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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 -70oC 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
| Patient Initials |
Age/Gender |
Disease Duration |
Disease Type |
HHV-6 Variant |
HHV-6 Genomes per ml |
| LC |
51y/F |
4 years |
RR1 |
ND3 |
9.3 X 103 |
| JU |
51y/F |
6 years |
SP2 |
ND |
1.1 X 105 |
| LJ |
47y/F |
6 years |
RR |
A |
4.2 X 105 |
| PW |
36y/F |
9 years |
RR |
A |
4.8 X 105 |
| JH |
41y/M |
3 years |
RR |
B |
2.2 X 105 |
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 of 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.
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