Cytomegalovirus reactivation in highstress environments - immunologic
mechanisms and implications
Keith R. Jerome, MD, PhD
University of Washington Department of Laboratory Medicine
Fred Hutchinson Cancer Research Center
March 26, 2013
Outline
• CMV history, biology, and replication cycle
• CMV disease
• Introduction to viral latency
• Laboratory assays for CMV
• CMV reactivation during spaceflight
• Implications for humanity in space
Discovery of cytomegalovirus
• 1904 – report of abnormal cells in lungs, kidney, liver
of infant thought to have died of syphilis (Jesionek)
Discovery of cytomegalovirus
• 1904 – report of abnormal cells in lungs, kidney, liver
of infant thought to have died of syphilis (Jesionek)
• Followed by a series of reports of similar cells from a
variety of tissues from older children
Discovery of cytomegalovirus
• 1904 – report of abnormal cells in lungs, kidney, liver
of infant thought to have died of syphilis (Jesionek)
• Followed by a series of reports of similar cells from a
variety of tissues from older children
• 1921 – term cytomegalia used
(Goodpasture/Talbot)
Discovery of cytomegalovirus
• 1904 – report of abnormal cells in lungs, kidney, liver
of infant thought to have died of syphilis (Jesionek)
• Followed by a series of reports of similar cells from a
variety of tissues from older children
• 1921 – term cytomegalia used
(Goodpasture/Talbot)
• 1926 – shown to be a filterable agent (Cole/Kuttner)
Discovery of cytomegalovirus
• 1904 – report of abnormal cells in lungs, kidney, liver
of infant thought to have died of syphilis (Jesionek)
• Followed by a series of reports of similar cells from a
variety of tissues from older children
• 1921 – term cytomegalia used
(Goodpasture/Talbot)
• 1926 – shown to be a filterable agent (Cole/Kuttner)
• 1954-55 – CMV isolated in cell culture (Smith, Wellner,
Cytomegalovirus
• AKA Human Herpesvirus 5 or HHV-5
• Betaherpesvirus (salivary gland tropism, slow growth
in culture, strict species tropism)
• dsDNA genome of 220-240 kb
• >200 ORFs
• Genetically distinct genotypes occur
andimblitar.blogspot.com; Wikimedia
CMV epidemiology and
transmission
• Worldwide distribution
• Infects all ages
• Seroprevalence increases with age; earlier in lower
socioeconomic groups
• Virus found in saliva, urine, breast milk, tears, stool,
blood, vaginal secretions, semen
• Transmission by close personal contact with person
shedding virus
CMV – Clinical Significance in
the immunocompetent
• Vast majority of infection in immunocompetent
children and adults are asymptomatic
• Can cause a mononucleosis syndrome with
prolonged fever, malaise, atypical lymphocytosis,
and mild hepatitis without heterphile antibody
• Associations with atherosclerosis and poor
outcomes in hospitalized immunocompetent
patients remain controversial
CMV – Clinical Significance in
neonates
• CMV infection occurs in 0.2 to 2.5% of newborns (the most
common congenital infection); transplacentally or during birth
• Risk of transmission and symptomatic disease much higher during
primary maternal infection
• Incidence of fetal damage highest when infection occurs at 1216 weeks gestation
• Symptoms seen in 10-15% of congenitally-infected newborns, can
include growth retardation, jaundice, hepatosplenomegaly,
thrombocytopenic purpura, myocarditis, pneumonitis, CNS
abnormalities, deafness, chorioretinitis
• Can be fatal; survivors often develop late neurological
manifestations (but not in perinatally infected)
• Infected infants who are asymptomatic at birth can still develop
hearing loss, visual impairment, psychomotor, and intellectual
impairment later in life
CMV in the immunosuppressed
• Most commonly seen in AIDS, cancer patients, solid-organ, or
hematopoietic stem cell transplants
• Associated with defects in cellular immunity (1-4 months post
transplant; CD4+ counts <50-100/uL)
• Fever, malaise, lethargy, muyalgia, leukopenia,
thrombocytopenia, hepatitis, retinitis (HIV), pneumonitis, GI
disease, delayed engraftment, organ rejection, GVHD,
bacterial/fungal superinfections
http://www.umm.edu/patiented/articles/000620.htm
Boeckh BBMT 2003
Therapy for CMV
• No vaccine currently available
• Several antivirals available (ganciclovir, valganciclovir,
foscarnet, cidofovir)
• Current drugs inhibit viral replication, but are not
curative
CMV replication cycle
Clin. Microbiol. Rev. January 2009 vol. 22 no. 1 76-98
Introduction to viral latency
•
Latency is a specialized form of viral persistence
•
Latently infected cells produce only a limited set
of viral transcripts, and few or no proteins
•
Hence, the latent virus is hidden from the
immune system
•
Virus does not replicate
•
Viral genome is maintained
•
Periodically, virus can reactivate
Viruses exploiting latency
•
HIV
•
HSV-1
•
HSV-2
•
VZV
•
EBV
•
CMV
•
HHV-8
•
HBV
•
HPV
Kane and Golovkina, JV 2010
CMV latency vs. persistence
• CMV can establish latency in lineage-committed myeloid
cells (progenitors for granulocytes, macrophages, dendritic
cells, maybe endothelial cells)
• DNA present in latently infected cells, but RNA expression is
restricted to a subset of transcripts (LATs)
• Proportion of cells latently infected is low (0.01 – 0.12% of
PBMC (Mocarski, Pass-Fields)
• Reactivation from latency promoted as cells differentiate in
the presence of activated immune cells
• Process of reactivation likely ongoing at all times
(persistence?), but controlled by immune response
Immune control of CMV
• NK cells (mouse lines with reduced NK function more
susceptible; human patient with genetic defect in NK had
severe CMV and other herpesviruses)
• Antibody (strong serologic response to infection; in infants
contracting CMV from blood products, maternal antibody
ameliorates disease)
• T cells, especially CD8+, are critical for control of CMV (severe
disease seen in advanced AIDS, transplantation)
• As much as 50% of CD8+ repertoire is CMV-specific in aged
individuals
Methods for monitoring CMV
• Serology – allows determination of infection
history of an individual; very limited utility in
following reactivation
• pp65 antigenemia – excellent for following
reactivation; technically demanding
• Quantitative PCR – highest sensitivity and
reproducibility; current state of the art
Quantitative real-time PCR
Loeb/Jerome Hepatology 2000
Impact of CMV monitoring on
transplant survival
Nesbitt/Jerome 2004
Gooley NEJM 2010
Physiologic stress as an
immunosuppressant
Stress and CMV reactivation
Yatim, Immunity 2011
Spaceflight and CMV reactivation
Spaceflight and reactivation of
other viruses - EBV
•
Testing 534 saliva specimens from 11 astronauts by
qualitative PCR, found EBV is shed more frequently in the
pre-flight period than during or afterward (Mehta 1999)
•
Sera taken before and after flight show increases in antiEBV antibodies consistent with reactivation (Stowe 2000)
•
Using 1398 saliva specimens from 32 astronauts by
quantitative PCR, confirmed EBV is shed more frequently
in the pre-flight period than during or afterward).
However, viral shedding during flight had 10x more virus,
and levels increased over the 2 week flight (Pierson 2005)
•
Testing peripheral blood B cells from 6 astronauts,
detected lytic gene transcription suggesting activation
from latency (Stowe 2011)
Spaceflight and reactivation of
other viruses – VZV
•
Testing 312 saliva specimens from 8 astronauts by
qualitative PCR, found VZV shedding increases from
~1% preflight to ~30% during and postflight (Mehta
2004)
•
VZV shed during flight is likely infectious (Cohrs 2008)
•
At least one clinically relevant VZV reactivation has
occurred during spaceflight
Spaceflight induces immune
abnormalities
•
Neutrophil counts increase 1.5x during flight, and
chemotactic response decreases 10x (Stowe 1999)
•
Lytic activity of NK cells decreases 40% (Mehta 2001)
•
CD8+ T cell function (including virus-specific T cells)
is decreased during and after flight (Crucian 2013)
•
Multiple cytokines are increased during flight
(Crucian 2013)
•
Generalized cytokine increase during flight is limited
to viral shedders (Mehta 2013)
Spaceflight and CMV reactivation
NASA/UW collaboration
•
Plasma and matched urine specimens from
spaceflight and pre/post flight periods
•
Evaluation by highly sensitive quantitative PCR
•
Numerous viruses being evaluated (CMV, HHV-6,
VZV…)
•
Goal is to build a quantitative temporal model of
viral shedding before, during, and after
spaceflight
What’s the best sample for
measuring CMV and other viruses?
What’s the best sample for
measuring CMV?
Dried blood spots for CMV
Limaye/Jerome 2013
CMV as a cause of
immune dysregulation
CMV as a cause of
immunosuppression
Effects on immune surveillance for other viruses or cancer?
CMV reactivation during
spaceflight - implications
• The observed reactivation may reflect serious underlying
immunosuppression
• Transmission to seronegative crew members?
• Primary CMV infection in spaceflight has not been studied (severe
manifestations?)
• CMV-induced immunosuppression may increase susceptibility to other
conditions (VZV, HSV, autoimmunity) during long-duration spaceflight
CMV and spaceflight unknowns
• About 530 humans have flown in space
• A cumulative total of just 77 years in space
The human presence in space is
about to change dramatically
…and the pace of change is likely
to accelerate
The human experience with CMV will soon
shift to space – questions to consider
•
What are the patterns of shedding during long-duration
orbital flights?
•
Are there additional risks with longer-term interplanetary
travel?
•
What is the frequency of CMV transmission during
spaceflight? How does it occur?
•
What does primary CMV disease look like in microgravity?
1/6 or 1/3 g?
•
Are the long-term effects of CMV the same in space as on
Earth?
•
What are the effects of congenital CMV in space?
Reactivation of CMV and other viruses in
space – a pathway forward
•
We need to develop a comprehensive understanding of
reactivation of latent and persistent viruses in space (HSV,
VZV, EBV, CMV, HHV-6, -7, -8, BKV, JCV, HPV, HBV),
including their interactions and long-term effects
•
We need much more extensive sampling of various body
sites during flight, including blood, saliva, urine
•
This will require development of minimally invasive
sampling techniques, and mass- and volume-optimized
sample storage and transport formats
•
Earth-based shedding studies and efforts to optimize
patient satisfaction should inform these efforts
Acknowledgments
Meei-Li Huang
Tracy Santo
Satish Mehta
Duane Pierson
University of Washington Molecular Virology Laboratory
Cytomegalovirus
• Air Force Major Arthur "Kit" Murray piloted the X-1A
to 90,400 feet; unofficial altitude record. (USA)
• Suborbital rocket flights with dogs (USSR)
Immune control of CMV
T cells, especially CD8+, are critical for
control of CMV
As much as 50% of CD8+ repertoire is
CMV-specific in aged individuals
Kern, Surel Nat Med 1998