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.