HLA alleles modulate EBV viral load in multiple sclerosis

Multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), is the most common cause of neurological disability in adults, and it affects over 2.5 million people worldwide [1]. The etiopathogenesis of the disease is still unclear and is believed to include both environmental and genetic factors, whose interplay leads to chronic activation of immune cells and neuronal damage. The major human leukocyte antigens (HLA) locus (6p21.3), in particular, was identified as the chromosomal region most strongly linked to MS [2], and a number of epidemiological studies identified HLA alleles that correlate with an increased risk of developing MS and that can influence disease progression in patients. Notably, the realization that different HLA alleles play a role in MS led to the understanding that this disease is, above all, an antigen-specific autoimmune disease [3]. To date, HLA-DRB1*15 is the strongest risk factor for the development of MS [4]. HLA-DRB1*15 is nevertheless not the only HLA allele related to MS: HLA-B*07+ patients are characterized by higher disease burdens and by a more evident degree of brain atrophy [5]. The presence of the HLA-A*02 allele, on the other hand, seems to be a protective factor against MS [6] and, in patients with the disease, it results in a more favorable clinical course, with a slower progression and a lower disease burden [7].

Amongst environmental factors, chronic viral infections, including those supported by Epstein Barr virus (EBV), are suspected to be involved in the initiation of MS [8]. EBV (also called human herpesvirus 4, HHV-4) is a ubiquitous human virus with a double stranded DNA genome (172 kbp long) wrapped in a protein capsid belonging to the Herpesviridae family, Gammaherpesvirinae subfamily, Lymphocryptovirus genus [9]. The virus is a worldwide pathogen and is persistently harbored by almost all adults regardless of health or geographic location. In western countries, with high standard of living, primary EBV infection occurs commonly in adolescence, and might cause infectious mononucleosis (IM) in a minority of cases. EBV replicates in oropharyngeal epithelial cells during primary disease and subsequently establishes a latent infection in circulating B lymphocytes.

IM and elevated titers of antibodies against EBV nuclear antigens (EBNA) have been associated to an increased risk to develop MS and, on the other hand, lack of EBV infection correlates with a lower risk to develop the disease [8]. Other epidemiologic results suggesting a role for EBV in the pathogenesis of MS include the observations that EBV-infected B cells and plasma cells accumulate in the brain of MS patients [10] and a strong EBV specific cell-mediated immune responses are seen in these patients [11, 12].

Moreover, evidences of EBV DNA or transcripts in brain or cerebrospinal fluid of MS individuals remains controversial [13], as reported by some papers [10, 14], but not confirmed by other authors [15‐17].

Confliting results are reported on possible correlations between HLA alleles, EBV infection, and MS [18, 19]. Thus, an interaction between HLA-class I alleles and reactivity to EBV-related epitopes was recently shown, suggesting that the mechanism through which HLA genes influence the risk of developing MS may involve EBV-specific immune responses [18, 20]. Other data showed that HLA-B*7 allele is associated with increased EBV-specific Ab titers, higher disability scores, and a more compromised MRI pattern in MS patients [21]. We verified if correlations could be established between protective/risk HLA-class I alleles and EBV-specific parameters in MS; results suggest that this is indeed the case.

The etiology of MS is still unknown, but it is widely accepted that interactions between environmental and genetic factors shape the susceptibility to this multifactorial disease. Thus, whereas the correlation between certain HLA alleles and disease susceptibility has been definitely established, the possible association between EBV infection and MS has been repeatedly suggested but is still in need of convincing and definitive conclusions.

Herein we initially analyzed EBV infection parameters in a MS study population; our results confirmed previous data indicating that higher anti-EBNA-1 IgG serum titers are present in MS patients compared to HC [8, 26]. We also observed that, in MS patients compared to controls: (1) EBV DNA in blood is more frequently detected, and (2) EBV viral loads are significantly increased, although these viral loads are not comparable to those seen in EBV-associated malignancies [27]. These data confirm previously published results [28, 29], even if not all authors confirm such findings [30, 31], and could be the consequence of increased rates of EBV replication, more frequent reactivations, or of higher frequencies of circulating EBV-infected B lymphocytes [32]. However, it is important to note that, because in our experiments DNA was extracted from whole blood, viral DNA originating from B cells could not be discriminated from that originating from virions.

No relations were observed between EBV-specific antibody titers and EBV viral load in the study population. These results are partially in agreement with recent results indicating that, whereas VCA-specific Ab titers do correlate with EBV viral load, EBNA-1-specific Ab do not [33]; analyses on wider cohorts of individuals will be needed to clarify this aspect.

Having covered the virological side, we next analyzed whether polymorphisms in the HLA region known to modulate MS susceptibility [34] and its progression could associate with virologic parameters. Several lines of evidence indicate the presence of possible interactions between EBV infection-associated parameters, usually EBNA-1 antibody titers, and HLA profiles in MS; these evidences are nevertheless not unequivocal [18‐21, 35, 36]. Whereas in our cohort we did not observe any correlations between anti-EBV antibody titers and HLA antigens, possibly because of the small number of individuals analyzed, clear correlations emerged between HLA alleles and EBV viral loads. Thus, results showed that patients carrying the MS risk allele B*07 (HLA-B*07+) in the presence of the risk allele DRB1*15 and in the absence of the protective allele A*02 (HLA-B*07+/DRB1*15+/A*02−) were characterized by the presence of the highest EBV viral loads. These findings were confirmed by the observation that the lowest EBV viral loads were seen in MS patients showing the opposite HLA profile (HLA-B*07−/DRB1*15−/A*02+). The same trend remained when HLA-B*07, DRB1*15 and HLA-A*02 alleles were analyzed singularly. To note, analyzing the effects of HLA alleles altogether by multiple regression analysis, HLA-B*07 allele appears to have the strongest role on EBV DNA viral load. It is noteworthy that HLA-B*07 belongs to the same HLA haplotype carrying the HLA-DRB1*15 allele, whose association with MS susceptibility is well-established. Recently, a role of B*07 in EBV infection in MS patients has been proposed [12]. It is also important to observe that these correlations disappeared when the analyses were performed in healthy controls.

Merging the virologic and the genetic results presented herein allows the speculation that the increased blood viral loads seen in our patients are the consequence of a suboptimal control of EBV replication/reactivation by HLA-B*07+ restricted CD8⁺ T cell. Notably, HLA-B*07 antigen was recently shown to only weakly present viral epitopes to cytotoxic T lymphocytes [11]. This would allow more frequent EBV lytic replication/reactivation and the infection of an ampler pool of naïve B lymphocytes.

The suboptimal control of EBV replication/reactivation observed in HLA-B*07+ MS subjects may have had an impact on disease severity, but interestingly, no differences in EBV viral load were observed between HLA-B*07+ restricted RR- and P-MS patients. Finally, it could be expected that the frequency and/or the potency of EBV-specific cytotoxic T cell would be different in MS patients expressing (or lacking) the different HLA alleles analyzed in this study. We are currently enrolling patients and controls selected according to their HLA profiles to verify this hypothesis. Interestingly, these results are in line with several analyses performed in other diseases in which the etiologic role of EBV is suspected, or has been definitely confirmed. Thus, the presence of the HLA-A*02 allele was shown to be protective in nasopharyngeal carcinoma and in Hodgkin’s lymphomas [37, 38]. Taken together results herein reinforce the hypothesis that the role of HLA-class I antigens in the pathogenesis of MS could be at least partially mediated by their ability to control EBV infection and persistence.