http://www.ivpresearch.org/AAN_2002.htm

K.K. Knox¹, L.J. Lobeck², E.F. Maas², and D.R. Carrigan¹.
Institute for Viral Pathogenesis¹, 10437 Innovation Drive, Milwaukee, WI 53226
Wisconsin and Department of Neurology, Medical College of Wisconsin², 9200 W Wisconsin Ave, Milwaukee, WI 53226
Presented at the 54th American Academy of Neurology Annual Meeting; April 2002
 
Abstract

Several laboratories, including our own, have presented data linking the pathogenesis of MS to active HHV-6 infections. Blood samples were obtained at the time of new clinical relapse in patients with relapsing-remitting MS and assessed for active HHV-6 infection by plasma PCR.  Then, several weeks later (mean interval:68 days), a second blood sample was obtained from the same patients and assessed for active HHV-6 infection.  Patients' changes of Expanded Disability Status Scale (EDSS) from the relapse and treatments at the time samples were obtained were noted.  Five of 39 (13%) patients had at least one sample positive for active HHV-6 infection. Variant typing of the positive samples was possible with 3 of the 5 positives, and 2 were HHV-6 variant A.  Four of the five (80%) positive samples were obtained at the time of relapse whereas only one (20%) positive was observed in a patient after relapse.  The HHV-6 positive patients suffered a larger change in their EDSS (mean EDSS change of 1.4) than the HHV-6 negative patients (mean EDSS change of 0.7). It was also found that the patients receiving either beta interferon or glatirimar acetate (copaxone) were less likely to be HHV-6 positive (2/30; 7%) than the patients receiving no treatment 3/9; 33%).  Since the majority (>75%) of the patients on therapy were receiving  beta interferon, the decreased positivity for active HHV-6 may reflect the known antiviral properties of beta interferon.

Introduction

• In a recently published study, Knox et al. (Clin Infect Dis 2000; 31:894-903) identified, by means of a rapid culture assay, active HHV-6 viremia in 54% (22/41) of patients with MS.  HHV-6 viremia was not detected in 61 healthy controls.
• Additional studies [Lobeck et al.; American Academy of Neurology, 2000] demonstrated a significant relationship between active HHV-6 viremia, as detected by a rapid culture assay, and disease relapse in patients with relapsing/remitting MS.MS patients.
• Based on these findings and the tropism of HHV-6 for blood leukocytes, the possibility was raised that the CNS lesions of MS are the result of invasion of the CNS by HHV-6 from the peripheral blood.

Patients and Controls

• Thirty-nine patients, with relapsing remitting MS had blood samples drawn at the time of a clinical relapse.
• No patients were receiving corticosteroids when the specimens were drawn.  Most patients received corticosteroid treatment after onset of their disease relapse.
• A second blood specimen was drawn from 36  of the same patients six to ten weeks later after the relapse had resolved.
• Any treatment that the patients were receiving at the time of the first blood specimen was noted.  Also, the changes in the patients' Expanded Disability Status Scale (EDSS) score due to the disease relapse were determined.
• 46 control plasma specimens were obtained from six healthy donors over a mean period of 140 days (range 62 days to 183 days).

Detection of Active HHV-6 Infections by Plasma PCR

• DNA was purified from plasma samples by means of a commercially obtained kit [QIAmp DNA Blood Mini Kit; QIAGEN Inc.; Valencia, California].
• DNA PCR was performed using a hotstart taq DNA polymerase system [TaqBead Hot Start Polymerase; Promega Corp.; Madison, Wisconsin]. The HHV-6 specific primer pair used has been described in detail previously (Drobyski et al; NEJM 1994; 330:1356-1360).  This PCR system can detect approximately 200 viral genomes per ml of plasma (5 genomes per PCR reaction).  Quantitation of the PCR reaction was accomplished by amplification of a quantitative DNA target control.  The HHV-6 variant involved in the positive samples was determined by means of variant specific restriction enzymes.

• Incidence of Active HHV-6 Infections in MS Patients at Time of Relapse Compared with Healthy Control Individuals.
• Results are summarized in figure below.
• Significantly increased incidence of active HHV-6 infection was observed with both rapid HHV-6 culture and plasma PCR.
• Rapid culture was significantly (p < 0.004) more sensitive than plasma PCR at detected the active HHV-6 infections.

• Patients 1 through 4 were positive for active HHV-6 infection at the time of disease relapse.
• HHV-6 positive patients did not significantly differ from HHV-6 negative patients with respect to age, gender, disease duration or disease type.
• Viral loads varied from 9000 genomes per ml to 500,000 genomes per ml.
• Two of the three viruses typed as to variant were HHV-6 variant A.

• Patients with MS have significantly elevated levels of TNFa mRNA in their peripheral blood leukocytes (p < 0.0001) and TNFa protein in their plasma (p < 0.005) compared to healthy controls.
• No relationship was observed between TNFa mRNA or protein and active HHV-6 infection.

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 plasma 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.

© 2001, 2002 Institute for Viral Pathogenesis