Epstein-Barr virus infection after adolescence and human herpesvirus 6A as risk factors for multiple sclerosis.

BACKGROUND AND PURPOSE: Infections with human herpesvirus 6A (HHV-6A) and Epstein-Barr virus (EBV) have been linked to multiple sclerosis (MS) development. For EBV, late infection has been proposed as a risk factor, but serological support is lacking. The objective of this study was to investigate how age affects the EBV and HHV-6A associated risks of developing MS. METHODS: In this nested case-control study, Swedish biobanks were accessed to find pre-symptomatically collected blood samples from 670 individuals who later developed relapsing MS and 670 matched controls. A bead-based multiplex assay was used to determine serological response against EBV and HHV-6A. Conditional logistic regression was used to calculate odds ratios and 95% confidence intervals. RESULTS: Seropositivity against EBV exhibited a pattern where associations switched from a decreased risk of developing MS in the group below 20 years of age to an increased risk amongst individuals aged 20-29 and 30-39 years (p for trend 0.020). The age of transition was estimated to be 18.8 years. In contrast, HHV-6A was associated with increased MS risk in all age groups (total cohort odds ratio 2.1, 95% confidence interval 1.6-2.7). CONCLUSIONS: This study suggests EBV infection after adolescence and age independent HHV-6A infection as risk factors for MS.

10th European immunogenicity platform open symposium on immunogenicity of biopharmaceuticals.

Therapeutic proteins and emerging gene and cell-based therapies are attractive therapeutic tools for addressing unmet medical needs or when earlier conventional treatment approaches failed. However, the development of an immune response directed against therapeutic agents is a significant concern as it occurs in a substantial number of cases across products and indications. The specific anti-drug antibodies that develop can lead to safety adverse events as well as inhibition of drug activity or accelerated clearance, both phenomena resulting in loss of treatment efficacy. The European Immunogenicity Platform (EIP) is a meeting place for experts and newcomers to the immunogenicity field, designed to stimulate discussion amongst scientists across industry and academia, encourage interactions with regulatory agencies and share knowledge and the state-of-the-art of immunogenicity sciences with the broader scientific community. Here we report on the main topics covered during the EIP 10th Open Symposium on Immunogenicity of Biopharmaceuticals held in Lisbon, 26-27 February 2019, and the 1-d training course on practical and regulatory aspects of immunogenicity held ahead of the conference. These main topics included immunogenicity testing, clinical relevance of immunogenicity, immunogenicity prediction, regulatory aspects, tolerance induction as a mean to mitigate immunogenicity and immunogenicity in the context of gene therapy.

Association of female sex and positive rheumatoid factor with low serum infliximab and anti-drug antibodies, related to treatment failure in early rheumatoid arthritis: results from the SWEFOT trial population.

Objective: Infliximab-treated patients with rheumatoid arthritis (RA) may respond insufficiently due to low serum infliximab (sIFX) levels, caused by anti-drug antibodies (ADAs). However, monitoring of sIFX and ADAs is not routinely implemented, and levels for optimal outcome have not been validated. We searched for predictors for sIFX < 0.2 µg/mL and ADA development in a randomized setting. Methods: In the SWEFOT trial, of 128 patients randomized to methotrexate + IFX therapy, 101 had serum samples at 3, 9, and 21 months that were analysed for sIFX [enzyme-linked immunosorbent assay (ELISA)] and ADAs [ELISA, and precipitation and acid dissociation (PandA) when sIFX > 0.2 µg/mL]. The primary and secondary outcome measures were low disease activity [LDA = 28-joint Disease Activity Score (DAS28) ≤ 3.2] and remission (DAS28 < 2.6). Baseline characteristics were assessed as potential predictors of sIFX < 0.2 µg/mL or ADA positivity, using logistic regression. Results: Categorization of sIFX levels into < 0.2, 0.2-2.9, 3.0-7.0, and > 7.0 µg/mL showed a dose-response association with LDA (30%, 64%, 67%, and 79%, respectively, p = 0.008) and remission (10%, 45%, 39%, and 66%, p = 0.004) at trial cessation (21 months). Female patients had sIFX < 0.2 µg/mL more often than males (35% vs 7%, p = 0.006), with a similar trend for rheumatoid factor (RF)-positive vs RF-negative patients (34% vs 16%, p = 0.059). ADA positivity showed similar patterns, also after adjustment for potential confounders (female sex: p = 0.050; RF positivity: p = 0.067). PandA captured four highly ADA-reactive patients with sIFX > 0.2 µg/mL, of whom three were ADA positive at other time-points, all with high DAS28 at follow-up. Conclusion: In early RA patients receiving IFX as a second-line agent, sIFX < 0.2 µg/mL and ADA development were associated with treatment failure and were more common in females, with a similar trend for RF positivity. Our findings support the use of therapeutic drug monitoring, and PandA in ADA-negative non-responders. Trial registration: SWEFOT NCT00764725 ( https://clinicaltrials.gov/ct2/show/NCT00764725 ).

The Viral Hypothesis of Mesial Temporal Lobe Epilepsy - Is Human Herpes Virus-6 the Missing Link? A systematic review and meta-analysis.

PURPOSE: Mesial temporal lobe epilepsy (MTLE) is a common epileptic disorder. Although likely multifactorial, the mechanisms underlying the etiology and pathogenesis of the disease remains unknown in majority of patients. Viruses, particularly Human Herpes Virus 6A and B (HHV-6), two neurotropic herpes viruses, have been implicated in MTLE due to their ubiquitous nature and ability to establish lifelong latency with risk of reactivation. However, the results of studies investigating this relationship are conflicting. This systematic review and meta-analysis was conducted to determine the relationship between HHV-6 DNA (not specifying if A or B) in brain tissue and MTLE based on the current evidence. METHOD: Two independent assessors carried out a comprehensive electronic search to identify all relevant studies. Both fixed- and random-effects models were used to determine the overall odds ratio. RESULTS: A total of 10 studies met the inclusion criteria for the systematic review and eight for the meta-analysis. In 19.6% of all MTLE patients HHV-6 DNA was detected in brain tissue compared to 10.3% of all controls (p >0.05). The pooled odds ratio of HHV-6 positive cases in MTLE patients was 2.016 [95%-CI: 1.16-3.50] in the fixed effect model. CONCLUSION: The results of this meta-analysis indicate an association between HHV-6 DNA and MTLE surgically resected tissue samples, unspecified if A or B or both. However, the casual relationship and possible pathological role of HHV-6 in MTLE are yet to be elucidated. This study's results provide a basis for future studies continuing the investigation into pathological implications of HHV-6.

Antidrug Antibodies: B Cell Immunity Against Therapy.

Chronic inflammatory diseases are now treated with a range of different biopharmaceuticals, often requiring lifelong parenteral administrations. This exposure to drugs is unnatural and can trigger the immune system and result in the formation of antidrug antibodies. Drug-specific antibodies will, if of sufficiently high titre and affinity, block the intended effect of the drug, increase its clearance and make continued treatment worthless. We expect the immune system to react towards therapies against which tolerance has never been established, which is the case for factor VIII treatment in patients with haemophilia A. However, even biopharmaceutical molecules that we should be tolerant against can elicit antidrug antibodies, for instance in treatment of multiple sclerosis patients with recombinant human interferon-beta. Possible immunological mechanisms behind the breaking of tolerance against drugs, the impact this has on continuous treatment success, clinical practice and drug development, will be discussed in this review.

Standardizing terms, definitions and concepts for describing and interpreting unwanted immunogenicity of biopharmaceuticals: recommendations of the Innovative Medicines Initiative ABIRISK consortium.

Biopharmaceuticals (BPs) represent a rapidly growing class of approved and investigational drug therapies that is contributing significantly to advancing treatment in multiple disease areas, including inflammatory and autoimmune diseases, genetic deficiencies and cancer. Unfortunately, unwanted immunogenic responses to BPs, in particular those affecting clinical safety or efficacy, remain among the most common negative effects associated with this important class of drugs. To manage and reduce risk of unwanted immunogenicity, diverse communities of clinicians, pharmaceutical industry and academic scientists are involved in: interpretation and management of clinical and biological outcomes of BP immunogenicity, improvement of methods for describing, predicting and mitigating immunogenicity risk and elucidation of underlying causes. Collaboration and alignment of efforts across these communities is made difficult due to lack of agreement on concepts, practices and standardized terms and definitions related to immunogenicity. The Innovative Medicines Initiative (IMI; www.imi-europe.org), ABIRISK consortium [Anti-Biopharmaceutical (BP) Immunization Prediction and Clinical Relevance to Reduce the Risk; www.abirisk.eu] was formed by leading clinicians, academic scientists and EFPIA (European Federation of Pharmaceutical Industries and Associations) members to elucidate underlying causes, improve methods for immunogenicity prediction and mitigation and establish common definitions around terms and concepts related to immunogenicity. These efforts are expected to facilitate broader collaborations and lead to new guidelines for managing immunogenicity. To support alignment, an overview of concepts behind the set of key terms and definitions adopted to date by ABIRISK is provided herein along with a link to access and download the ABIRISK terms and definitions and provide comments (http://www.abirisk.eu/index_t_and_d.asp).

Smokers run increased risk of developing anti-natalizumab antibodies.

BACKGROUND: Smoking may contribute to the induction of neutralizing antibodies to interferon ß-1a. OBJECTIVE: In this study, we aimed to investigate the influence of smoking on the risk of developing antibodies to natalizumab, another biological drug in the treatment of multiple sclerosis. METHODS: This report is based on 1338 natalizumab-treated multiple sclerosis patients included in either of two Swedish case-control studies in which information on smoking habits was collected. Using logistic regression, patients with different smoking habits were compared regarding risk of developing anti-natalizumab antibodies, by calculating odds ratios with 95% confidence intervals. RESULTS: Compared with nonsmokers, the odds ratio of developing anti-natalizumab antibodies was 2.4 (95% CI 1.2-4.4) for patients who smoked at the time of screening, and a significant trend showed higher risk of developing antibodies with higher intensity of smoking. When smoking within two years prior to screening was considered, the odds ratio of developing anti-natalizumab antibodies was 2.7 (1.5-5.1). INTERPRETATIONS: The finding strengthens our hypothesis of the lungs as immune-reactive organs on irritation in relation to autoimmune responses, and may also be of clinical relevance since antibodies against natalizumab abrogate the therapeutic effect of the treatment.

Characterization of anti-natalizumab antibodies in multiple sclerosis patients.

BACKGROUND: A small proportion of multiple sclerosis (MS) patients treated with natalizumab develop anti-drug antibodies. OBJECTIVE: The objective of this paper is to characterize the anti-natalizumab antibody response and to investigate differences between persistently and transiently antibody-positive patients. METHODS: Screening for anti-natalizumab antibodies was performed using a standardized bridging ELISA. Antibody-positive samples were further analyzed for IgM and IgG1-4 antibodies using ELISA and ImmunoCAP®. RESULTS: Anti-natalizumab antibodies developed in 57 of 1379 (4.1%) treated patients after a median treatment duration of three months. Of the positive patients, 20 (35%) patients reverted to negative, 19 (33%) patients were confirmed persistently positive and 18 (32%) patients were unconfirmed positive. Significantly higher anti-natalizumab antibody levels were detected in persistently compared to transiently positive patients. A cutoff value predicting persistence of antibodies could be determined with a sensitivity of 0.84 and a specificity of 0.80. IgM and IgG4 antibody levels were significantly higher in persistently compared to transiently positive patients, and IgG1, IgG2 and IgG4 increased significantly over time. CONCLUSIONS: The level of total anti-natalizumab antibodies in a first positive sample can be used to predict patients at risk for persisting antibody positivity. However, neither IgM nor IgG1-4 antibodies could be used to discriminate between transiently and persistently positive patients.

Interleukin-2 gene expression in different phases of episodic cluster headache--a pilot study.

BACKGROUND: The pathophysiology of cluster headache (CH) is still largely unknown. Immunological mechanisms have been suggested to be of importance. AIM: This study aimed to investigate cytokine interleukin-2 (IL-2) as a possible marker of immune system involvement in the pathophysiology of CH. METHODS: Eight episodic patients with CH and 16 healthy headache-free control subjects matched for age and gender were studied. Venous blood samples were drawn from the patients with CH on three occasions; during active period between headache attacks, during an attack and in remission. Venous blood samples were drawn once from each control subject. We analysed IL-2 gene expression, using quantitative real-time polymerase chain reaction. RESULTS: Patients with CH had significantly increased relative IL-2 gene expression levels between headache attacks during active CH period (median 9.9 IL-2 cDNA/glyceraldehyde-3-phosphate dehydrogenase cDNA; IQR 6.2-10.3) compared to during attacks (median 2.8; IQR 0.7-3.2, P = 0.012), remission (median 1.6; IQR 0.9-1.8, P = 0.017) and controls (median 0.9; IQR 0.6-1.9, P = 0.0001). CONCLUSION: The increment of IL-2 found during the active CH period may support a role for this cytokine and subsequently for the immune system in the pathophysiology of CH. An expansion of this study to a broader group of cytokines and a larger patient cohort is warranted.

Multiple sclerosis patients lacking oligoclonal bands in the cerebrospinal fluid are less likely to develop neutralizing antibodies against interferon beta.

Multiple sclerosis patients without cerebrospinal fluid oligoclonal IgG bands have been proposed to constitute an immunogenetically distinct subgroup of multiple sclerosis that may also differ in terms of prognosis. A proportion of patients with multiple sclerosis receiving IFNbeta develop neutralizing antibodies, which interfere with treatment efficacy. Evidence suggests that the likelihood of developing neutralizing antibodies is partly genetically determined. Here, we hypothesized that absence of oligoclonal IgG bands reflects a property of B-cell responses in oligoclonal IgG band-negative patients characterized by a lessened propensity to develop neutralizing antibodies. We aimed to compare the development of neutralizing antibodies against IFNbeta between oligoclonal IgG band-negative and oligoclonal IgG band-positive multiple sclerosis patients. Treatment, oligoclonal IgG band and neutralizing antibody information was obtained for 2219 patients from the Swedish multiple sclerosis registry and the Swedish neutralizing antibody registry. Additional data on genotype was available for 532 patients. A correlation was found between oligoclonal IgG band negativity and neutralizing antibody negativity (p = 0.02). This difference was confined to neutralizing antibodies against IFNbeta-1a, since oligoclonal IgG band-negative patients were, to a lesser extent, neutralizing antibody positive compared with oligoclonal IgG band-positive patients if treated with IFNbeta-1a (12% vs. 23%; p = 0.005). No difference was observed for IFNbeta-1b-treated patients (44% vs. 46%). We propose that oligoclonal IgG band-negative patients differ immunologically from oligoclonal IgG band-positive patients, potentially influenced by distinct HLA-DRB1 alleles.

Neutralizing antibodies against interferon ß: fluctuation is modest and titre dependent.

BACKGROUND AND PURPOSE: Neutralizing antibody (NAb) titres may vary over time and thereby some patients can either lose or regain therapeutic effect of interferon beta (IFNbeta). We assessed NAb titres in a large sample of multiple sclerosis (MS) patients to identify the pattern of fluctuation during 1-3 years. METHODS: Data from IFNbeta-treated MS patients who had been tested for NAbs twice (n = 822) were analysed. NAb titres were compared between the first and the second samples across the critical NAb level of 150 TRU/ml, which we have previously reported to correlate with loss of bioactivity. RESULTS: Of patients with NAb titres high enough to indicate loss of bioactivity (>150 TRU/ml) in the first analysis 15% showed titres low enough to indicate a regained bioactivity. Conversely, 6% of those without detectable NAbs or NAb titres below the critical level of 150 TRU/ml had shifted to titres above this limit, indicating potential loss of bioactivity. Fluctuation did not differ between IFNbeta preparation used, treatment duration or sampling interval. CONCLUSION: A first NAb test is prognostic for the NAb status during the coming 1-3 years. Choice of IFNbeta preparation had no influence on the chance of reverting to lower levels once NAb titres are high.

Impression of clinical worsening fails to predict interferon-beta neutralizing antibody status.

The development of neutralizing antibodies (NAbs) against interferon-beta(IFNbeta) reduces clinical efficacy and markers of bioactivity in patients with multiple sclerosis (MS), although it has also been shown that a poor response to IFNbeta coincided with unexpectedly low NAb levels. To try and resolve this incoherency, this study investigated 2822 patients referred to a NAb testing facility. The reason for NAb testing was indicated for 2506 patients: routine testing (76%), worsening of disease (14%) and other reasons (10%). Overall, 31% of patients were NAb positive and 17% had titres high enough to obliterate IFNbeta bioactivity. The frequency of NAbs was similar in patients in the routine testing group compared with the worsening group. Samples showing high titres failed to be associated with worsening of symptoms. The study failed to show low NAb levels in patients responding poorly to IFNbeta. It is concluded that it is not possible to predict NAb status by clinical impression of treatment response. This is likely to be an effect of the partial efficacy of IFNbeta. Thus routine testing for NAbs must be carried out in order to identify NAb status in patients with MS.

Comparative study of four different assays for the detection of binding antibodies against interferon-beta.

BACKGROUND: Binding antibodies (BAB) against interferon-beta (IFNbeta) are often determined as screening assays before performing an expensive and elaborate neutralizing antibody (NAB) test. METHODS: In this study, we compared four BAB tests, a western blot (WB), a direct binding enzyme-linked immunosorbent assay (ELISA) (dELISA), a capture ELISA (cELISA), and a commercial enzyme immuno-assay (EIA) in 325 multiple sclerosis patients with and without neutralizing antibodies to evaluate the sensitivity and specificity to detect NAB by receiver operating characteristics analysis. RESULTS: The area under the curve (AUC) values were 0.907 for the dELISA, 0.925 for the cELISA, and 0.776 for the EIA (P < 0.0001 for all). At a sensitivity of 95%, the specificity was approximately 30% in the dELISA, 55% in the cELISA, and 13% in the EIA. The WB as a qualitative BAB detection method had a given sensitivity of 97% and a specificity of 55%. There was a strong and significant correlation between high NAB titers (>500 neutralizing units [NU]) and titers obtained by all quantitative BAB assays. However, low to medium NAB titers (20-500 NU) did not significantly correlate with BAB titers. CONCLUSION: We conclude that the cELISA seems to be most suitable for NAB screening, but BAB titers cannot reliably predict NAB titers.

In vivo bioactivity of interferon-beta in multiple sclerosis patients with neutralising antibodies is titre-dependent.

BACKGROUND: Development of neutralising antibodies (NAbs) against recombinant interferon-beta (IFNbeta) is a significant clinical problem in the treatment of multiple sclerosis (MS). Several methods are available to assess NAbs, but there is a lack of consensus on how the different NAb titre levels interfere with the efficacy of the drug, especially in the individual patient. METHODS: NAb titres were measured with an in vitro MxA induction assay and the in vivo IFNbeta response was assessed by measuring MxA mRNA expression using real-time PCR. RESULTS: We identified titre levels of NAbs at which the IFNbeta biological activity was reduced or abrogated. Patients with NAb titres of up to 150 TRU/ml (ten times reduction units per ml) still had retained IFNbeta bioactivity, whereas greatly reduced levels of IFNbeta bioactivity were found in patients with NAbs of 150-600 TRU/ml. Titres above 600 TRU/ml were associated with loss of IFNbeta bioactivity. Similar results were obtained when TRAIL mRNA was used as a marker of the in vivo response to IFNbeta. CONCLUSION: There is a stepwise loss of IFNbeta bioactivity with increasing NAb titres and it is possible to identify functionally critical NAb titre levels that are useful to support treatment decisions at the individual patient level.

Interferon beta preparations for the treatment of multiple sclerosis patients differ in neutralizing antibody seroprevalence and immunogenicity.

Development of neutralizing antibodies (NAbs) reduces the clinical efficacy of interferon beta (IFNbeta) treatment in multiple sclerosis (MS) patients. The aim of this study was to evaluate NAb seroprevalence (frequency of patients with NAbs) and immunogenicity (titer levels) of IFNbeta preparations in a clinical setting. We analysed 1115 consecutive MS patients, treated with one of the three available IFNbeta preparations, for an average of 40 months (1-120 months), for the presence of NAbs with the MxA protein induction assay. Overall, 32% of patients were positive for NAbs with neutralizing titers above 10. The frequency of NAbs, ie, the seroprevalence, was 13% in Avonex-treated patients, 43% for Betaferon, 39% for Rebif22 and 30% for Rebif44. In addition, the potential to induce high titer levels, ie, the immunogenicity, was observed to differ between preparations. Avonex, showing the lowest seroprevalence, also showed low immunogenicity and typically induced low titers. Betaferon, showing the highest seroprevalence when inducing NAbs, induced lower titers compared to Rebif22 and Rebif44. Treatment duration over five years only marginally correlated with decreased seroprevalence and titer levels. In conclusion, NAbs to IFNbeta are common in a clinical setting and the IFNbeta preparations differ not only in NAb seroprevalence, but also in immunogenicity.

Confirmed primary HHV-6 infection in children with suspected encephalitis.

HHV-6 infection has been associated with neurological symptoms in children. Two variants of human herpes virus 6, HHV-6A and HHV-6B, have been identified. Their role in neurological infections is poorly understood. We studied 53 children with suspected encephalitis for HHV-6A (strain GS) and HHV-6B (strain Z29) antibodies using an indirect immunofluorescence test. Primary infection was separated from past infection by an IgG-avidity test. The identified primary infections were studied for HHV-6 specific DNA by PCR. Forty-one children of 53 had IgG antibodies to HHV-6. Six children had low avidity of HHV-6 IgG antibodies indicating acute primary infection; four to type A, one to B, and one to both types. By serology, HHV-6 viral etiology was suggested in 6/53 (11.3%) of cases. One of the six patients with primary infection had HHV-6 DNA in serum and two in CSF. The children with primary HHV-6 infection were significantly younger than the whole series, 2.3 years vs. 6.4 years. We conclude that primary HHV-6 infection appears to be an important associated or causative agent in neurological infections of young children, and it can be confirmed from a single serum specimen using the IgG-avidity test.

Co-purification of soluble membrane cofactor protein (CD46) and human herpesvirus 6 variant A genome in serum from multiple sclerosis patients.

The association of human herpesvirus 6 (HHV-6) and multiple sclerosis (MS) has been supported by several immunological and molecular studies. Recently, membrane cofactor protein (CD46) has been identified as the cellular receptor for the A and B variants of HHV-6. Elevated levels of soluble CD46 (sCD46) have been reported in the serum and CSF of MS patients. The aim of this study was to investigate a possible correlation between elevated levels of soluble CD46 and the presence of serum HHV-6 DNA in MS patients. An immunoaffinity column comprised of immobilized monoclonal antibodies to CD46 was developed to isolate sCD46 from cell free body fluids of MS patients and controls. After immunoaffinity purification, DNA was extracted from anti-CD46 column eluates and subjected to PCR amplification. Of the 42 MS samples tested, 4 serum samples were HHV-6 positive, 3 of which were typed as HHV-6A. The co-purification of sCD46 and HHV-6 DNA from MS sera indicates that HHV-6 is tightly connected to its receptor, CD46, in the serum of MS patients.

A NOTCH4 association with multiple sclerosis is secondary to HLA-DR*1501.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) with supposedly autoimmune features known to be associated with a specific HLA DR-DQ haplotype (DR15, DQ6, or HLDRB1*1501,DRB5*0101,DQA1*0102,DQB1*0602). We have previously reported that the associated haplotype extends to HLA-B and described an independent association with HLA-A alleles in MS. Owing to a complex situation with extensive linkage disequilibria, it is still unclear whether classical HLA genes are responsible or whether associations may be due to other genes in this region. Here, we analyzed an association in MS with the NOTCH4 and TNFalpha (tumor necrosis factor-alpha) genes, located between the HLA-DRB1 gene and the HLA-A gene. For NOTCH4, located 0.4 Mb telomeric to HLA-DRB1, an SNP at position -25 and a trinucleotide repeat were investigated in 181 MS patients, and 180 controls also typed P = 0.027 for HLA-DRB and HLA-A. A modest association was observed (OR = 3.44) with the C-25 allele. However, two-locus analysis revealed that this association was secondary to the classical association with HLA-DRB1. For TNF, located 0.7 Mb telomeric of NOTCH4, SNPs at positions -308 and -238 were studied in the same dataset. We found no association between these TNFalpha gene polymorphisms and MS in this dataset, although there was linkage disequilibrium (LD) between DRB1 and TNF and between HLA-A and TNF. We conclude that alleles of the NOTCH4 and TNFalpha genes are unlikely to be of importance for the susceptibility to MS, although specific alleles of these genes are often carried on the same haplotype as DR15, DQ6.

Detection of human herpesvirus-6 in mesial temporal lobe epilepsy surgical brain resections.

BACKGROUND: Human herpesvirus-6 (HHV-6), a ubiquitous beta-herpesvirus, is the causative agent of roseola infantum and has been associated with a number of neurologic disorders including seizures, encephalitis/meningitis, and multiple sclerosis. Although the role of HHV-6 in human CNS disease remains to be fully defined, a number of studies have suggested that the CNS can be a site for persistent HHV-6 infection. OBJECTIVE: To characterize the extent and distribution of HHV-6 in human glial cells from surgical brain resections of patients with mesial temporal lobe epilepsy (MTLE). METHOD: Brain samples from eight patients with MTLE and seven patients with neocortical epilepsy (NE) undergoing surgical resection were quantitatively analyzed for the presence of HHV-6 DNA using a virus-specific real-time PCR assay. HHV-6 expression was also characterized by western blot analysis and in situ immunohistochemistry (IHC). In addition, HHV-6-reactive cells were analyzed for expression of glial fibrillary acidic protein (GFAP) by double immunofluorescence. RESULTS: DNA obtained from four of eight patients with MTLE had significantly elevated levels of HHV-6 as quantified by real-time PCR. HHV-6 was not amplified in any of the seven patients with NE undergoing surgery. The highest levels of HHV-6 were demonstrated in hippocampal sections (up to 23,079 copies/10(6) cells) and subtyped as HHV-6B. Expression of HHV-6 was confirmed by western blot analysis and IHC. HHV-6 was co-localized to GFAP-positive cells that morphologically appeared to be astrocytes. CONCLUSIONS: HHV-6B is present in brain specimens from a subset of patients with MTLE and localized to astrocytes in the absence of inflammation. The amplification of HHV-6 from hippocampal and temporal lobe astrocytes of MTLE warrants further investigation into the possible role of HHV-6 in the development of MTLE.