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