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

XMRV, A New Human Pathogenic Retrovirus: Detection
In Chronic Diseases
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Picture of cell art
Picture of cell under a microscope
Daniel L. Peterson, MD
Whittemore Peterson Institute
Daniel L. Peterson, MD
Whittemore Peterson Institute
October 29th, 2009
Chronic Fatigue Syndrome (CFS)
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A debilitating disorder of unknown etiology that is estimated
A debilitating disorder of unknown etiology that is estimated
to affect 17 million people worldwide
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
or relapsing
fatigue
of 6 mo
longerdiagnosis
in duration,
Other Persistent
known medical
conditions
excluded
byor
clinical
generally with a distinct onset
Patients have at least 4 of the following symptoms:
Impaired
memory
or concentration
Other
known
medical conditions excluded by clinical diagnosis
CDC Criteria (Fakuda, 1994)
Sore throat
at least
of the following symptoms:
TenderPatients
cervical orhave
axillary
lymph4 nodes
Muscle pain
 pain
Impaired memory or
 Multi-joint pain
Multi-joint
concentration
 New headaches
New headaches
 Sore
throat
Un-refreshing
sleep
 Un-refreshing sleep
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Tender
cervical
or
axillary
lymph
Post exertional malaise
 Post exertional malaise
nodes
lasting > 24 hrs
lasting > 24 hrs
 Muscle pain
CFS Clinical Research Findings
CFS is a heterogeneous, multi-system disorder manifested by
• CFS is a heterogeneous,
multi-system disorder
inflammatory
sequelae including:
manifested by inflammatory sequelae including:
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antiviral enzyme (RNase L) dysfunction
 low natural killer (NK) cell numbers and function
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antiviral enzyme (RNase L) dysfunction
increased
numbers
activated
T cells and function
low 
natural
killer
(NK)ofcell
numbers
 increased production of inflammatory
innate immune
activation
cytokines/chemokines
 innate immune activation
– increased numbers of activated T cells
– increased production of inflammatory
Could these patients’ immune cells be infected with XMRV?
Could these patients’ immune cells be infected with
XMRV?
Gamma (Type-C) retrovirus XMRV
Envelope proteins
p15E
gp70
• Picture of Envelope proteins
Core proteins
and core proteins
p30
p15
p12
p10
Core proteins
Envelope 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:
Budding XMRV from a
– HIV, HTLV-1 (bothcell
complex
•XMRV is a simple retrovirus—it encodes only for structural
proteinsand XMRV
retroviruses)
•Retroviruses are NOT ubiquitous and NOT benign
Budding XMRV from a cell
•They are all associated with disease such as cancer and neurological disease
•There are three known human exogenous retroviruses:
4
•HIV, HTLV-1 (both complex retroviruses) and XMRV
Presence of XMRV Sequences in
Human DNA
• These results are
representative of the 101
patients tested.
• They reflect the presence of
virus (DNA PCR).
68/101 (67%)
• 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
12
/320 (3.75%)
and the on in CFS
These results are representative of the 101 patients tested.
They reflect the presence of virus (DNA PCR).
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)
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)
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
•Not a mouse contaminant
•XMRV is a new human retrovirus
•Not prostate cancer XMRV
Detection of Infectious Xenotropic MuLV-Related Virus
(XMRV)in Blood Cells From Patients With Chronic
Fatigue Syndrome
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University Of Nevada, Reno
Vincent Lombardi
Judy Mikovits
Daniel Peterson
Kathryn Hagen
Max Pfost
Cleveland Clinic – Bob Silverman and Javdip Das Gupta
University of Nevada, Reno
• National Cancer Institute
• Sandra Ruscetti
• Frank Ruscetti
• Rachel Bagni
Sandra
• Ruscetti
Cari Petrow-Sadowski
Frank
• Ruscetti
Bert Gold
Rachel Bagni
• Michael Dean
Cari Petrow-Sadowski
Bert Gold
Michael Dean
Vincent Lombardi
Judy Mikovits
Daniel Peterson
Kathryn Hagen
Max Pfost
Bob Silverman
Jaydip Das Gupta
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?
XMRV Protein Expression in Purified Activated T and B
Lymphocytes from CFS Patients
• 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
Rat α-MuLV
p30 Gag mAbto purified viral
proteins
Transmission of XMRV from Activated PBMCs of CFS
Patients to the Human Prostate Cancer Cell Line LNCaP
• Prostate cancer cell line
Cell Free Transmission of XMRV from CFS Patients
Plasma to 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%)
Transmission Electron Micrograph of C-type Retrovirus
Particles Transmitted from CFS patient T-Cells 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
Antibodies in CFS Patients Plasma to XMRV Env
WB: αSFFV Env
mAb
SFFV
Env
BaF3-ER
BaF3-ER-SFFV Env
BaF3-ER
BaF3-ER-SFFV 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
XMRV Expression in NC/FL Cohort
Since
the submission
to Science, we to
haveScience,
replicated atwe
the NCI/CC
findings in other
Since
the submission
have our
+-replicated
cohorts not in the WPI repository.
at the NCI/CC our findings in other cohorts not in the
WPI repository.
(60%) positive for XMRV gag DNA from fresh
• 9/15
9/15
PBMC (60%) positive for XMRV gag DNA from fresh
PBMC
(86.7%)
positive by
western forby
XMRV
Env
• 13/15
13/15
(86.7%)
positive
western
for XMRV Env
and Gag upon co-culture of plasma or PBMC with LNCaP
• and Gag upon co-culture of plasma or PBMC with
LNCaP
8/15 (53%) plasma samples contain antibody to XMRV Env
8/15
(53%) plasma samples contain antibody to XMRV
Env
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
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4/7 ( 57%) positive for serum Antibody to XMRV Env
• Gulf War Illness : Not Tested
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Samples were taken from family members of XMRV
positive CFS subjects with these neuroimmune diseases.
TCR g Clonality in Nevada CFS Cohort
• ~ 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,
WPI Repository CFS Subjects with Cancer
ID#
XMRV status
Clonal TCRg
Lymphoma/cancer
• XMRV status determined by the WPI since the
submission to Science.
XMRV status
1103
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
determined by
the WPI since
the submission
to Science.
WPI-1125 CFS Diagnosis One Decade
Prior to MCL
• 1988---Seen
1988- seen
atforNIH
at NIH
CFS for CFS
to decrease aggressiveness.
• 1998---Splenectomy
1998- Splenectomy
to decrease
 2000---Seen at NIH for mantle cell lymphoma. Given Rituxan
aggressiveness.
and Velcade
with at
adult
stemfor
cellsmantle cell lymphoma.
• 2004---BMT
2000- seen
NIH
 2008---Blast crisis MCL … death
Given Rituxan and Velcade
• 2004- BMT with adult stem cells
• 2008- Blast crisis MCL…. death
MCL cell line developed 2008
Neither XMRV infection nor a diagnosis of CFS correlate
to RNase L genetic variation R462Q
• Relationship between RNaseL variant
genotype and XMRV expression in CFS
patients
Dysfunction of Natural Killer Activity in a
Family with Chronic Fatigue Syndrome
Hypothesis of XMRV disease progression: NK
dysregulation
Relative time-scale of the virological and
immunological events during XMRV infection
CFS develops
Env antigen
Relative time-scale of the virological and immunological events during XMRV infection
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
Preliminary results from 8 study subjects
• Ampligen significantly amplifies the degranulation of
Ampligen significantly amplifies the degranulation of NK cells
NK cells
Ampligen may increase the ability of CD56+ cells to degranulate when
– Ampligen
may increase the
encountering
a target
ability of CD56+ cells to
degranulate when encountering a target
• Ampligen significantly increases the production of
Ampligen
significantly
the production of Perforin and GranzymeB
Perforin
andincreases
GranzymeB
Ampligen may promote synthesis of cytotoxic proteins- perforin and
– Ampligen may promote synthesis of cytotoxic proteinsGranzymeB
perforin and GranzymeB
• Ampligen may regulate NK and inflammatory
signaling
molecules
Ampligen
may regulate
NK and inflammatory signaling molecules
Anti-viral
as wellas
as immune-stimulating
cytokines are upregulated
– Anti-viral
well as immune-stimulating
cytokines are
upregulated
Preliminary Results Cont.
XMRV copy number is modulated by Ampligen
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When treated with Ampligen, qRT-PCR indicates a decrease in some patients and an
increase in others.
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This may suggest why Ampligen worsens some CFS patients and not others.
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Stratification for Ampligen treatment must consider XMRV status.
There is a highly significant
• Infectious
XMRV was detected
in lymphocytes
association
between
the and
plasma from >75% of CFS patients
XMRV retrovirus and Chronic Fatigue
• CFS-XMRV can be transmitted cell-free from patient
Syndrome
plasma to human prostate
and T cell lines and to
primary
T cells
Infectious XMRV was detected in lymphocytes and plasma from >75%
of CFS
patients response to the virus was detected in a
• An
immune
CFS-XMRV can
beCFS
transmitted
cell-free from patient plasma to human
majority
of
subjects
prostate and T cell lines and to primary T cells
• XMRV
inresponse
CFS and
cancer
closely related
An immune
to theprostate
virus was detected
in a are
majority
of CFSform
subjectsa distinct phylogenetic branch
and
XMRV in CFS and prostate cancer are closely related and form
a distinct phylogenetic branch
Acknowledgements:
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Cancer and Inflammation Program:
Frank
Ruscetti
Cancer and
Inflammation
Program:
Mike Dean
Frank
Ruscetti
Bert Gold
Mike Dean
Dan Bertolette
Bert
Ying Gold
Huang
Dan Bertolette
Ying Huang
Laboratory
of Cancer Prevention:
Sandra Ruscetti
Charlotte
Hanson
Laboratory
of Cancer
Prevention:
Jami
Troxler
Sandra Ruscetti
Charlotte Hanson
Jami Troxler
Cari Petrow-Sadowski
Rachel Bagni
Kunio Nagashima
Cari Petrow-Sadowski
Rachel Bagni
Kunio Nagashima
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From
Judy AWPI
Mikovits
Judy
A Mikovits
Vincent Lombardi
Vincent Lombardi
Max Pfost
Max
Pfost
Kathryn
Hagen
Kathryn Hagen
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From Cleveland Clinic
Robert Silverman
Robert
Silverman
Jaydip
Das Gupta
Jaydip
Das Gupta
Robert
Silverman
Robert
Silverman
Jaydip
Das Gupta
Jaydip Das Gupta
The CFS patients in the US