Human Herpesvirus Six and Multiple Sclerosis: Role of Cytokines

National Multiple Sclerosis Society Grant PPO751

Final progress report

November 15, 2001

Konstance K. Knox, Ph.D. and Donald R. Carrigan, Ph.D.
Institute for Viral Pathogenesis
Milwaukee, Wisconsin

Scientific Summary of Research Progress

Several laboratories have presented data linking the pathogenesis of multiple sclerosis (MS) to active human herpesvirus six (HHV-6) infections. Different diagnostic technologies have been used in these studies including immunohistochemical staining of tissues, polymerase chain reaction analysis of serum samples and isolation of the virus from blood samples. The majority of our work has involved staining of central nervous system (CNS) and lymphoid tissues for HHV-6 antigens and isolation of the virus from peripheral blood leukocytes of MS patients by a rapid culture procedure (Knox et al.; Clin Infect Dis 2000; 31:894-903). In the studies supported by this pilot grant, we have used two additional diagnostic procedures [HHV-6 specific serum PCR and HHV-6 specific reverse transcriptase PCR (RT-PCR)], to analyze specimens from patients with MS for the presence of active HHV-6 infections. In addition, we used serum PCR to assess the patient samples for the presence of active Epstein-Barr virus (EBV), another virus that has been implicated in MS. The virological findings were correlated with the clinical outcomes of disease relapses, the therapy status of the patients, the level of tumor necrosis alpha (TNFa) mRNA in peripheral blood leukocytes (PBL), and the level of TNFa protein in the patients' serum.

Specifically, blood and serum samples were obtained from 39 patients with definite MS at the time of new relapses of their disease. Then, after an interval of time (mean of 68 days; range 23 to 213 days) during which the disease relapse clinically resolved, second samples of blood and serum were obtained from the same patients. Numbers of samples in the procedures described below vary since not all samples were available for all patients. PBL were purified from the blood samples by density gradient centrifugation and frozen at -70^oC until processed for RNA purification. Purification of total RNA from the PBL was accomplished by means of a commercially obtained kit [PAXgene Blood RNA Kit; QIAGEN Inc.; Valencia, California].

RT-PCR analysis for HHV-6 mRNA was performed using a commercially obtained RT-PCR system (Access RT-PCR System; Promega Corp.; Madison, Wisconsin) and used a DNA primer pair that detects both the A and B variants of the virus. The RT-PCR product spans several introns of the appropriate viral gene, which encodes a structural glycoprotein of the virus. However, specificity for the mRNA rather than for the genomic sequence is conferred by variant specific capture probes in the RT-PCR product detection system. This product detection system utilizes a 96 well microplate format which gives quantitative results expressed as optical density (OD).

RT-PCR analysis for TNFa mRNA using a TNFa specific DNA primer pair was performed by means of the same commercially obtained system. To assure that genomic TNFa DNA does not amplify in this system, the sense DNA primer was designed to span an intron within the TNFa gene. The TNFa RT-PCR product was detected using a system similar to that used for the HHV-6 RT-PCR product. TNFa protein was detected in serum samples using a commercially obtained enzyme immunoassay (BD Biosciences; San Diego, California).

DNA was purified from serum samples by means of a commercially obtained kit [QIAmp DNA Blood Mini Kit; QIAGEN Inc.; Valencia, California].Standard DNA PCR was performed using a hotstart taq DNA polymerase system [TaqBead Hot Start Polymerase; Promega Corp.; Madison, Wisconsin]. An EBV specific DNA primer pair was designed using the genomic DNA sequence of the EBV LMP-1 gene. The HHV-6 specific primer pair used has been described in detail previously (Drobyski et al; NEJM 1994; 330:1356-1360). The HHV-6 variant involved in the positive samples was determined by means of variant specific restriction enzymes. These PCR systems used the same product detection system as was used with the RT-PCR systems.

When serum specimens were analyzed for all 39 patients with samples available by HHV-6 specific PCR, 5 (13%) were found to be positive. A summary of these five patients is shown in the table below. Consistent with work from our and other laboratories, the majority of the positive samples were HHV-6 variant A. When these samples were assessed for whether they were first (at relapse) or second (after relapse) for the patients it was found that the majority (80%, 4/5) were obtained at the time of relapse. Interestingly, when other characteristics of these HHV-6 PCR positive patients were compared with the other patients, two important observations were made. First, by comparing the extent of recovery of the patients from their disease relapse as measured by the increase in the patients' Expanded Disability Status Scale (EDSS), it was seen that they suffered a more severe and damaging relapse than the other negative patients (Figure 1). Second, when the various therapies that the patients were receiving when the serum specimens were obtained were compared, it was found that the HHV-6 positive patients were much more likely to be receiving either beta interferon or copaxone than the HHV-6 negative patients (Figure 2). Since the majority (>75%) of the patients receiving therapy were getting beta interferon, this decreased positivity for active HHV-6 may reflect the known antiviral properties of beta interferon.

 Summary of MS Patients Positive for Active HHV-6 Infection by Serum PCR

1 Relapsing/Remitting
2 Secondary Progressive
3 Not Determined due to low level of viral DNA present

Figure 1

Figure 2

Analysis of the patients' PBL samples by HHV-6 specific RT-PCR was uniformly negative, i.e., no HHV-6 specific mRNAs were detected. Beta actin mRNA, which is present at high levels in PBL, was detected in all samples. It is likely that these findings reflect a decreased sensitivity of the assay due to the suboptimal manner in which the PBL samples were stored prior to assay. RNAse degradation of the viral RNA could have occurred during the time interval between blood draw and specimen processing and also during thawing of the frozen PBL. Future studies using more optimal RNA preparation procedures should clarify these findings.

Serum PCR for EBV DNA was negative with all patient samples tested. Since the same purified DNA preparation was used in both the HHV-6 specific and EBV specific assays, if an active EBV infection was present in any patient the viral load in the serum must be much lower for EBV than for HHV-6.

High levels of TNFa mRNA were detected in all samples from patients with MS (Figure 3). No significant difference was observed between the first (at relapse) and second (after relapse) samples from the patients with MS. However, when compared to the results with sera from healthy control subjects, the samples from the MS patients showed a very significant elevation of TNFa mRNA, consistent with results reported by other investigators. Further, when the levels of TNFa mRNA were compared with the levels of TNFa protein present in the MS patients' sera, no relationship was observed (Figure 4) similar to the findings of other investigators. We observed a significantly increased positivity for serum TNFa protein in patients with MS compared to healthy control subjects (Figure 5). Finally, although the number of HHV-6 positive subjects was small, no relationship was observed between positivity for active HHV-6 by serum PCR and the level og TNFa mRNA in the PBL of MS patients.

Figure 3

Figure 4

Figure 5

The major findings of these studies can be summarized as follows:

• Cross sectionally, at least 13% of patients with definite MS have active systemic infections with HHV-6 as detected by serum PCR
• The proportion of MS patients with active systemic infection with HHV-6 is disproportionately high at the time of clinical relapse
• The majority of the active HHV-6 infections in MS patients involve the A variant of the virus
• The viral loads in the sera of the HHV-6 positive patients range from 10,000 to 500,000 viral genomes per milliliter
• Patients with active HHV-6 infections at the time of relapse show a greater degree of residual disability than HHV-6 negative patients
• Patients who are receiving beta interferon or copaxone therapies are at reduced risk for active HHV-6 infections
• Patients with MS have significantly increased levels of TNFa mRNA in their PBL compared to healthy controls
• Levels of PBL associated TNFa mRNA in patients with MS shows no correlation with levels of serum TNFa protein or with the presence of active HHV-6 infection as detected by serum PCR

For Release to the Public

The goals of these studies were:

• to use sophisticated molecular techniques to analyze blood samples from patients with MS for infections with two different herpesviruses, i.e., human herpesvirus six (HHV-6) and Epstein-Barr virus (EBV)
• to correlate the presence of one or both of these viruses with the disease and therapy status of the patients
• to assess blood white cells from patients with MS for the presence of a protein [tumor necrosis factor alpha (TNFa)] believed to be important in MS, and
• to correlate the presence of one or both of the viruses with the expression of that protein

The major findings of these studies were:

• at least 13% of patients with definite MS have active infections with HHV-6
• the proportion of MS patients with active HHV-6 infection is disproportionately high at the time of clinical relapse
• the majority of the active HHV-6 infections in the patients involve the little understood A variant of the virus
• patients with active HHV-6 infections at the time of relapse show a greater degree of residual disability than HHV-6 negative patients
• patients who are receiving beta interferon or copaxone therapies are less likely to have active HHV-6 infections
• active infections with EBV were not detected in the MS patients
• patients with MS have significantly increased levels of TNFa in their blood compared to healthy controls
• Levels of TNFa in the blood of patients with MS show no correlation with the presence of active HHV-6 infection.