XMRV, A New Human Pathogenic Retrovirus: Detection In Chronic Diseases

1. Picture of cell art Picture of cell under a microscope Daniel L. Peterson, MD Whittemore Peterson Institute XMRV, A New Human Pathogenic Retrovirus: Detection In Chronic Diseases Daniel L. Peterson, MD Whittemore Peterson Institute October 29th, 2009

2. A debilitating disorder of unknown etiology that is estimated to affect 17 million people worldwide CDC Criteria (Fakuda, 1994) Persistent or relapsing fatigue of 6 mo or longer in duration,generally with a distinct onset Other known medical conditions excluded by clinical diagnosis Patients have at least 4 of the following symptoms: Impaired memory or concentration Sore throat Tender cervical or axillary lymph nodes Muscle pain Multi-joint pain New headaches Un-refreshing sleep Post exertional malaise lasting > 24 hrs Chronic Fatigue Syndrome (CFS) Persistent or relapsing fatigue of 6 mo or longer in duration, generally with a distinct onset Other known medical conditions excluded by clinical diagnosis Patients have at least 4 of the following symptoms: • Impaired memory or concentration • Sore throat • Tender cervical or axillary lymph nodes • Muscle pain • Multi-joint pain • New headaches • Un-refreshing sleep • Post exertional malaise lasting > 24 hrs A debilitating disorder of unknown etiology that is estimated to affect 17 million people worldwide CDC Criteria (Fakuda, 1994)

3. CFS is a heterogeneous, multi-system disorder manifested by inflammatory sequelae including: antiviral enzyme (RNase L) dysfunction low natural killer (NK) cell numbers and function innate immune activation increased numbers of activated T cells increased production of inflammatory Could these patients’ immune cells be infected with XMRV? CFS Clinical Research Findings • CFS is a heterogeneous, multi-system disorder manifested by inflammatory sequelae including: • antiviral enzyme (RNase L) dysfunction • low natural killer (NK) cell numbers and function • innate immune activation • increased numbers of activated T cells • increased production of inflammatory cytokines/chemokines Could these patients’ immune cells be infected with XMRV?

4. Picture of Envelope proteins and core proteins XMRV is a simple retrovirus—it encodes only for structural proteins Retroviruses are NOT ubiquitous and NOT benign They are all associated with disease such as cancer and neurological disease There are three known human exogenous retroviruses: HIV, HTLV-1 (both complex retroviruses) and XMRV Budding XMRV from a cell Gamma (Type-C) retrovirus XMRV Envelope proteins gp70 p15E Core proteins p30 p15 p12 p10 Envelope proteins Core proteins Budding XMRV from a cell • XMRV is a simple retrovirus—it encodes only for structural proteins • Retroviruses are NOT ubiquitous and NOT benign • They are all associated with disease such as cancer and neurological disease • There are three known human exogenous retroviruses: • HIV, HTLV-1 (both complex retroviruses) and XMRV 4

5. The importance of this slide- in the normals 3.75% is 10 million Americans! This gel is representative gel- 11 of the 101 are shown Joy Das Gupta in the Silverman lab obtained full length sequence from 3 patient samples To explore the relationship between XMRV in prostate cancer and the on in CFS These results are representative of the 101 patients tested. They reflect the presence of virus (DNA PCR). Presence of XMRV Sequences in Human DNA 68/101 (67%) 12 /320 (3.75%) These results are representative of the 101 patients tested. They reflect the presence of virus (DNA PCR).

6. In latent infection- retroviral genome is present but is not transcribing viral genome or mRNA for structural proteins. Distinguished by qPCR (DNA) and qRT-PCR (RNA) Retroviral Life Cycle:Latent vs. Active infection In latent infection- retroviral genome is present but is not transcribing viral genome or mRNA for structural proteins. Distinguished by qPCR (DNA) and qRT-PCR (RNA)

7. Not a mouse contaminant XMRV is a new human retrovirus Not prostate cancer XMRV Phylogenetic analysis revealed that XMRV isolates from prostate cancer and CFS form a distinct branch within non ecotronic MLVs • Not a mouse contaminant • XMRV is a new human retrovirus • Not prostate cancer XMRV

8. University Of Nevada, Reno Cleveland Clinic – Bob Silverman and Javdip Das Gupta Vincent Lombardi Judy Mikovits DanielPeterson Kathryn Hagen Max Pfost National Cancer Institute Sandra Ruscetti Frank Ruscetti Rachel Bagni Cari Petrow-Sadowski Bert Gold Michael Dean Detection of Infectious Xenotropic MuLV-Related Virus (XMRV)in Blood Cells From Patients With Chronic Fatigue Syndrome University of Nevada, Reno Vincent Lombardi Judy Mikovits DanielPeterson Kathryn Hagen Max Pfost Sandra Ruscetti Frank Ruscetti Rachel Bagni Cari Petrow-Sadowski Bert Gold Michael Dean Bob Silverman Jaydip Das Gupta

9. CFS Study Cohort Reported in Science: Study cohort from the WPI national repository. Repository samples include samples from NV, CA, OR, FL, NC and NY as well as international CFS patients. Repository inclusion criteria: CFS diagnosis, regardless of severity 19-75 years of age Study characteristic: 67% women, reflecting gender incidence of CFS Mean age: 55 320 control samples from same geographic locations

10. Questions What cell types are infected? How is XMRV transmitted? Do infected individuals make an immune response? What are the interactions between XMRV and the innate immune system? Does XMRV infection alter the risk of cancer development in CFS? Can we develop immune based therapies for CFS based on XMRV?

11. See using a flow cytometry assay expression of 3 goat poly clonal antibodies to purified viral proteins in both T and B cells but not in T cells from a normal donor .. We have shown expression of XMRV proteins but next wanted to ask if that reflected the presence of infectious and transmissable virus 3 goat polyclonal antibodies to purified viral proteins XMRV Protein Expression in Purified Activated T and B Lymphocytes from CFS Patients Rat α-MuLV p30 Gag mAb

12. Prostate cancer cell line Transmission of XMRV from Activated PBMCs of CFS Patients to the Human Prostate Cancer Cell Line LNCaP

13. Using a technique known as spinoculation we transmitted virus from plasma and confirmed infectious particles by TEM next slide Cell Free Transmission of XMRV from CFS Patients Plasma to LNCap 21 positive of 25 (84%)

14. Early and late buds type C morphology Mature extracellular viral particles with condensed centrally located nucleoid surrounded by an outer membrane separated by an electron lucent area Having detected the presence of XMRV viral proteins and particles in the PBMC of CFS patients, we next asked if we could detect an immune response to XMRV in CFS patients Infectious whole virus budding from the cell membrane Transmission Electron Micrograph of C-type Retrovirus Particles Transmitted from CFS patient T-Cells to LNCaP Infectious whole virus budding from the cell membrane

15. Antibodies in CFS Patients Plasma to XMRV Env BaF3-ER BaF3-ER-SFFV Env BaF3-ER-SFFV Env BaF3-ER WB: αSFFV Env mAb BaF3-ER BaF3-ER-SFFV Env SFFV Env

16. Evidence for the presence of XMRV in 33 PCR Negative US CFS Patients 19/33 Antibodies in the plasma 30/33 Transmissable virus in the plasma 10/33 Protein expression in Decitibine (5Aza2DC) treated PBMC Thus, since the submission to Science we determined 99 of 101 US patients show evidence of XMRV infection

17. Since the submission to Science, we have +-replicated at the NCI/CC our findings in other cohorts not in the WPI repository. 9/15 (60%) positive for XMRV gag DNA from fresh PBMC 13/15 (86.7%) positive by western for XMRV Env and Gag upon co-culture of plasma or PBMC with LNCaP 8/15 (53%) plasma samples contain antibody to XMRV Env 9/15 (60%) positive for XMRV gag DNA from fresh PBMC 13/15 (86.7%) positive by western for XMRV Env and Gag upon co-culture of plasma or PBMC with LNCaP 8/15 (53%) plasma samples contain antibody to XMRV Env XMRV Expression in NC/FL Cohort Since the submission to Science, we have replicated at the NCI/CC our findings in other cohorts not in the WPI repository.

18. Familial Transmission of XMRV From Plasma from patients with Childhood Alzheimer’s Acute flulike infection in the family Mother/Father XMRV Env/Antibody positive (no active virus detected) XMRV envelope protein in LNCaP from children’s plasma

19. XMRV Associated Neuroimmune Diseases (XAND):Potential Candidates: Atypical MS: 3/3 positive for XMRV ENV protein and gag DNA Fibromyalgia: 12/20 (60%) positive for XMRV gag DNA Autism: 6/15 (40%) positive for XMRV gag DNA 4/7 ( 57%) positive for serum Antibody to XMRV Env Gulf War Illness : Not Tested Samples were taken from family members of XMRV positive CFS subjects with these neuroimmune diseases.

20. ~ 77 of the Nevada cohort have a clonal TCRg rearrangement More than 100 of these patients have clonal populations of gamma delta T cells Gamma delta T cells play an active role in the regulation and resolution of pathogen induced immune responses. They accumulate at sites of inflammation associated with viral, bacterial and parasitic infections and in auto immune diseases Interestingly, Gamma Delta T cells up-regulate MIP1lalpha and Beta , TNF alpha, TCR g Clonality in Nevada CFS Cohort

21. XMRV status determined by the WPI since the submission to Science. WPI Repository CFS Subjects with Cancer XMRV status determined by the WPI since the submission to Science.

22. 1988- seen at NIH for CFS 1998- Splenectomy to decrease aggressiveness. 2000- seen at NIH for mantle cell lymphoma. Given Rituxan and Velcade 2004- BMT with adult stem cells 2008- Blast crisis MCL…. death WPI-1125 CFS Diagnosis One Decade Prior to MCL • 1988---Seen at NIH for CFS • 1998---Splenectomy to decrease aggressiveness. • 2000---Seen at NIH for mantle cell lymphoma. Given Rituxan and Velcade • 2004---BMT with adult stem cells • 2008---Blast crisis MCL … death MCL cell line developed 2008

23. Relationship between RNaseL variant genotype and XMRV expression in CFS patients Neither XMRV infection nor a diagnosis of CFS correlate to RNase L genetic variation R462Q

24. Dysfunction of Natural Killer Activity in a Family with Chronic Fatigue Syndrome

25. Relative time-scale of the virological and immunological events during XMRV infection Hypothesis of XMRV disease progression: NK dysregulation Relative time-scale of the virological and immunological events during XMRV infection CFS develops Env antigen

26. NK cells kill targets that do not express HLA class I

27. Methods for addressing the NK cell dysregulation • PBMCs from XMRV infected patients with low NK cell function were activated with the mitogen PHA and treated with Ampligen • The effects on NK cell (CD56+) phenotype were determined by flow cytomentry • Signaling changes due to the treatment were detected via cytokine analysis • The change in XMRV copy number was detected with qRT-PCR

28. Ampligen significantly amplifies the degranulation of NK cells Ampligen may increase the ability of CD56+ cells to degranulate when encountering a target Ampligen significantly increases the production of Perforin and GranzymeB Ampligen may promote synthesis of cytotoxic proteins- perforin and GranzymeB Ampligen may regulate NK and inflammatory signaling molecules Anti-viral as well as immune-stimulating cytokines are upregulated Preliminary results from 8 study subjects • Ampligen significantly amplifies the degranulation of NK cells • Ampligen may increase the ability of CD56+ cells to degranulate when encountering a target • Ampligen significantly increases the production of Perforin and GranzymeB • Ampligen may promote synthesis of cytotoxic proteins- perforin and GranzymeB • Ampligen may regulate NK and inflammatory signaling molecules • Anti-viral as well as immune-stimulating cytokines are upregulated

29. Preliminary Results Cont.XMRV copy number is modulated by Ampligen • When treated with Ampligen, qRT-PCR indicates a decrease in some patients and an increase in others. • This may suggest why Ampligen worsens some CFS patients and not others. • Stratification for Ampligen treatment must consider XMRV status.

30. Infectious XMRV was detected in lymphocytes and plasma from >75% of CFS patients CFS-XMRV can be transmitted cell-free from patient plasma to human prostate and T cell lines and to primary T cells An immune response to the virus was detected in a majority of CFS subjects XMRV in CFS and prostate cancer are closely related and form a distinct phylogenetic branch There is a highly significant association between the XMRV retrovirus and Chronic Fatigue Syndrome Infectious XMRV was detected in lymphocytes and plasma from >75% of CFS patients CFS-XMRV can be transmitted cell-free from patient plasma to human prostate and T cell lines and to primary T cells An immune response to the virus was detected in a majority of CFS subjects XMRV in CFS and prostate cancer are closely related and form a distinct phylogenetic branch

31. From WPI Judy A Mikovits Vincent Lombardi Max Pfost Kathryn Hagen From Cleveland Clinic Robert Silverman Jaydip Das Gupta Robert Silverman Jaydip Das Gupta Cancer and Inflammation Program: Frank Ruscetti Mike Dean Bert Gold Dan Bertolette Ying Huang Laboratory of Cancer Prevention: Sandra Ruscetti Charlotte Hanson Jami Troxler Cari Petrow-Sadowski Rachel Bagni Kunio Nagashima Acknowledgements: Cancer and Inflammation Program: Frank Ruscetti Mike Dean Bert Gold Dan Bertolette Ying Huang Laboratory of Cancer Prevention: Sandra Ruscetti Charlotte Hanson Jami Troxler Cari Petrow-Sadowski Rachel Bagni Kunio Nagashima Judy A Mikovits Vincent Lombardi Max Pfost Kathryn Hagen Robert Silverman Jaydip Das Gupta Robert Silverman Jaydip Das Gupta The CFS patients in the US