015). The progression of MS was also more severe in male MS patients (55.1%) than females (34.8%). This might suggest a rare immuno-modulatory sex-influenced genetic variant(s) that has attenuated expression in females, but a profound effect in males. Such genetic factors have been found in other autoimmune diseases where the variant is found on the X chromosome or Xinfluenced genes. An example is SLE where two X-linked genes' variants were found to associate with increased incidence of SLE in females (Libert et al., 2010). Such genetic factors are still elusive for MS, however several mechanisms have been proposed to drive gender bias as more gender specific neuro- and immuno-physiological differences are discovered (Bordon, 2014). These differences might provide a survival advantage against pathogenic insults but can also enhance the susceptibility of females to autoimmunity and neuro-inflammation. Whereas males would have a more severe outcome as seen with the MS male cohort. Our findings suggest that male MS patients are more suitable candidates for efficacy studies of anti-viral therapeutics or autologous CD8+ T-cell therapy to attenuate MS symptomatic progression (Pender and Burrows, 2014). One of the strongest genetic factors associated with MS incidence is the HLA DRB1*1501 haplotype which is estimated to contribute to an approximately 24-fold increase in MS risk (Sundstrom et al., 2009). While this association was primarily replicated in some European
populations where the association remained constant, in some other populations the association was not as strong (Marrosu et al., 1998; Schmidt et al., 2007; Wu et al., 2009). Similarly, in our population such association was not found. However, this does not negate the association of other HLA haplotypes, nor does it fully dispute the association of HLA-DRB1*1501 as the SNP we used is a tagSNP that might have deviated from the linkage in our population than that seen in European populations. In conclusion, our findings corroborate other studies associating an altered immune response to EBV infection with MS differentiated by the type of EBV antigen and gender basis. In addition, HLA-DRB1*501 did not associate with MS risk or influence the immune response against EBV antigens in our Kuwaiti MS cohort. Finally, testing antibody titers related to EBV infections and assigning infection stage may be implicated in predicting the disease progression of male MS patients which would provide valuable prognostication to the treating physicians. Acknowledgments This project was funded by Kuwait University research sector, grant no. MG02/12. We wish to thank nurse Fatma Al-Kandari at Dasman Diabetes Institute for her efforts in securing the samples and Ms Anwar AlEnzi for her help in collecting healthy control samples. References Al-Din, A.S., Khogali, M., Poser, C.M., Al-Nassar, K.E., Shakir, R., Hussain, J., Behbahani, K., Chadha, G., 1990. Epidemiology of multiple sclerosis in Arabs in Kuwait: a comparative study between Kuwaitis and Palestinians. J. Neurol. Sci. 100, 137–141. Alroughani, R., Ahmed, S.F., Behbahani, R., Khan, R., Thussu, A., Alexander, K.J., Ashkanani, A., Nagarajan, V., Al-Hashel, J., 2014. Increasing prevalence and incidence rates of multiple sclerosis in Kuwait. Mult. Scler. 20, 543–547. Barcellos, L.F., Sawcer, S., Ramsay, P.P., Baranzini, S.E., Thomson, G., Briggs, F., Cree, B.C., Begovich, A.B., Villoslada, P., Montalban, X., Uccelli, A., Savettieri, G., Lincoln, R.R., Deloa, C., Haines, J.L., Pericak-Vance, M.A., Compston, A., Hauser, S.L., Oksenberg, J.R., 2006. Heterogeneity at the HLA-DRB1 locus and risk for multiple sclerosis. Hum. Mol. Genet. 15, 2813–2824.
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Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Gender influence in EBV antibody response in multiple sclerosis patients from Kuwait Rabeah Al-Temaimi a,⁎, Raed Alroughani b,c, Sindhu Jacob d, Fahd Al-Mulla d a
Human Genetics Unit, Department of Pathology, Faculty of Medicine, Kuwait University, Kuwait Division of Neurology, Department of Medicine, Amiri Hospital, Kuwait, Kuwait Neurology Clinic, Department of Medicine, Dasman Diabetes Institute, Kuwait d Molecular Pathology Unit, Department of Pathology, Faculty of Medicine, Kuwait University, Kuwait b c
a r t i c l e
i n f o
Article history: Received 2 March 2015 Received in revised form 18 May 2015 Accepted 20 May 2015 Available online xxxx Keywords: Multiple sclerosis Kuwait Epstein–Barr virus EBNA1 VCA HLA-DRB1*1501
a b s t r a c t Background: Epstein–Barr virus (EBV) infection is implicated with multiple sclerosis (MS) risk, exacerbation, and progression. The HLA-DRB1*1501 haplotype is a strong MS risk factor consistently documented in MS populations. There are no studies of EBV infections and HLA-DRB1*1501 haplotype associating with MS from Kuwait where MS prevalence has increased significantly. Objectives: To determine the association of EBV infection with MS incidence, and to investigate HLA-DRB1*1501 as a potential genetic risk factor for MS in Kuwait. Methods: This is a case–control study involving 141 MS patients and 40 healthy controls. Antibody titers against EBV antigens' viral capsid antigen (VCA) and Epstein–Barr nuclear antigen 1 (EBNA1) were measured using enzyme-linked immunosorbent assays. HLA-DRB1*1501 haplotype assessment was done using rs3135005 TaqMan genotyping assay. Results: Antibody titers against EBV were significantly elevated in MS patients compared to healthy controls (anti-EBNA1, p = 0.008; anti-VCA, p = 0.028). MS males had higher antibody titers to EBNA1 than healthy male controls (p = 0.005) and female MS patients (p = 0.03). HLA-DRB1*1501 haplotype genotypes failed to generate a risk association with MS or EBV antibody titers (p = 0.6). Conclusion: An increased immune response to EBV infection is associated with MS incidence influenced by the type of antigen and sex. HLA-DRB1*1501 haplotype is not associated with MS risk in our Kuwaiti MS cohort. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Multiple sclerosis (MS) is an inflammatory demyelinating disorder resulting from an autoimmune reaction against myelin and myelin associated antigens in the central nervous system (CNS), however the exact etiology remains unknown. Repeated demyelination events in the white matter result in subsequent neuro-axonal degeneration and oligodendrocyte cell death in the CNS manifesting primarily as lesions in the white matter of the brain and spinal cord causing perturbed central sensory and motor nerve conduction. MS incidence is 2:1 female:male ratio at a typical age of onset between 20–40 years of age (Liguori et al., 2000). MS is considered a multi-factorial complex disorder where genetic and environmental factors play a role in MS pathogenesis and relapse risk (Milo and Kahana, 2010). Kuwait has become a high-risk area for MS, its prevalence has increased from 4.4 in 1990 to 85 cases per 100,000 individuals in 2014 (Al-Din et al., 1990; Alroughani et al., 2014). One of the most consistently reported ⁎ Corresponding author at: Human Genetics Unit, Dept. of Pathology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait. E-mail address: [email protected] (R. Al-Temaimi).
http://dx.doi.org/10.1016/j.jneuroim.2015.05.021 0165-5728/© 2015 Elsevier B.V. All rights reserved.
association with MS risk is the association of viral infections, specifically Epstein–Barr virus (EBV) infection (Owens and Bennett, 2012; Lucas and Taylor, 2012). EBV is a human gamma-herpes virus that specifically infects nasopharyngeal epithelial cells and resting B-lymphocytes. EBV has the ability to activate or persist in a latent phase within the cells of infected individuals throughout their lives. EBV can mimic the stimuli of antigens and T-cell receptors in activating naïve B-lymphocytes into antigen specific memory B-cells. As infected memory B-cells differentiate into plasma cells EBV switches to lytic reproductive phase to produce new EBV particles (Laichalk and Thorley-Lawson, 2005). Human immune response against flourishing EBV production is to eliminate production houses; mainly EBV-infected plasma cells, effectively via the action of cytotoxic CD8+ T-cells (Hislop et al., 2007). EBV infection specificity to immune cells has resulted in its association with the incidence of several autoimmune disorders and malignancies in individuals predisposed to such disorders. Systemic lupus erythematosus, rheumatoid arthritis, Burkitt's lymphoma, and Hodgkin's lymphoma are examples of such disorders (Toussirot and Roudier, 2008; Saha and Robertson, 2011). In recent years, the role of EBV infections as an MS risk factor preluding MS incidence in susceptible individuals has become a field of
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interest in MS research (Owens and Bennett, 2012; Pender, 2011). Although EBV association with MS has become common knowledge in the field of MS research, no study has been reported from Kuwait investigating its association among Kuwaiti MS patients. Moreover, the most common known genetic risk factor associated with MS is the human leukocyte antigen (HLA) DRB1*1501 haplotype. HLA-DRB1*1501 has consistently proven its association in many case–control and familial genetic studies of MS risk and inheritance. (Hillert et al., 1994; Barcellos et al., 2006; Schmidt et al., 2007). However, the exact mechanism of how this haplotype increases susceptibility to MS is still unknown, but some evidence suggests its association in antigen detection, specifically EBV antigens. (Sundqvist et al., 2012; Kumar et al., 2014). Here we investigated the association of EBV antibody titers in Kuwaiti MS case–control study in relation to gender, antibody titers, HLA-DRB1*1501 genotype, infection stage, and other clinical variables pertinent to MS disease course. 2. Materials and methods 2.1. Patient and healthy control recruitment Blood samples from 141 Kuwaiti MS patients and 40 healthy control individuals were collected. Patients were recruited at the MS clinic in the Dasman Diabetes Institute (DDI). Information of procedures to be performed was fully explained to patients prior to procurement of their informed consent in agreement with the Joint Committee for the Protection of Human Subjects at Kuwait's Health Sciences Center (HSC), and in agreement with the Ethical Review Committee (ERC) of DDI. The inclusion criteria were as follows: 1) Patients aged 14–60 years, 2) patients with clinically definite multiple sclerosis (CDMS) with clear clinical course (RRMS, SPMS, PPMS, Benign), 3) availability of a detailed clinical history (demographics, age of onset, disease duration, Expanded Disability Status Scale (EDSS) score, and treatments received), 4) being born in Kuwait and have resided in Kuwait from birth to at least early adult life, and 5) willingness to provide a blood sample. Exclusion criteria included patients who have Clinically Isolated Syndrome (CIS), possible MS (not yet confirmed) or other demyelinating disorders such as acute disseminated encephalomyelitis/neuromyelitis optica, and patients with incomplete data or those who are unable to provide informed consents. Progression index (PI) was computed for each patient to determine the rate of disease progression by dividing EDSS score over disease duration since diagnosis. 2.2. EBV ELISA Blood samples were centrifuged at 2500 ×g for 5 min in a swinging bucket centrifuge at room temperature. Blood phases were isolated into plasma, buffy coat, and compact red blood cell pellet fractions. Plasma fractions were subjected to three ELISA assays for the presence of antibodies against specific viral antigens associated with EBV infection stage. Immunoglobulins against viral coat antigens (VCA), and Epstein–Barr nuclear antigen 1 (EBNA1) titers were assayed using GenWay's EBV VCA IgG, and EBNA1 IgG kits (GenWay Biotech Inc., CA, USA). The manufacturer's protocols were followed throughout the procedure. In brief, diluted plasma samples and ready-to-use standards were pipetted into the wells of microtiter plates pre-coated with respective EBV antigens. Plasma antibodies binding to immobilized EBV antigens were facilitated by incubation for 1 h at room temperature. The plate was rinsed with diluted wash solution to remove unbound material. The ready-to-use anti-human-IgG peroxidase conjugate was added and incubated at room temperature. After a second washing step, the substrate TMB solution was added and incubated at room temperature in the dark. The color development was terminated by the addition of a stop solution. The resulting dye was measured spectrophotometrically at 450 nm wavelength. The concentration of antibodies is directly proportional to the intensity of the color, and can be inferred in arbitrary
units (U/mL) from the standard curve plotted using ready-to-use standards. Positive EBV EBNA1 and VCA antibody titers were recorded as positive if titers were N20 U/mL, whereas ≤20 U/mL titers were recorded as negative. Segregation of the antibody titers into high (N50 U/mL) and low (50–20 U/mL) was performed to reach a grouped view of the non-normally distributed variation. Viral infection stage was determined according to De Paschale and Clerici, by categorization of EBV infection stage according to comparative assessment of antibody titers of both antigens into: acute infection, reactivation, past infection, and negative (De Paschale and Clerici, 2012). 2.3. HLA-DRB*1501 genotyping Blood samples were collected following patient and control consent. An additional 7 patients and 15 healthy controls provided frozen blood samples that were omitted from plasma antibody analyses. Approximately 3–4 mL of blood was collected from each individual and an aliquot of 200 μL of blood was retained for DNA extraction prior to blood fractionation. Genomic DNA was extracted using QIAamp DNA blood mini kit (Qiagen, CA, USA). The manufacturer's protocol was followed with minor modifications. In brief, whole blood samples were added to 25 μL of proteinase K and 200 μL of lysis buffer. Incubation at 56 °C was increased to 30 min to maximize lysis. DNA was precipitated in 200 μL absolute ethanol and bound to a filtered spin column. The column was washed twice in two different concentrations of wash buffer, followed by a dry spin at maximum speed for 1 min. DNA was eluted with nuclease free water. Genomic DNA yield and quality were ascertained using a NanoDrop spectrophotometer. HLA-DRB1*1501 genotyping was performed using tagSNP rs3135005 shown previously to correlate with the HLA-DRB1*1501 polymorphism (r2 = 0.93) and is in complete linkage disequilibrium with it (De Jager et al., 2008). TaqMan® rs3135005 genotyping assay (Life Technologies, CA, USA) was used for genotyping according to standard protocols and analyzed using the ABI 7500 Fast Real-time PCR system (Life technologies, CA, USA). 2.4. Statistical analysis All statistical analyses were performed using GraphPad prism 6.0 software. None of the ELISA results was normally distributed. Mann– Whitney rank sum test was used to compare groups with a two-tailed significance of p b 0.05. Hodges–Lehmann relative risk (RR) was used at 95% confidence interval (CI) for differences between median comparisons, and p b 0.05 was considered significant. In addition, Fisher exact test and Chi-square test with Yate's corrections and Student t-test were used when appropriate for the effect of HLA-DRB1*1501 locus on EBV antibody titers, infection stage, interferon treatment, and MS PI index. Positive HLA-DRB1*1501 genotype was assigned to homozygous/heterozygous allele A, while ancestral allele G homozygosity was assigned negative. 3. Results 3.1. EBV related antibody titers in MS patients A total of 141 MS patients were compared to 40 healthy controls' plasma EBV related antibody titers, patient and control demographics and clinical characteristics are depicted in Table 1. Most MS patients had RRMS course. Female to male ratio were at ~2:1 in both MS patients and healthy controls to account for gender difference in disease incidence. Five healthy controls and five MS patients were negative (no immunity) for antibodies against both VCA and EBNA1, whereas the remaining samples had detectable titers. Total anti-EBNA1 titers in MS patients differed significantly from healthy controls with higher positive response in MS patients than that of healthy individuals (p = 0.008, RR 22.59, 95% CI: 5.07–40.45). Grouping of the anti-EBNA1 titer into high
R. Al-Temaimi et al. / Journal of Neuroimmunology 285 (2015) 57–61 Table 1 Demographics and clinical characteristics of the studied cohort. Characteristic
MS cohort, n = 141
Healthy control cohort, n = 40
Nationality Sex [n (%)] Female Male Age [n (%)] ≤20 21–30 31–40 41≤ MS type (RR/SP/PP) Duration of disease (mean ± SD) EDSS [median (interquartile range)] Type of treatment [n (%)] IFNβ 1a (Avonex, Rebif) IFNβ 1b (Betaferon) Other treatments (Gilenya, Tysabri) Not on treatment EBNA1 IgG positive ≥ 50 U/mL [n (%)] EBNA1 IgG titers (median) VCA IgG positive ≥ 50 U/mL [n (%)] VCA IgG titers (median) EBV infection stage [n (%)] No immunity Acute infection Reactivation Past infection HLA-DRB1*1501 [n (%)] Positive Negative
Kuwaiti
Kuwaiti
93 (66) 48 (34)
27 (67.5) 13 (32.5)
11 (7.8) 58 (41.1) 50 (35.5) 22 (15.5) 138/2/1 4.83 ± 4.76 2 (1–3)
1 (2.5) 27 (67.5) 9 (22.5) 3 (7.5) NA NA NA NA
23 (16.3) 15 (10.6) 84 (59.6) 19 (13.5) 104 (73.7) 79.32 U/mL 104 (73.7) 80.58 U/mL
19 (47.5) 47.7 U/mL 23 (57.5) 55.4 U/mL
5 (3.5) 29 (20.6) 65 (46.1) 42 (29.8) N = 148 41 (27.7) 107 (72.3)
5 (12.5) 12 (30) 13 (32.5) 10 (25) N = 59 18 (30.5) 41 (69.5)
(N50 U/mL) and low (50–20 U/mL) resulted in a significant high response to EBNA1 antigen among MS patients when compared to healthy controls (p = 0.003). Similarly, anti-VCA titers among MS patients versus healthy controls were significantly different, albeit with non-significant risk association (p = 0.028, RR 20.42, 95% CI: 0.59– 39.66). Anti-VCA titer stratification into high and low was not significant. Categorization of EBV infection stage was performed according to comparative assessment of antibody titers of both antigens into: acute infection, reactivation, past infection, and negative (De Paschale and Clerici, 2012). This stratification allowed for EBV infection stage comparison among healthy controls and MS patients. There was a significant association between reactivation of EBV infection and MS when compared to healthy individuals (p = 0.02). Considering reactivated EBV infections' EBNA1 titers between MS and healthy controls, no significant association was revealed. However, when past infection titers of persistent immune surveillance was examined, anti-EBNA1 titers differed significantly (p = 0.046) indicative of a stronger immune surveillance in MS patients. The MS cohort was divided into four groups based on their treatment history: patients currently receiving any form of interferon beta treatment (Avonex, Betaferon, or Rebif) (26.9%), patients with previous history of interferon beta therapy in which the change of treatments was due to increased aggressiveness of disease or side effects (43.3%), patients on other treatments (16.3%), and patients without any treatment (13.5%). Anti-EBNA1 and anti-VCA titers did not differ significantly between patients currently on interferon beta treatments when compared to patients on other treatments or no treatment. 3.2. Gender differences in MS Our MS and healthy control cohorts were reflective of gender ratio associating with MS diagnoses (1.9:1 F:M). EDSS scores and progression index was computed for each MS patient. In total, 12 patients were excluded from EDSS and PI association analyses among male and female MS patients because they lacked an EDSS score (score of zero), or/and were recently diagnosed (disease duration = 0). There was no
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significant difference in the mean EDSS score between male and female MS patients since both genders had similar EDSS score distribution ranging from mild (0–2.5), intermediate (3–5.5) to severe (6–8). When comparing MS progression index classified as slow (PI ≤ 0.5) or fast (PI N 0.5), we found a significant difference between male and female patients (p = 0.006, RR 0.66, 95% CI: 0.52–0.86) (Fig. 1a). AntiEBNA1 titers between female MS patients and female healthy controls did not show any significant differences. However, when male MS patients are compared to male healthy controls, titers were significantly higher in MS males (p = 0.005, RR 41.2, 95% CI: 12.7–67) (Fig. 1b). Furthermore, male to female healthy controls' anti-EBNA1 titers did not reveal any significant differences, whereas male to female MS patients' titers showed a significant increase that was evident in MS males (p = 0.03) (Fig. 1c). Anti-VCA titers similarly showed a significant difference in male MS patients' VCA IgG titers compared to male healthy controls (p = 0.0003), whereas female MS patients' titers did not differ from female healthy controls (Fig. 1d). Female to male MS patients' VCA IgG titers did not differ significantly. There were no differences in EBV infection stage between MS male and female patients. 3.3. HLA-DRB*1501 and EBV immune response HLA-DRB*1501 haplotype was assayed in 141 MS patients, and 40 healthy controls, in addition to 15 healthy controls and 7 MS patients' DNA that were added to strengthen the power of our analysis; using the tagSNP rs3135005. Resultant rs3135005 genotypes are presented in Table 1. Of the 148 MS patients 11 were homozygous A and 30 were heterozygous AG and were assigned positive for HLA-DRB*1501 haplotype, whereas 2 healthy controls were homozygous A, and 16 were heterozygous AG. We found no association between positive HLA-DRB1*1501 genotype and MS risk. Stratifying MS patients into HLA-DRB1*1501 positive and negative did not result in any significant association with EBV antibody titers separately, or based on infection stage. In addition, no relationship was found between HLA-DRB1 positivity and EDSS, PI, or response to INF-beta treatment. 4. Discussion Viral infectious agents are the most convincing environmental factors to be associated with MS risk and incidence, primarily due to their ability to modulate the immune response. Studies in different populations reported the association of EBV infection with MS onset along with other viruses (Santiago et al., 2010; Virtanen and Jacobson, 2012). Different parameters of EBV infection have been analyzed to arrive at a more specific association, such as EBV infection stage, EBV cycle (lytic/lysogenic), EBV specific antibody titers, EBV viral load, EBV genotypes, and EBV infection in MS interacting with other environmental factors such as vitamin D deficiency and smoking (Kakalacheva et al., 2011; Lucas and Taylor, 2012; Pender and Burrows, 2014). Here we have shown that EBV related antibody response is altered in pharmaceutically managed MS patients when compared to healthy controls. More specifically, this alteration is determined by the type of EBV antigen and sex. Anti-EBNA1 had a stronger association with MS incidence which could be explained by the antigenic similarities between EBNA1 and myelin (Lunemann et al., 2008; Mameli et al., 2014). It is possible that EBNA1 would incite an amplified immune response to the one already in progress under the influence of MS neuro-inflammation. The resultant increase in cross-reacting antibody titers can be of diagnostic value for measuring neuro-inflammation if specific thresholds were to be identified. In addition, MS patients were shown to have increased B-cell transformation events albeit with similar kinetics to healthy individuals suggesting an increased propensity for having latent-EBV infected B-cells (Torring et al., 2014). This suggests higher EBV reactivation events in MS patients due to the presence of more latent EBV infected B-cells. We found such increased EBV reactivation to be significantly evident in MS patients. Moreover, past infections' anti-EBV titers were
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Fig. 1. Significant associations of gender influenced characteristics assayed in MS patients. (a) Progressive MS clinical course was significantly higher in MS males than females (p = 0.006); (b) Anti-EBNA1 titers were higher in MS males that healthy males (p = 0.005) and higher than MS females (p = 0.03) (c). (d) Similarly, anti-VCA titers were higher in MS males than healthy males (p = 0.0003). In (b–d); the line in the middle represents the median value, the box represents the interquartile range with whiskers representing the minimum and maximum values, and (*) represents the degree of p-value significance.
significantly higher in MS patients possibly due to increased immune surveillance driven by the autoimmune response to cross-reactive myelin antigens. The risk of developing MS is 2-times higher in females compared to males. However, very little is understood about the underlying mechanisms that contribute to this gender bias. This phenomenon is not restricted to MS, it is extended to other autoimmune disorders such as systemic lupus erythematosus (SLE) and rheumatoid arthritis. The reasons for gender bias in MS are unclear. The immune response in male MS patients to EBV antigens VCA and EBNA1 was significantly higher when compared to male healthy controls and female MS patients. Similar finding in a Lebanese study was reported for anti-EBNA1 IgG but not for anti-VCA IgG (Mouhieddine et al., 2015). The progression of MS was also more severe in male MS patients (55.1%) than females (34.8%). This might suggest a rare immuno-modulatory sex-influenced genetic variant(s) that has attenuated expression in females, but a profound effect in males. Such genetic factors have been found in other autoimmune diseases where the variant is found on the X chromosome or Xinfluenced genes. An example is SLE where two X-linked genes' variants were found to associate with increased incidence of SLE in females (Libert et al., 2010). Such genetic factors are still elusive for MS, however several mechanisms have been proposed to drive gender bias as more gender specific neuro- and immuno-physiological differences are discovered (Bordon, 2014). These differences might provide a survival advantage against pathogenic insults but can also enhance the susceptibility of females to autoimmunity and neuro-inflammation. Whereas males would have a more severe outcome as seen with the MS male cohort. Our findings suggest that male MS patients are more suitable candidates for efficacy studies of anti-viral therapeutics or autologous CD8+ T-cell therapy to attenuate MS symptomatic progression (Pender and Burrows, 2014). One of the strongest genetic factors associated with MS incidence is the HLA DRB1*1501 haplotype which is estimated to contribute to an approximately 24-fold increase in MS risk (Sundstrom et al., 2009). While this association was primarily replicated in some European
populations where the association remained constant, in some other populations the association was not as strong (Marrosu et al., 1998; Schmidt et al., 2007; Wu et al., 2009). Similarly, in our population such association was not found. However, this does not negate the association of other HLA haplotypes, nor does it fully dispute the association of HLA-DRB1*1501 as the SNP we used is a tagSNP that might have deviated from the linkage in our population than that seen in European populations. In conclusion, our findings corroborate other studies associating an altered immune response to EBV infection with MS differentiated by the type of EBV antigen and gender basis. In addition, HLA-DRB1*501 did not associate with MS risk or influence the immune response against EBV antigens in our Kuwaiti MS cohort. Finally, testing antibody titers related to EBV infections and assigning infection stage may be implicated in predicting the disease progression of male MS patients which would provide valuable prognostication to the treating physicians. Acknowledgments This project was funded by Kuwait University research sector, grant no. MG02/12. We wish to thank nurse Fatma Al-Kandari at Dasman Diabetes Institute for her efforts in securing the samples and Ms Anwar AlEnzi for her help in collecting healthy control samples. References Al-Din, A.S., Khogali, M., Poser, C.M., Al-Nassar, K.E., Shakir, R., Hussain, J., Behbahani, K., Chadha, G., 1990. Epidemiology of multiple sclerosis in Arabs in Kuwait: a comparative study between Kuwaitis and Palestinians. J. Neurol. Sci. 100, 137–141. Alroughani, R., Ahmed, S.F., Behbahani, R., Khan, R., Thussu, A., Alexander, K.J., Ashkanani, A., Nagarajan, V., Al-Hashel, J., 2014. Increasing prevalence and incidence rates of multiple sclerosis in Kuwait. Mult. Scler. 20, 543–547. Barcellos, L.F., Sawcer, S., Ramsay, P.P., Baranzini, S.E., Thomson, G., Briggs, F., Cree, B.C., Begovich, A.B., Villoslada, P., Montalban, X., Uccelli, A., Savettieri, G., Lincoln, R.R., Deloa, C., Haines, J.L., Pericak-Vance, M.A., Compston, A., Hauser, S.L., Oksenberg, J.R., 2006. Heterogeneity at the HLA-DRB1 locus and risk for multiple sclerosis. Hum. Mol. Genet. 15, 2813–2824.
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