T-ce ll Immunity to the Hepatitis C Virus During and After Pregnancy
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Transcript of T-ce ll Immunity to the Hepatitis C Virus During and After Pregnancy
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T-cell Immunity to the Hepatitis C Virus During and After Pregnancy
BCMM AND VACCINES & IMMUNITY JOINT MEETING
Sept 2, 2011
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C E1 E2 p7NS2 NS3 aNS4b aNS5b
≈3000 aa
EnvelopeGlycoproteins
Core Serine proteaseHelicase
ProteaseCofactor
RNA dependentRNA polymerase
F
Hepatitis C Virus• Small, positive-stranded RNA virus
• Prototype virus within the Hepacivirus genus of the Flaviviridae
• 6 Genotypes, multiple subtypes
• Genotype 1 is the most common and the most resistant to treatment
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Adapted from Lauer G & Walker B. NEJM 2001;345:41-52
≥ 30 years Slow ProgressionFemale sex, young age
at infection
Rapid ProgressionAlcohol use, coinfection
≤ 20 years
Normal Liver
Chronic Hepatitis
Cirrhosis (20%)
Carcinoma (1-4% per year)
Acute Infection
Resolved Infection
(25%)
Chronic Infection
(75%)
- HCV related liver diseases – now the leading cause for liver transplantation in developed world
Outcome of HCV Infection
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~25%
~75%
Transaminase (liver cell death)
2 6 12 24
Vire
mia
CD
4+ T
cel
ls (b
lood
)C
D8+
T c
ells
(blo
od)
serum antibodies
weeks years
serum antibodies
Vire
mia
CD
4+ T
cel
ls (b
lood
)C
D8+
T c
ells
(blo
od)
2 6 12 24
T-cell Immunity to HCV in Acute Resolving vs Persisting Infection
RapidResolution
Persistent Infection
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T-Lymphocytes: Adaptive Immune Cells
Randomly generated receptors
- Repertoire of 2*107 distinct T-cell receptors in peripheral blood
Recognize foreign peptides presented by MHC molecules on cell surfaces
CD8+ Cytotoxic T-cells: Target peptides from endogenous proteins on infected cells
Kill infected cells
CD4+ Helper T-cells:Target peptides from exogenous proteins presented by professional antigen presenting cells
Secrete antiviral cytokines and augment function of CD8+ T-cells and B-cells
(Nikolich-Zugich et al. Nat Rev Imm. 2004; 4:123-132)
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HCV-specific CD8+ T-cell
HCV-Infected Hepatocyte
Class I MHC Molecule
HCV Peptide
T-cell ReceptorAntiviral cytokines
inhibit viral replication &
cytotoxic chemicals kill infected cells
T-cell Success in Resolving HCV
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T-cell Failure in Chronic HCV
Poor Proliferation
Reduced cytotoxicity and
antiviral cytokine secretion
Low Frequency
Inhibitoryco-receptors
(PD-1, CTLA4, Tim-3)
Mutated HCV
Epitope
Escape mutation T-cell exhaustion
HCV-specific CD8+ T-cell
HCV-Infected Hepatocyte
HCV-specific CD8+ T-cell
HCV-Infected Hepatocyte
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Weak HCV-specific T-cell responses in chronic infection
Core E1 E2 P7 NS2 NS3 aNS4b aNS5b
CMVpp65
DMSOcontrol
HCV 1b Peptide Set
Ex-vivo IFN-γ ELISpot200,000 PBMCs/well
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OSU-NCH Hepatitis C Virus Immunity in Women and Children Study
AntepartumMaternal Samples
infant Samples
18 moDelivery 6 mo3 mo
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Influence of Pregnancy on Hepatitis C Viral Load
M001: Genotype 2b. Age at 1st delivery: 26 yrs. Estimated duration of infection prior to 1st delivery: 12 yrs.
M003: Genotype 1a. Age at 1st delivery: 34 yrs. Estimated duration of infection prior to 1st delivery: 0.6 yrs.
M016: Genotype 2a. Age at 1st delivery: 24 yrs. Estimated duration of infection prior to 1st delivery: 4.7 yrs.
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Years
Vire
mia
CD
8+ F
unct
ion
CD
4+ F
unct
ion
Prepregnancy Pregnancy Postpartum
Influence of Pregnancy on Hepatitis C Viremia
Hypothesis: Resurgent HCV-specific T-cell immunity after delivery mediates the
drop in viremia.
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Postpartum Viral Load Decline Associated with Broadening of HCV-Specific T-cell Response after Delivery
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Resurgence of Polyfunctional CD4+ T-cells
Subject M001
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Define the Immunological Signature of Postpartum Viral Control
Pregnancy Postpartum
Postpartum Viral Control
Stable Viremia
1) Function & Phenotype of HCV-specific T-cells (Proliferation, Cytokine Secretion, Survival and Inhibitory Receptor Expression)
2) HCV-specific T-cell Receptor Analysis(Diversity, Avidity)
3) Serum Cytokine Profile4) Gene-expression profile of HCV-specific T-cells
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Influence of Pregnancy on HCV Evolution
HCV genome mutates readily– 1012 virions produced daily– RNA-dependent RNA polymerase lacks proofreading function
HCV mutates to escape CD8+ T-cell pressure– 50-70% of targeted class I epitopes mutate to escape T-cell responses – Appearance of escape mutations linked to failure to clear viremia– Some escape mutations impair replicative fitness and revert to wild-type when transferred to other
individuals
Hypothesis: Enhanced CD8+ T-cell pressure after delivery will cause accelerated viral evolution
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Viral Sequencing Through Consecutive Pregnancies
Subject M003
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Study week
-6
0
25
40
56
66
86
152
Preg #1
Preg #2
Viral Sequencing Through Consecutive Pregnancies
Vertical lines represent amino acid substitutions relative to week -6 consensus sequence.Height of lines proportionate to fraction of clones bearing mutant residue.
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Viral Evolution Accelerates After Both Pregnancies
Preg
nanc
y #1
(wk.
..
Posp
artu
m #
1 (w
...
Late
Pos
tpar
tum
...
Preg
nanc
y #2
(w...
Post
part
um #
2 (..
.0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Amino Acid Substitution Rates: Non-Structural Region
Transient substi-tutions
Persisting substi-tutions
New
am
ino
acid
su
bstit
ution
s pe
r wee
k
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Study week
-6
0
25
40
56
66
86
152
Preg #1
Preg #2
Reversion of Mutation During 2nd Pregnancy
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Mutations in overlapping T-cell epitopes permit escape from T-cell pressure
M003 1395/9 M003 1402/9
(*Adjusted for transfection efficiency)
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H77S.3/AG L1403F L1403V K1398R & A1409T
0
50
100
150
200
250
300
350
FFU/
ml
24 48 72 960
10
20
30
40
50
60
H77S.3/AGL1403FL1403VK1398R & A1409TH77S/AAG
Hrs after transfection
GLuc
acti
vity
Infectious HCV cell culture virus H77S.3 bearing “wild-type” week -6 sequence is more replicatively fit than virus bearing
the escape mutant sequences.
RNA Replication Infectious Virus Production
“wk -6 wild-type sequence”
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Influence of Pregnancy on HCV Evolution
YearsVi
rem
iaCD
8+ S
elect
ion
Pres
sure
Vira
l Rep
licat
ive
Fitn
ess
Prepregnancy Pregnancy Postpartum
A
B
C
A: Reversion of escape mutations
B: Re-emergence of previous +/- appearance of new escape mutations
C: Compensatory mutations
Aim 2. Define patterns of HCV evolution during and after pregnancy. - Determine if reduced virus replication after pregnancy is associated with an increased frequency of
escape mutations in class I epitopes. - Determine the fitness cost of non-synonymous viral escape mutations that are lost during pregnancy.
Rationale. Provides an independent virologic readout of intrahepatic CD8+ T-cell selection pressure to address the important question of whether HCV-specific T-cells can be functionally restored
Provide insight into the replicative fitness of viral quasispecies passed vertically in mother to child transmission, the most common route of pediatric HCV infection
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Pregnancy and other Persistent Viral Infections
HBV• Mean HBV DNA levels rise in
pregnancy and fall in the postpartum period
– ter Borg et al. J Viral Hepat 2008; 15:37-41
• 5 of 31 HBeAg+ women became HBeAg- in the postpartum period compared to 0 of 30 non-pregnant women.
– Lin et al. J Med Virol 1989; 29:1-6
HIV• No significant change in viral load
during or after pregnancy– Burns et al. Am J Obstet Gynecol
1998; 178:355-9– Melvin et al. J Acquir Immune Defic
Syndr 1997; 14:232-236– Garcia et al. N Engl J Med
1999;341:394-402
T3 2 mo PP 12 mo PP 24 mo PPT1 or
T2
ter Borg et al. Burns et al.