t-cell immunity to the hepatitis c virus during and after pregnancy bcmm and vaccines & immunity...
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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
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
Adapted from Lauer G & Walker B. NEJM 2001;345:41-52
≥ 30 years Slow Progression
Female sex, young age at infection
Rapid Progression
Alcohol 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
~25%
~75%
Transaminase (liver cell death)
2 6 12 24
Vire
mia
CD
4+
T c
ells
(b
loo
d)
CD
8+
T c
ells
(b
loo
d)
serum antibodies
weeks years
serum antibodies
Vire
mia
CD
4+
T c
ells
(b
loo
d)
CD
8+
T c
ells
(b
loo
d)
2 6 12 24
T-cell Immunity to HCV in Acute Resolving vs Persisting Infection
RapidResolution
Persistent Infection
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)
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
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
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 ELISpot
200,000 PBMCs/well
OSU-NCH Hepatitis C Virus Immunity in Women and Children Study
AntepartumMaternal Samples
infant Samples
18 moDelivery 6 mo3 mo
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.
Years
Vir
emia
CD
8+ F
un
ctio
nC
D4+
Fu
nct
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.
Postpartum Viral Load Decline Associated with Broadening of HCV-Specific T-cell Response after Delivery
Resurgence of Polyfunctional CD4+ T-cells
Subject M001
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 Profile
4) Gene-expression profile of HCV-specific T-cells
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
Viral Sequencing Through Consecutive Pregnancies
Subject M003
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.
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 substitu-tions
Persisting substitu-tions
New
am
ino
acid
su
bstit
ution
s pe
r wee
k
Study week
-6
0
25
40
56
66
86
152
Preg #1
Preg #2
Reversion of Mutation During 2nd Pregnancy
Mutations in overlapping T-cell epitopes permit escape from T-cell pressure
M003 1395/9 M003 1402/9
(*Adjusted for transfection efficiency)
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”
Influence of Pregnancy on HCV Evolution
YearsVi
rem
iaC
D8+
Sel
ectio
n Pr
essu
reVi
ral R
eplic
ativ
e Fi
tnes
s
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
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.