Antiviral Immunity andViral Escape Mechanisms

Barbara Rehermann, MD

Immunology Section and Liver Diseases Branch, NIDDKNational Institutes of Health, DHHS,

Bethesda, Maryland, USA

Time after Infection [Weeks] [Years]

Anti-HCV

Chronic InfectionAcute PhaseInnate

2220

Per

cent

of M

axim

um

0

20

40

60

80

100

0 2 4 6 8 10 12 14 16 18 24 26 28 30 10 20 30

HCV RNAALT

2220

Per

cent

of M

axim

um

0

20

40

60

80

100

0 2 4 6 8 10 12 14 16 18 24 26 28 30 10 20 30

Anti-HCV

Time after Infection [Weeks] [Years]

ALTHCV RNA

Recovery = Protective Immunity?

T cell responses weak

T cell responsesreadily detectable

Induction of Antiviral State

IFN-β

STA

T 1

STA

T 2

PP

IRF9

IFN-stimulated genes:OAS, PKR, IRF7 and others

Jak1Tyk2

HCVcoreIFN-β

IFN-αIFN-β

ISGF3

IRF3-P

RIG-I: CARD helicase

CARDIF

TLR3

TBK1/IKKε

TBK1/IKKε

IFN-β

HCV replication:dsRNA

Cytokines Chemokines

HCV

IRF7-P

IKKα/β

NF-κBrelease

NF-κBrelease

IKKα/β

Sensing of dsRNA

TRIF

HCVNS3/4A

HCVNS3/4A

HCV NS5A/IL-8

SOCS-1/-3

HCV E2 / NS5A

Induction of PP2Ainhibits

STAT 1 function

NS5A

Hepatocytes

HCV and Innate Immune ResponsesINNATE

IFN-α/β ↓

HCV

NS3/4

Hepato-cytes

antigen uptake

DC

NK

IL-10TGF-β

CD81

HCVenv proteins?

KIR

DCDendriticcells

Naturalkiller cells

E2

NS5

In VitroIn Vitro HCV Infection Model HCV Infection Model

Wakita et al. 2005 Nat Med 11:791Zhong et al. 2005 PNAS 102:9294Lindenbach et al. 2005 Science 309:623

C E1 E2 NS2 NS3 NS4B NS5A NS5B7JFH1 Virus (genotype 2a) 5’NTR 3’NTR

JFH1 (genotype 2a)

4A

Chimeric genotype 1a / 2a virus

H77S (genotype 1a) JFH1 (genotype 2a)

5’NTR 3’NTRC E1 E2 NS2 NS3 NS4B NS5A NS5B7 4A

In VitroIn Vitro HCV Infection Model HCV Infection Model

105

0 7 14 21 28

HC

V R

NA

tite

r in

cel

l cul

ture

supe

rnat

ant [

RN

A c

opie

s / m

l]

Time after infection [days]

108

107

106

Chimeric virus

JFH1 virus

HCV RNA Titer

1.5

2.0

2.5

3.0

LDH

lev

el in

inf

ecte

d cu

lture

s re

lativ

e to

uni

nfec

ted

cultu

res

0.0

1.0

0 7 14 21 28Time after infection [days]

3.5

LDH Release

0

20

40

60

80

100

120

% v

iabl

e ce

lls in

infe

cted

cul

ture

s re

lativ

e to

uni

nfec

ted

cultu

res

0 7 14 21 28

Time after infection [days]

Cell Viability

Yoon et al., Hepatology 2008, in pressShiina et al., Hepatology 2008

HCV Inhibits IFN-α Production of Dendritic Cells

1. HCV inhibits IFN-α production of plasmacytoid DC viadirect interaction that does not require replication.

Shiina et al., Hepatology 2008

2. Influenza A virus is able to override the inhibition.

3. HCV does not inhibit myeloid DCs and monocyte-derived DCs, but vaccinia virus does.

4. Uptake of HCV-infected or uninfected apoptotic hepatoma cells does not result in differential activation of dendritic cells.

Does HCV Inhibit NK Cell Function ?

Crotta et al., J Exp Med 2002Tseng et al., J Exp Med 2002

HCV E2 protein (1-10 µg/ml)

CD81NK

Published:

HCV envelope protein

• E2 in natural configuration• lower concentration

?NK

HCV virion

Question:

HCV particles?

Yoon et al., Hepatology 2008 in press

HCV Does Not Inhibit NK CellsHCV Does Not Inhibit NK CellsCD

25+

NK c

ells

(%)

0

20

40

60

p < 0.01

+ hi

gh co

nc.

HCV(

JFH1

)

α CD

16α

CD16

α CD

16

α CD

16

+ α

CD8

1

+ high

con

c.

HCV(

H-NS

2/3-J

)

CD25 Expression

CD69

+ NK

cel

ls (%

)

0

20

40

60

p < 0.05

+ hi

gh co

nc.

HCV(

JFH1

)

α C

D16

α CD

16

α CD

16

+ α

CD8

1

+ high

con

c.

HCV(

H-NS

2/3-J

)

α CD

16

CD69 Expression

0

500

1000

1500

2000

+ hi

gh co

nc.

HCV(

JFH1

)

α CD

16

α CD

16

α CD

16

+ α

CD8

1

+ high

con

c.

HCV(

H-NS

2/3-J

)

α CD

16

IFN-γ

(pg/

mL)

IFN-γ Secretion

Yoon et al., Hepatology in press

Khakoo SI et al., Science 2004

Odds Ratio of HCV Clearance

0 1 2 3 4

KIR2DL3 + HLA-C1homozygous

KIR2DL3 + HLA-C1heterozygous

KIR2DL3 + HLA-C1null

X2 for trend = 12.23P=0.0004

Epidemiologic Study:HLA and NK Cell Genes Associated with HCV Clearance

Hsu et al., Immunological Reviews 2002

Killer Cell Immunoglobulin-Like Receptor (KIR) Alleles

Ligand: HLA-C

NK Cell

Target Cell

KIR

HLA

Tolerance

HLA⇓

Inflammation

Regulation of NK Cell Activation by KIR / HLA

HLA-C Dimorphism

Ser77Asp80

Cw1 Cw3 Cw7 Cw8 Cw12Cw13Cw14

HLA-C1 group

KIR2DL3/2DL2 NK cellinhibition

HLA-C2 groupAsp77Lys80

Cw2 Cw4 Cw5 Cw6 Cw15Cw17

KIR2DL1

Role of KIR/HLA-C in Liver Disease

HLA-C1/C1(KIR2DL3) HLA-C2/C2

(KIR2DL1)

Chronic infectionPartial protection

Increased riskLonger graft survival

HCV infectionPrimary sclerosing

cholangitisHepatocellular carcinoma

Liver transplantation

Spontaneous recovery

Partial protection

Liver disease

NK cell response weaker

NK cell response

stronger

Does KIR2DL3/HLA-C1 homozygosity lead to stronger NK

cell responses than KIR2DL1/HLA-C2 homozygosity?

Study Cohort

All donors have KIR haplotype A

(KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR2DS4)

HLA-C Group 1 homozygous HLA-C Group 2 homozygous(n = 12) (n = 10)

Frequency of KIR-Expressing NK Cells

HLA-C1/C1 positive subject

HLA-C2/C2 positive subject

0

10

20

30

40

50

60

70

HLA-C1/C1Subjects

HLA-C2/C2Subjects

p = 0.0105

Freq

uenc

y of

HLA

-C in

hibi

ted

NK

cel

ls in

PB

MC

(%)

Ahlenstiel, J Clin Invest 2008

others

KIR2DL3 - FITC

CD

56-P

eCy7

0 103 104 105

0

103

104

105 55.9 44.1

KIR2DL3+

KIR2DL1 - APC

0 103 104 105

0

102

103

104

105 75.9 24.1

CD

56-P

eCy7

others KIR2DL1+

Analysis of NK Cell Responses in a Viral Infection Model

PBMC

CD3+ T cells

Influenza A virus Infection

3, 6, 9, 12, 15 h

Cytotoxicity:- % IFN-γ+ NK cells- Released IFN-γ

IFN-γ production:% Degranulating

NK cells

KIR2DL3+ NK Cells Degranulate more inResponse to Virus than KIR2DL1+ NK Cells

0 3 6 9 12 15300

350

400

450

500

550

600

HLA-C1/C1HLA-C2/C2

time (h)

HLA-C specific NK cells

Δ M

FI

others

0 103 104 105

0

103

104

105 55.9 44.1

0 103 104 105

0

102

103

104

105 75.9 24.1

KIR2DL3+

others KIR2DL1+

0 3 6 9 12 15 18300

500

700

900

1100

1300

1500

HLA-C1/C1

HLA-C2/C2

time (h)

Other NK cells

Δ M

FI

Ahlenstiel et al., J Clin Invest 2008

KIR2DL3+ NK Cells Respond Faster to Influenzathan KIR2DL1+ NK Cells

KIR2DL3 - FITC

HLA-C1/C1 positive subject

HLA-C2/C2 positive subject

KIR2DL1 - FITC

IFN

-γ -

PE

IFN

-γ -

PE

no infection IAV infection

no Infection IAV infection

8.1 5.3

33.453.2

0.4 0.3

37.262.1

0.2 0.1

23.875.9

10.5 1.9

24.263.40 3 6 9

0

1

2

3

4

5 2DL3+ NK cells (C1/C1)

Time (h)

Rat

io o

f IFN

-γ+

NK

cel

ls b

etw

een

indi

cate

d tim

e po

int &

17h

tim

e po

int

Ahlenstiel et al. J Clin Invest 2008

2DL1+ NK cells (C2/C2)

Summary: HCV and Innate Immune Responses

NK cell responsiveness is influenced by KIR/HLA

compound genotypes and contributes to viral clearance.

HCV does not directly inhibit natural killer cells.

HCV does blocks interferon-α production

- by hepatocytes: requires infection

- by plasmacytoid dendritic cells: direct interaction,not infection.

1. Innate Immune Responses

2. Adaptive Immune Responses

3. Protective Immunity?

ADAPTIVE IMMUNE RESPONSES

Resistance toeffect of

type I IFN?

HCV Modulates Host Immune Responses

Resistance ?

E2, NS5A

Infected hepatocytes

INNATE

IFN-α/β

HCV

?

antigen uptake ↓?

DC

NK

IL-10TGF-β

CD81

Inhibition by HCVenvelope proteins?

KIRHLA-C

DC

NS3/4

HCV quasispecies, mutations

HCV

CXCR3+CCR5+

CD4

CD8

Replication,HCV Titer

Late peak responsesHCV Resistance

CD4

ReducedT cell

primingDC

IL-2↓

IL-12↓

HCVcore

C1qR

C1qR

CD8

CD4CD25

Foxp3

Reducedproliferation

Priming not optimal

WeakCD4 T cell

help

IL-10

HCV

CD8

CXCR3+CCR5+

CD4

HCV persistence

Impairedfunctions

Rehermann and Nascimbeni, Nat. Rev. Immunol. 2005

1. Innate Immune Responses

2. Adaptive Immune Responses

3. Protective Immunity?

Outline

Antibodies decrease toundetectable levels 10-20 yearsafter recovery.

Immune Status after HCV Clearance

Takaki et al., Nature Medicine 2000

T cell responses remain readilydetectable in the blood.

Delayed HCV Clearance in the Absence of CD8+ T Cells

Weeks Post Infection

72

3

4

5

6

7

2

3

4

5

6

0 2 124 6 8 10 222014 16 18

HCV Infection

Primary

Secondary

Chimpanzee 1

Chimpanzee 2

Shoukry et al., J Exp. Med., 2003

HC

V R

NA

[log

GE/

ml p

lasm

a]

After CD8 depletion

Acknowledgements

Golo AhlenstielEui-Cheol Shin Suresh VeerapuAintzane ZabaletaSukanya RaghuramanSu Hyung ParkMaria Quasdorff

Jonathan StoltzfusBirgit EdlichFrances Kigel

Mary Carrington

Liver Diseases Branch, NIDDK, NIH, USA

Theo HellerMarc GhanyJay HoofnagleT. Jake LiangHarvey Alter

Collaborators

SAIC FrederickMaureen Martin

Alumni

University of Texas

National Institute of In-fectious Diseases, Japan

Stanley Lemon

Takashi Wakita

Rockefeller UniversityCharles Rice

Masaaki ShiinaJoo Chun Yoon Fareed RahmanChristina Weiler-Normann