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National Multiple Sclerosis Society 733 Third Avenue New York, NY 10017-3288 (212) 986-3240			(NMSS use only) Application Number PP
Application for a Pilot Research Award (Please submit original and two (2) copies of application and all supporting material)
Project Title (Do not exceed 80 characters): Human Herpesvirus Six and Multiple Sclerosis: Molecular Mimicry
Applicant’s name and degree(s): Konstance K. Knox, Ph.D.			Title or position Director of Research
Institutional address Department: School: Institution: Institute for Viral Pathogenesis Street: 10437 Innovation Drive City: Milwaukee State: WI Postal Code: 53226 Country : USA							Phone: (414) 774-8305 FAX: (414) 302-0646 Email: kknox@ivpresearch.org
Will Human Subjects or Materials be Used? Yes No			Will project use vertebrate animals? Yes No
Requested start date: June 1, 2003			Total amount requested :
Name, title & address of Financial Officer (to whom award check will be mailed if an award results from this application): Julie S. Krier Institute for Viral Pathogenesis 10437 Innovation Drive Milwaukee, Wisconsin 53226 Phone: (414) 774-8305			Name, title and address of the Director of Sponsored Research: Konstance K. Knox, Ph.D. Director of Research 10437 Innovation Drive Milwaukee, Wisconsin 53226 Phone: (414) 774-8305
Make award check payable to: Institute for Viral Pathogenesis
By the act of submitting an application for a grant, the applicant and the applicant’s institution agree that: 1) funds awarded as a result of this request will be expended for the purpose(s) set forth in the application, and in accordance with the research Policies and Procedures of the National Multiple Sclerosis Society; 2) the information contained herein is true and complete to the best of our knowledge; 3) the grant may be revoked in whole or in part at any time by the Board of Directors of the National Multiple Sclerosis Society, except that such revocation shall not include any amount obligated previous to the effective date of revocation, if such obligations were made solely for the purpose(s) set forth in this application; 4) all reports of investigations supported by a grant made as a result of this application will acknowledge such support; 5) if patentable discoveries or inventions are made in the course of work aided by a grant made as a result of this application, the applicant and the institution will conform to the patent policy of the National Multiple Sclerosis Society

		Applicant’s signature Date				Signature of official authorized to sign for institution
				Name and title of official signing for institution: Shawn G. Rice Chairman of Board of Directors Institute for Viral Pathogenesis
	Applicant’s full name and degree(s) Konstance K. Knox, Ph.D.				Application number (NMSS use only)

Project description. Briefly describe your project, its relation to multiple sclerosis, why it is novel, and techniques you will use. Do NOT exceed two (2) pages. You may include references in an appendix.

Human herpesvirus six (HHV-6) is a beta herpesvirus that has been implicated in the pathogenesis of multiple sclerosis.^1,2,3 Active HHV-6 infections have been demonstrated within peripheral blood leukocytes and lymphoid and CNS tissues of many patients with MS,³ and it has been postulated that this infection eventually leads to an autoimmune response against myelin with the CNS.³ The mechanisms responsible for the induction of this viral associated autoimmune response are unclear, but molecular mimicry may be involved.⁴

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.^5,6 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.⁷ 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.⁸ Interestingly, U94 is a protein that is unique to HHV-6 since there are no homologous proteins in any other members of the herpesvirus family.^9,10

Recent work by Caselli et al.¹¹ demonstrated that patients with MS have statistically significant higher prevalence and titers of serum antibodies reactive with U94 than healthy controls. The simplest explanation for this observation is that patients with MS have unusually high levels of cells carrying latent HHV-6.^7,12 This increased load of latent HHV-6 may result from recurrent reactivations of the virus to full, active replication.^3,12

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.^13,14,15 When the sequence alignments were analyzed, a strong homology was observed between a peptide of the U94 protein and a particular region of MBP.

This region of MBP is of special interest since it contains an immunodominant epitope known to be a major target of autoantibodies in patients with MS^13.14 as well as an important T cell epitope.¹⁴ This MBP peptide is also known to be bound by MS related HLA-DR2 subtypes (DRB1*1501 and DRB1*1602).¹⁴ Further, this peptide is immediately adjacent to a triproly peptide that is highly encephalitogenic in animal models of experimental allergic encephalomyelitis (EAE).^16,17

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. When the predicted secondary structures of the two proteins in this region were compared, they were found to be essentially identical. Further, using a major histocompatability complex (MHC) binding prediction program¹⁸ (accessible at http://www.imtech.res.in/raghava/propred), the U94 peptide is predicted to be bound by HLA Class II molecule DRB1*1501. Therefore, we hypothesize that this cross reacting epitope plays an important role in the induction of MBP reacting autoantibodies in patients with MS.

In the proposed project we will:

1) use 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) use synthetic peptides in an ELISA to determine the prevalence of U94 reactive antibodies in the sera and cerebrospinal fluids of MS patients and healthy controls

3) use the same synthetic peptides in cross absorption studies to determine whether the detected antibody activities represent cross reactive antibodies that recognize both U94 and MBP, e.g. can preabsorption of MBP autoantibodies with U94 peptide block their reaction with MBP and vice versa.

The patient materials available in our laboratory for these studies include:

1) 50 matched serum and CSF specimens from a cross sectional sample of patients with MS

2) 15 matched serum and CSF specimens from patients with other inflammatory, neurologic diseases

3) 18 matched serum and CSF specimens from healthy control subjects

4) 36 serum samples from patients with MS obtained at the time of clinical relapse and 36 serum samples obtained from the same patients after the relapse had resolved

5) 39 serum samples from a well characterized group of patients with MS obtained at the time of clinical relapse

6) serum samples from 56 healthy control subjects.

Appendix: Include literature citations and graphs or illustrations of data supporting the research proposal.

Literature Citations

1. Challoner PB, Smith KT, Parker JD, MacLeod DL, Coulter SN, Rose TM, Schultz ER, Bennett JL, Garber RL, Chang M, Schad PA, Stewart PM, Nowinski RC, Brown JP and Burmer GC. Plaque associated expression of human herpesvirus 6 in multiple sclerosis. Proc Natl Acad Sci 1995; 92:7440‑7444.

2. Soldan SS, Berti R, Salem N, Secchiero P, Flammand L, Calabresi P, Brennan MB, Maloni HW, McFarland HF, Lin HC, Patnaik M and Jacobson S. Association of human herpes virus 6 (HHV‑6) with multiple sclerosis: increased IgM response to HHV‑6 early antigen and detection of serum HHV‑6 DNA. Nature Med 1997; 3:1394‑1397.

3. Knox KK, Brewer JH, Harrington DJ, Henry JM, and Carrigan DR. Human herpesvirus six and multiple sclerosis: systemic active infections in patients with early disease. Clin Infect Dis 2000; 31:894-903.

4. Wucherpfennog KW and Strominger JL. Molecular mimicry in T cell mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell 1995; 80:695-705.

5. Luppi M, Barozzi P, Maiorana A, Marasca R and Torelli G. Human herpesvirus 6 infection in normal brain tissue. J Infect Dis 1994; 169:943-944.

6. Knox KK and Carrigan DR. Active human herpesvirus six viremia in patients with multiple sclerosis. In Genes and Viruses in Multiple Sclerosis; Elsevier Science B.V. Amsterdam, The Netherlands, 2001; pp. 185-194.

7. Rotola A, Ravaioli T, Gonelli A, dewhurst S, Cassai E and Di Luca D. U94 of human herpesvirus 6 is expressed in latently infected peripheral blood mononuclear cells and blocks virus gene expression in transformed lymphocytes in culture. Proc Natl Acad Sci 1998; 95:13911-13916.

8. Turner S, Di Luca D and Gompels UA. Characterization of a human herpesvirus 6 variant A 'amplicon' and replication modulation by U94-Rep 'latency gene'. J Virol Meth 2002; 105:331-341.

9. Dominguez G, Dambaugh TR, Stamey FR, Dewhurst S, Inoue N and Pellett PE. Human herpesvirus 6B genome sequence: coding content and comparison with human herpesvirus 6A. J Virol 1999; 73:8040-8052.

10. Gompels UA, Nicholas J, Lawrence G, Jones M, Thomson BJ, Martin ME, Efstathiou S, Craxton M and Macaulay HA. The DNA sequence of human herpesvirus‑6: structure, coding content, and genome evolution. Virology 1995; 209:29‑51.

11. Caselli E, Boni M, Bracci A, Rotola A, Cermelli C, Castellazzi M, Di Luca D and Cassai E. Detection of antibodies directed against human herpesvirus 6 U94/REP in sera of patients affected by multiple sclerosis. J Clin Microbiol 2002; 40:4131-4137.

12. Carrigan DR. Human herpesvirus six (HHV-6) and bone marrow transplantation. Blood 1995; 85:294-295.

13. Warren KG, Catz I, and Steinman L. Fine specificity of the antibody response to myelin basic protein in the central nervous system in multiple sclerosis: the minimal B cell epitope and a model of its features. Proc Natl Acad Sci 1995; 92:11061-11065.

14. Wucherpfennig KW, Catz I, Hausmann S, Strominger JL, Steinman L and Warren KG. Recognition of the immunodominant myelin basic protein peptide by autoantibodies and HLA-DR2 restricted T cell clones from multiple sclerosis patients. J Clin Invest 1997; 100:1114-1122.

15. Chamczuk AJ, Ursell M, O'Conner P, Jackowski G and Moscarello MA. A rapid ELISA based serum assay for myelin basic protein antibodies in multiple sclerosis. J Immunol Meth 2002; 262:21-27.

16. Fritz RB and McFarlin DE. Encephalitogenic epitopes of myelin basic protein. Chem Immunol 1989; 46:101-125.

17. Potter NT, Hashim GA and Day ED. Identification of an antigenic determinant within the phylogenetically conserved triprolyl region of myelin basic protein. J Immunol 1986; 136:561-520.

18. Singh Harpreet and Raghava GPS. ProPred: prediction of HLA-DR binding sites. Bioinformatics 2001; 17:1236-1237.