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Laboratory Correlations of Leukotropic Herpesvirus Infections
in Patients with Chronic Immuno-Metabolic Disease

Konstance K. Knox, Ph.D.1
Donald R. Carrigan, Ph.D.1
Daniel L. Peterson, M.D.2

Wisconsin Viral Research Group1
10437 Innovation Drive
Milwaukee, Wisconsin 53226
(414) 774-8612
kknox@wisconsinlab.com

Sierra Internal Medicine Associates2
865 Tahoe Boulevard
Incline Village, Nevada 89451
(775) 832-0989

Executive Summary

In preliminary studies we have found that approximately 25% of patients with chronic immuno-metabolic diseases (CIMD) have ongoing, active infections with human cytomegalovirus (HCMV)active. The specific aims of the proposed studies are to:

  1. Assess a cohort 180 patients for the existence of active HCMV infections.
  2. Perform detailed chart reviews of the patients to determine correlates of active HCMV infections. Specifically, we will assess (a) whether the HCMV positive patients have an increased frequency of abnormal T lymphocyte clones in their blood, (b) whether the HCMV positive patients have an increased occurrence of autoantibodies in their blood and (c) whether the HCMV positive patients have been treated with the antiviral drug visitide.
  3. Frozen samples (derived from the BioRepository of Wisconsin Viral Research Group) obtained on the cohort of 180 patients will be assessed by the following laboratory tests in order to determine further correlates of active HCMV infections:
(a) cell culture detection of active human herpesvirus six (HHV-6) infection
(b) detection of serum IgM antibodies specific for HCMV and Epstein-Barr virus (EBV)
(c) detection of anti-pancreas autoantibodies in order to determine whether HCMV infections can lead to diabetes, as has been reported in the scientific literature.

We project that the proposed studies will have four specific milestones (% effort):

(1) chart reviews to identify patients in the HCMV tested cohort that are positive for abnormal T lymphocyte clones, have autoantibodies in their blood and have received treatment with visitide (40%).
(2) testing of frozen samples for active HHV-6 infections (20%)
(3) testing of frozen samples for anti-EBV IgM antibodies, anti-HCMV IgM antibodies and anti-pancreatic autoantibodies (20%)
(4) data analysis and preparation of manuscripts for publication (20%)

Background

Chronic immuno-metabolic diseases (CIMD) have been linked to infections by leukotropic herpesviruses, especially human cytomegalovirus (HCMV), human herpesvirus six (HHV-6) and Epstein-Barr virus (EBV) by both molecular and classical virological methods. The pathogenetic mechanisms involved in viral associated CIMD (VACIMD) are likely to be complex and may include direct destruction of CNS and lymphoid tissues, the dysregulation of important cytokines and chemokines, stimulation of pathologic clonal expansions of lymphocytes, and interference with various metabolic pathways such as insulin production or regulation. Several illustrative examples of such mechanisms include:

HCMV

  • Abnormal T lymphocyte clonal expansions are induced during HCMV mononucleosis (1).
  • HCMV infection has been documented as a risk factor in type 2 diabetes (2).
  • HCMV infections are known to induce the production of a variety of autoantibodies (3,4).
  • HCMV infections are an important cause of diabetes in patients following organ or stem cell transplantation (5).
  • The major DNA binding protein of HCMV (UL57) contains a protein sequence that is recognized by clonal T cells specific for the diabetes specific autoantigen glutamic acid decarboxylase (GAD 65) (6).
EBV
  • EBV has been strongly indicted as being involved in the pathogenesis of systemic lupus erythematosus and rheumatoid arthritis (7,8), and molecular mimicry of EBV proteins has been shown to be responsible for many of the autoantibodies involved in those diseases (9).
  • EBV is known to cause neoplasms of B lymphocytes, T lymphocytes and natural killer (NK) cells (10,11).

HHV-6

  • HHV-6 has been associated with multiple sclerosis by a number of laboratories (12,13,14), and the U24 protein of the virus contains an amino acid sequence identical to a highly encephalitogenic peptide of myelin basic protein , and T lymphocytes reactive with the peptide of MBP also reacted with the HHV-6 peptide (15).

Preliminary Data

In a study performed in collaboration with Anthony Komaroff (Harvard Medical School; Boston, Massachusetts), serum samples from 19 patients with CIMD and 19 healthy control subjects were assessed for IgM antibodies specific for HHV-6 and EBV by immunofluorescence. Six patients (32%) were positive for HHV-6 specific IgM compared to 0 (0%) of the controls (p<0.02 by Fisher's exact test). Five of the patients (26%) were positive for EBV specific IgM compared to only 1 control subject (p<0.1) by Fisher's exact test. Interestingly, 4 of the IgM positive samples were positive for both HHV-6 and EBV while 2 samples were positive for only HHV-6 and 2 were positive for only EBV specific IgM antibodies (p<0.003 by Fisher's exact test).
Summary: HHV-6 IgM and EBV IgM sort significantly with CIMD patients.

Thirty three CIMD patients were assessed for abnormal T lymphocyte clones and for active HHV-6 infection by polymerase chain reaction (PCR) of serum or plasma samples. Six of the 33 patients had one or more T lymphocyte clones and five (83%) of them were also positive for active HHV-6 infection. In contrast only two (7%) of the T lymphocyte clone negative patients were HHV-6 infection positive. This difference was statistically significant (p<0.02 by Fisher's exact test).
Summary: Abnormal T lymphocyte clones sort significantly with HHV-6 infection.

In a study similar to the one just described, 30 CIMD patients were assessed for active HCMV infections by cell culture and for abnormal T lymphocyte clones. Sixteen T lymphocyte clone positive patients were identified and 7 (44%) of them were positive for active HCMV infection. Of the 14 T lymphocyte clone negative patients, only one (7%) was positive for active HCMV infection. This difference was statistically significant (p<0.04 by Fisher's exact test).
Summary: Abnormal T lymphocyte clones sort significantly with HCMV infection.

In a preliminary study aimed at estimating the frequency of autoantibodies in patients with CIMD, serum samples from 36 such patients were submitted to the Laboratory Corporation of America (LabCorp; https://www.labcorp.com) for testing with their standard autoantibody panel. This panel included the following antigens: (1) parietal cell, (2) smooth muscle actin, (3) thyroglobulin, (4) cell nuclei, (5) Sjogrens anti SS DNA, (6) striation antigen, (7) thyroid peroxidase, (8) GM1, (9) GDb1, (10) asialo GM, (11) mitochondria, (12) reticulum, (13) scleroderma, (14) rheumatoid factor, and (15) complement. In total autoantibodies were detected in 16 (44%) of the serum samples.

The number of different autoantibodies per serum sample is summarized in the table below.

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Number of Different Autoantibodies

1

2

3

4

Number of Serum Samples

8

4

3

1

In summary, autoantibodies are frequently present in the sera of patients with CIMD, and in approximately 50% of the patients more than one autoantigen is involved. Of special interest, the autoantibody panel used in this study did not included any of the three autoantibodies most specific for diabetes, i.e. anti-insulin, anti-GAD65 or anti-tyrosine phosphatase [also known as insulinoma-associated antigen (IA-2)].

Prospective Study of HCMV in CIMD Patients

In a prospective study aimed at determining the incidence of active HCMV infections in CIMD patients, the peripheral blood leukocytes (PBL) from 180 such patients were analyzed by coculture with human fibroblasts and by an antigenemia assay (see Appendix 1). Transfer of an active HCMV infection to the fibroblasts and the presence of HCMV infection in the PBL were detected by immunofluorescent staining with murine monoclonal antibodies specific for the major immediate early protein of HCMV. A summary of the standard protocol for the assay is presented in the diagram below.

The results of the study are summarized in the figure below.

In both assays approximately one forth of the patients were positive for an on-going HCMV infection in their PBL. The autoantibody status, T lymphocyte clonal status, and what percentage of this patient cohort are actively infected with HHV-6 or EBV are unknown

Hypothesis

We postulate that infection with one or more of HCMV, HHV-6 and EBV:

  • triggers the expansion of abnormal T lymphocyte clones in the peripheral blood of the patient,
  • causes an immunological imbalance that leads to the production of one or more autoantibodies,
  • may be the causes of autoimmune diseases such as diabetes mellitus, thyroiditis, and multiple sclerosis.

Proposed Studies

The major goal of the studies proposed here is to confirm and extend the preliminary findings described in the preceding section. The study population will be the cohort of the 180 CIMD patients who were the subjects of the prospective study of HCMV infections. For each patient in the cohort, the following archived specimens are available for study.

  • Human fibroblast shell vial for immunofluorescent staining with a rabbit antiserum specific for the major immediate early protein of HHV-6 (see Appendix 1). These shell vials are stored at -70oC.
  • One to two milliliters of plasma for evaluation of antiviral IgM antibodies and for detection of a variety of autoantibodies, specifically those antibodies known to be associated with diabetes. These material are stored at -30oC)
  • Pellet of patient PBL prepared from purified PBL from one milliliter of blood (-70oC) for potential use in viral microarray analysis .

Specifically, we will:

  • assay for active HHV-6 infection using the archived patient PBL inoculated fibroblast shell vials (see Appendix 1)
  • analyze patient plasma samples for IgM antibodies specific for HHV-6, HCMV and EBV using FDA approved commercial kits (Meridian Bioscience, Inc.; Cincinnati, Ohio)
  • screen patient plasma samples for anti-pancreatic islet specific autoantibodies using a commercially available screening system (Islet Cell Antibody Test System; IMMCO Diagnostics, Inc.; Buffalo, New York)
  • perform detailed reviews of patient charts for the following information
(1) longitudinal changes in EBV IgG titers--viral capsid antigen (VCA) and early antigen (EA)
(2) detection of autoantibodies specific for a variety of antigens
(3) existence or appearance of abnormal T lymphocyte clones in peripheral blood
(4) implementation, duration and virological effects of antiviral therapies
(5) IgG subclass deficiencies and other immunologic abnormalities
(6) clinical correlations with virological findings (e.g., CNS, liver and gastrointestinal diseases)
(7) occurrence of paraneoplastic or neoplastic disease in association with abnormal
T lymphocyte clones or changes in EBV infection status
  • use a number of clinical assessment tools to measure the status of the patients involved in the studies. These tools will include:

(1) SF36 questionnaire
(2) depression index
(3) anxiety level
(4) Canadian consensus definition
(5) patient demographics
(6) CDC consensus definition

References

  1. P Mathew, SD Hudnall, MT Elghetany et a.  T-gamma gene rearrangement and CMV mononucleosis.  Amer J Hematol 2001; 66:64-66.
  2. BW Roberts and I Cech.  Association of type 2 diabetes mellitus and seroprevalence for cytomegalovirus.  South Med Assoc 2005; 98:686-693.
  3. AE Gharavi, SS Pierangeli and EN Harris.  Viral origin of antiphospholipid antibodies: endothelial cell activation and thrombus enhancement by CMV peptide-induced APL antibodies.  2003;  Immunobiology 207:37-42.
  4. A Rahbar, L Boström and  C Söderberg-Naucler.  Detection of cytotoxic CD13-specific autoantibodies in sera from patients with ulcerative colitis and Crohn's disease. 2006; J Autoimmun. 26:155-164.
  5. J Hjelmesaeth, F Muller, T Jenssen et al.  Is there a link between cytomegalovirus infection and new-onset posttransplantation diabetes mellitus?  Potential mechanisms of virus induced B-cell damage.  2005; Nephrol Dial Transplant 20:2311-2315.
  6. BO Roep, HS Hiemstra, NC Schloot et al .  Molecular mimicry in type 1 diabetes.  Immune cross-reactivity between islet autoantigen and human cytomegalovirus but not coxsackie virus.  2002;  Ann NY Acad Sci 958:163-165.
  7. JA James, JB Harley and R Hal Scofield.  Epstein-Barr virus and systemic lupus eryythematosus.  2006; Curr Opin Rheumatol 18:462-467.
  8. E Toussirot and J Roudier.  Epstein-Barr virus in autoimmune diseases.  2008; Best Pract Res Clin Rheumatol. 2008;  22:883-896.
  9. BD Poole, RH Scofield, JB Harley and JA James.  Epstein-Barr virus and molecular mimicry in systemic lupus eryythematosus.  2006; Autoimmunity 39:63-70.
  10. AJ Ferreri, I Ernberg and C Copie-Bergman.  Infectious agents and lymphoma development: molecular and clinical aspects.  2009;.J Intern Med 265:421-438.
  11. JI Cohen, CM Bollard, R Khanna and S Pittaluga.  Current understanding of the role of Epstein-Barr virus in lymphomagenesis and therapeutic approaches to EBV-associated lymphomas.  2008; Leuk Lymphoma 49 Suppl 1:27-34
  12. A Rotola, I Merlotti, L Caniatti et al.  Human herpesvirus 6 infects the central nervous system of multiple sclerosis patients in the early stages of the disease.  2004; Multiple Sclerosis 10:348-354.
  13. R Alvarez-Lafuente, R Martin-Estefania, V de LasHeras et al.  Active human herpesvirus 6 infection in patients with multiple sclerosis.  2002; Arch Neurol 59:929-933.
  14. KK Knox, JH Brewer, DJ Harrington, JM Henry and DR Carrigan.  Human herpesvirus six and multiple sclerosis: systemic active infections in patients with early disease.  2000; Clin Infect Dis, 31:894-903.
  15. MV Tejada-Simon, YC Zang, J Hong et al.  Cross-reactivity with myelin basic protein and human herpesvirus 6 in multiple sclerosis.  2003; Ann Neurol 53:189-197.