• 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, MDWhittemore Peterson InstituteOctober 29th, 2009

• 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

A debilitating disorder of unknown etiology that is estimated to affect 17 million people worldwide

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

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:

Chronic Fatigue Syndrome (CFS)

• 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 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?

CFS Clinical Research Findings

• 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 XMRVBudding XMRV from a cell

Core proteins Envelope proteins

Core proteins

Envelope proteinsgp70p15E

p30p15p12p10

Gamma (Type-C) retrovirus XMRV

4

• 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

• 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).

68/101 (67%) 12 /320 (3.75%)

These results are representative of the 101 patients tested.They reflect the presence of virus (DNA PCR).

Presence of XMRV Sequences in Human DNA

• 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)

• Not a mouse contaminant• XMRV is a new human retrovirus• Not prostate cancer XMRV

• 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

• University Of Nevada, Reno Cleveland Clinic – Bob Silverman and Javdip Das Gupta• Vincent Lombardi• Judy Mikovits• Daniel Peterson• Kathryn Hagen• Max Pfost

• National Cancer Institute • Sandra Ruscetti• Frank Ruscetti• Rachel Bagni• Cari Petrow-Sadowski• Bert Gold• Michael Dean

University of Nevada, Reno

Sandra RuscettiFrank RuscettiRachel BagniCari Petrow-SadowskiBert GoldMichael Dean

Bob SilvermanJaydip Das Gupta

Vincent LombardiJudy Mikovits

Daniel PetersonKathryn Hagen

Max Pfost

Detection of Infectious Xenotropic MuLV-Related Virus (XMRV)in Blood Cells From Patients With Chronic

Fatigue Syndrome

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

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?

• 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

Rat α-MuLV p30 Gag mAb

XMRV Protein Expression in Purified Activated T and B

Lymphocytes from CFS Patients

• Prostate cancer cell line

Transmission of XMRV from Activated PBMCs of CFS Patients to the Human Prostate Cancer Cell Line LNCaP

• Using a technique known as spinoculation we transmitted virus from plasma and confirmed infectious particles by TEM next slide

21 positive of 25 (84%)

Cell Free Transmission of XMRV from CFS Patients Plasma to LNCap

• 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

Infectious whole virus budding from the cell membrane

Transmission Electron Micrograph of C-type Retrovirus Particles Transmitted from CFS patient T-Cells to LNCaP

BaF3-ER-SFFV Env

BaF3-ER

SFFV Env

WB: αSFFV Env mAb

BaF3-ER BaF3-ER-SFFV Env

BaF3-ER BaF3-ER-SFFV Env

Antibodies in CFS Patients Plasma to XMRV Env

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

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

LNCaP8/15 (53%) plasma samples contain antibody to XMRV

Env

XMRV Expression in NC/FL Cohort

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

Since the submission to Science, we have replicated at the NCI/CC our findings in other cohorts not in the WPI repository.

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

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.

• ~ 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

• XMRV status determined by the WPI since the submission to Science.

ID# XMRV status Clonal TCRg Lymphoma/cancer1103 positive positive MCL

1109 positive negative Thymoma

1118 positive negative myelodysplasia

1125 positive Positive + IGH MCL

1186 positive positive Lymphoma

1199 positive positive Previous Lymphoma

1150 positive positive Lymphoma

1320 Not tested Not tested Thymoma

1321 Not tested Not tested MCL

1174 positive positive Thymoma

1205 positive Not tested lymphoma

1172 positive positive MCL

1135 positive positive suspicious

1204 positive Positive + IGH suspicious

1113 positive positive CLL

1322 Not tested Not tested MCL

1181 positive Not tested CLL

1188 positive positive CLL

1189 positive positive MCL

1190 positive positive suspicious

XMRV status determined by the WPI since the submission to Science.

WPI Repository CFS Subjects with Cancer

• 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

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

WPI-1125 CFS Diagnosis One Decade Prior to MCL

• 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

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

Relative time-scale of the virological and immunological events during XMRV infection

Relative time-scale of the virological and immunological events during XMRV infection

CFS develops

Env antigen

Hypothesis of XMRV disease progression: NK

dysregulation

NK cells kill targets that do not express HLA class I

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

• 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

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.

• 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

XMRV in CFS and prostate cancer are closely related and form a distinct phylogenetic branch

An immune response to the virus was detected in a majorityof CFS subjects

CFS-XMRV can be transmitted cell-free from patient plasma to humanprostate and T cell lines and to primary T cells

• 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

Judy A Mikovits

Vincent Lombardi

Max Pfost

Kathryn Hagen

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

Robert Silverman Jaydip Das GuptaRobert Silverman Jaydip Das Gupta

The CFS patients in the US

Acknowledgements: