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Human Herpesvirus Six and Multiple Sclerosis: Molecular Mimicry

National Multiple Sclerosis Society Grant PP0919

Principal Investigator: Konstance Knox, Ph.D.

Final Progress Report

Human herpesvirus six (HHV-6) is a beta herpesvirus that has been implicated in the pathogenesis of multiple sclerosis.  As a herpesvirus, HHV-6 establishes a life-long latent infection in its host, and CNS and lymphoid tissues are well established sites of HHV-6 latency. Within cells latently infected with HHV-6 viral gene activity is limited to a single protein designated U94 which is constitutively expressed in the infected cell.  The U94 protein's function appears to be the establishment of "active latency" of HHV-6, i.e. as long as it is expressed in the infected cell, the virus remains in it's latent or inactive state.  Therefore, in patients with MS, a viral protein, U94, is constitutively expressed in both CNS and peripheral tissues that is apparently highly immunogenic.  The possibility of the HHV-6 U94 protein playing a role in the induction of autoantibodies reactive with myelin is raised.

                In order to address the possible role of U94 in a molecular mimicry mechanism in MS, its amino acid sequence was compared to that of human myelin basic protein (MBP), a major constituent of myelin that is known to be a target of autoantibodies in patients with MS.  When the sequence alignments were analyzed, a strong homology was observed between peptide 296 - 311 of the U94 protein and the 78 to 98 region of MBP.  This region of MBP is of special interest since it contains an immunodominant epitope (designated by a box in the figure) known to be a major target of autoantibodies in patients with MS as well as an important T cell epitope.

The marked similarity (63% identity with 25% conservative amino acid changes) of the U94 peptide and the ENPVVHFF peptide of MBP suggests the existence of a cross reacting epitope in the two proteins.

            In the study that we have now completed, we:

1)     used synthetic peptides in an enzyme linked immunoassay (ELISA) to determine the prevalence of MBP reactive autoantibodies in the sera and cerebrospinal fluids (CSF) of MS patients and healthy controls

2)     used synthetic peptides in an ELISA to determine the prevalence of U94 reactive antibodies in the sera and cerebrospinal fluids (CSF) of MS patients and healthy controls, and

3)  determined the prevalence of dual positivity for antibodies reactive with the MBP and U94 peptides in MS patients and healthy controls.

Summary of Patient Samples and Specimens from Healthy Control Individuals

            One set of  serum and CSF samples were obtained from Drs. George Ellison and Lawrence Myers of the Department Neurology of the University of California at Los Angeles ("UCLA" samples).  These were:

• 32 matched (i.e. both CSF and serum samples were from the same patient) serum and CSF samples from patients with MS
• one unmatched CSF sample, and
• 18 matched serum and CSF samples from healthy control individuals.

            A second set of samples were obtained from Dr. Lorri Lobeck of the Medical College of Wisconsin and Froedtert Memorial Lutheran Hospital (FMLH) in Milwaukee, Wisconsin.  These were 37 serum samples obtained from MS patients at the time of clinical relapse ("first samples") and 35 serum samples obtained from the same patients after the disease relapse had resolved (mean interval of 68 days) ("second samples").  Second samples were not obtained from two patients because they were lost to follow-up.

            A third set of samples were comprised of 47 serum samples obtained from healthy laboratory personnel or purchased from Analytical Biological Services, inc.; Wilmington, Delaware.

Development of Peptide Specific Enzyme Immunoassay (PSEIA)

The basic design of the PSEIA system used can be summarized as follows:

• the Multiscreen Millipore Vacuum Manifold system was used for specimen application and washing during the immunoassay
• Millipore Immunobilon-P Membrane 96 well plates were used due to their low intrinsic backgrpound and high protein binding characteristics
• the IgG concentration of all serum and cerebrospinal fluid (CSF) specimens was determined using an in-house EIA system, and all samples were analyzed at an IgG concentration of 10 ugper milliliter (CSF samples having IgG concentrations below 10 ug/ml were analyzed undiluted)
• it was empirically determined that optimal coating of the assay plates occurred at a peptide input of 3 ug per well in a volume of 100 microliters
• all samples were run in duplicate
• binding of IgG to bound peptides was detected by treatment with a peroxidase labeled anti-human IgG antibody with tetramethylbenzidine (TMB) as a substrate.  Optical density of the reaction product in each well was determined by means of an ELX800 Universal Microplate Reader.

             Initially, detection of antibodies reactive with the MBP and U94 peptides (and their random sequence counterparts) was attempted using the optical density (OD) and the end result.  representative  results obtained with serum samples and the MBP peptide are summarized in Table 1.

Table 1.  Optical density results of PSEIA analysis of serum sample reactivity with MBP specific peptide.

All four sets of samples gave similar OD results, and all gave a broad range of values.  Similar results were obtained for all four peptides, i.e. MBP, MBP random, U94, and U94 random.  Also, it was observed that each serum sample gave similar OD vaues for each of the four peptides.

             It was therefore determined that, for each sample, the ratio between the OD obtained with a specific peptide and its randomized counterpart would serve as a better detection system.  Representative data obtained for the MBP peptide and its randomized counterpart using healthy control serum samples are shown in Figure 1. 

In the figure showing data from all patients (left figure), it can be seen that all but 5 subjects gave ratios lying within the two standard deviations.  When these individuals were excluded and the mean and standard deviation were recalculated, the results in the right hand figure were obtained.  It was concluded that a ratio of 1.15 could appropriately be used as an upper cutoff value, i.e. samples giving a ratio above 1.15 would be considered positive for antibodies reactive with the MBP peptide.  A similar analysis of the U94-U94 randomized peptide system indicated that it's ratio cut-off value should be 1.20.

Analysis of Serum and CSF Samples from MS patients and Controls for Antibodies Reactive with MBP and U94 Peptides

When this PSEIA system was applied to the various sets of samples, the results summarized in Table 2 were obtained.

 Table 2. Positivity Rates for the MBP and HHV-6 U94 Peptide Specific Antibodies

Positivity rates for reactivity with the MBP peptide ranged from 3% to 16% with no significant differences being observed among the different sets of samples, and no significant differences were seen between serum and CSF samples.  Similarly, the positivity rates for the HHV-6 U94 peptide ranged from 3% to 14% with no significant differences being observed among the various groups.

            When the matched serum and CSF samples from UCLA were considered, three showed concordant positivity.  One control subject was positive for MBP peptide antibodies in both serum and CSF, and one control subject was positive for U94 peptide antibodies in both serum and CSF.  Also, one MS patient had MBP peptide reactive antibodies in both serum and CSF.  Interestingly, this patient had markedly high ratios for both samples, i.e. serum ratio of 1.29 and CSF ratio of 1.81 (see Table 2).

             When the first and second samples from the FMLH patients were analyzed, three were seen to be concordant.  Two patients had both their first and second serum samples positive for MBP peptide reactive antibodies , while the third patient had both serum samples positive for U94 peptide specific antibodies.  Simultaneous positivity for both the MBP peptide and the U94 peptide was observed in only one subject, a healthy control. 

Conclusions

• The PSEIA system developed in these studies is effective, and the use of Peptide to randomized control peptide ratio as the major end point provides a high degree of specificity in the detection of peptide specific antibodies.

• Antibodies reactive with both the MBP and HHV-6 U94 peptides are present in serum and CSF samples from both MS patients and healthy controls.

• Positivity rates for MBP and U94 peptides do not differ between healthy controls and MS patients.

• Dual positivity for the MBP peptide and the U94 peptide in the same serum or CSF sample is rare and was observed in only one serum from a healthy control subject.

• Molecular mimicry between the ENPVVHFF peptide of MPB and HHV-6 U94 protein is unlikely to be of pathogenic importance.