日本神户大学医学研究院微生物学
教授,神户大学医学院国际医学研究
和治疗中心主任。
研究领域:微生物学
学术兼职:在日本病毒学会、日本
肝病学会、日本细菌学会、日本免疫
学会、日本癌症协会等多个团体任职。
Hak HOTTA 教授
丙肝发病机理及治疗进展
Hak Hotta, MD, PhD
Professor, Division of Microbiology
Kobe University Graduate School of Medicine
丙肝病毒
基因结构:单股, 正链RNA (9.6kb)
病毒结构:球形,有包膜, 55〜65 nm
黄病毒科,丙型肝炎病毒属
E2
E1
C (core)
RNA
Envelope
(lipid bilayer)
C E1 E2 NS2
NS4A
NS4B NS5A NS5B
p7
serine proteasesignal peptidase
signal peptide
peptidase
NS3
Protein HVR
zinc protease
C E15’ E2/NS1 NS2 NS3 NS4 NS5 3’
Genome
E2
E1
C
genome RNA
Virion
p7 : ion channel
NS2 : zinc protease
NS3 : serine protease
RNA helicase
NS4A: cofactor for NS3
NS5A: phosphoprotein
NS5B: RNA polymerase
HCV基因组, 蛋白质及病毒结构
structural proteins non-structural (NS) proteins
丙肝病毒吸附及进入细胞模拟图
CD81SR-B1 CLDN1LDLR
LDLR : LDL receptor
SR-B1 : Scavenger receptor class B1
CD81 :
CLDN1 : Claudin 1
( phagocytosis )
( envelope fusion )
( viral adsorption )
( low pH ) Moradpour et al. Nat Rev Microbiol 5:453-463, 2007
丙肝病毒生活周期图
( protein synthesis )
( genome replication )( virion maturation )
( virion release )
( viral entry )
Moradpour et al. Nat Rev Microbiol 5:453-463, 2007
这些“病毒学”的资料来自以下文献:
1) Wakita et al. Nat Med 11:791-796, 2005.
2) Lindenbach et al. Science 309:623-626, 2005.
3) Zhong et al. Proc Natl Acad Sci USA 102:9294-9299, 2005.
通过体外模拟,我们发现丙肝感染引起细胞凋亡是由BAX激发
的,线粒体介导的,并依赖半胱天冬酶的通路实现的。
(Deng et al. J Virol, in press)
Active caspase-3 HCV antigens
Mock HCV
A
c
t
i
v
a
t
e
B
a
x
Mock HCV
M
i
t
o
S
o
x
丙肝病毒进化树
1
2
3
4
5
6
1a
1b
1c
2b
2a
6 genotypes
>60 subtypes
Subtype Prevalent area
1a USA, Europe
1b East Asia〜Worldwide
1c Indonesia
2a Worldwide
2b Worldwide
2c – 2f
3a Thailand, UK, Brazil
3b Thailand
3c – 3f Nepal
3g Indonesia
4a – 4h Egypt, Middle East
5a South Africa
6a – 6k Thailand, Viet Nam
Japan: 1b (70%), 2a (20%), 2b (10%)
Doi et al. J Clin Microbiol 34:569-574, 1996
HCV-1c
Type 6
HCV-1b
丙肝病毒亚型亚洲分布图
Hotta et al. SEA J. Trop. Med. Public Health 28(Suppl 3): 23-31, 1997
Type 3
HCV感染人数:
全球:1.7亿
日本:150万
Prevalence of anti-HCVAb at different age
携带率(日本):
携带率 1.0%
携带率随年龄增高而增高
(过去传染率高)
Male
Female
Total
携带率(中国):
3.0% ~ 4.1% (Wkly Epidemiol Rec 72:341-348, 1997; 74:421-428, 1999)
9.6% (Zhang et al. Emerg Infect Dis 11:17-21, 2005)
Japan
丙肝流行病学
Chronic hepatitis
Cirrhosis
HCC
AH AC
Yearly 7%
30 y20 yCure
60-80%
Clinical course of HCV infection
丙肝临床
现
非持续性丙肝:
(CD4反应好)
急性肝炎
持续性丙肝:
(CD4反应差)
无症状携带者
慢性肝炎
非活动性
活动性
肝硬化
肝癌
丙肝与肝癌
丙肝和肝癌
日本每年超过2.7万人因肝癌死亡
(80%来自丙肝)
中国每年约超过80万
丙肝和肝硬化
日本每年有1.2万因肝硬化死亡
(70%来自丙肝)
中国每年约有36万
丙肝诱导宿主基因变异及肿瘤
Oxidative stress iNOS
DNA repair by DNA polymerase ζ and ι
mutation-prone
(10-times higher)
mutations of anti-oncogenes and proto-oncogenes
Tumor development
chromosomal DNA break (dsDNA break)
( Superoxide, NO, etc )
activation-induced
cytidine deaminase (AID)
activation
mutation-prone
Machida et al. PNAS 101:4262-4267, 2004
Machida et al. J Virol 79:8079-8089, 2005
HCV infection
1 转基因小鼠肝癌发生机理
2 体外培养细胞恶变及过度生长机理
3 分子机理
胞嘧啶脱氨酶激酶激活
Ras信号通路激活
MAP激酶通路激活
NF-kB激活
细胞转录失调(激活和受抑)
细胞周期紊乱(p21Waf1)
脂质代谢紊乱(APO II)
丙肝病毒核心蛋白致癌机理
(肝癌形成的分子机理)
Cellular changes
Immune escape
Immune disturbance
IFN escape
宿主与丙肝病毒相互作用
Immune system
HCV
Autoimmune diseases
・Hepatocellular carcinoma
・B lymphoma
・Fatty liver
・Insulin resistance
・CPE (cell death)
IFN system
Chronic infection
丙肝与干扰素
内源性表达干扰素:非特异性免疫
外源性给予干扰素:治疗
丙肝病毒能逃避干扰素的作用吗?
怎样逃避……?
TLR-3
IFN-α
IRF-3
(inactive)
IRF-3
(active)
IRF-7
IFN-β
TLR3 和 RIG-I是 dsRNA 的受体,可以监测病毒复制情况,
激发IFN-β产生
IFN-β induction
IFN-α induction
RIG-I
MAVS/IPS-1
(mitochondria)
TBK1/IKKε
( NF-κB )
TRIF
dsRNA
dsRNA
cf. Yoneyama et al. Nat Immunol 5:730-737, 2004
TLR-3
IFN-α
IRF-3
(inactive)
IRF-3
(active)
IRF-7
IFN-β
HCV NS3/4A 可以剪切 MAVS以抑制 RIG-I受体诱导的 IFNβ合
成,但是 TLR3诱导 IFNβ 产生通路不受影响
IFN-βinduction
IFN-αinduction
RIG-I
MAVS/IPS-1
(mitochondria)
TBK1/IKKε
( NF-κB )
TRIF
NS3/4A
0
0.5
1.0
1.5
Co
nt
NS
3
NS
3/4
A(
H0
5-5
)
NS
3/4
A(
H1
7-2
)I F
N
β
p
r
o
m
o
t
e
r
a
c
t
i
v
i
t
y
* *
Goodbourn et al., 2000
IFN-α/β
JAK1TYK2
STAT1 STAT2
IRF9 ISGF3
ISRE
IFN受体介导信号转导通路
IFNAR
Modified from Vilcek & Sen, Fields Virology 1996
PKR
2,5OAS
eIF2α
RNaseL
IFN
JAK/STAT
MxA
IFN介导产生抗病毒蛋白通路
Inhibition of protein synthesis
HCV感染抑制:
1)RIG-I介导 IFNβ产生 (NS3/4A)
2) JAK/STAT 信号通路 (Core; NS3/4A)
3) dsRNA激活蛋白激酶 (PKR) (NS5A)
4) 2’,5’-二磷酸腺苷合成酶 (NS5A)
HCV逃避IFN
C E1 E2
p7
NS2
NS4A
NS4B NS5A NS5BNS3
目前推荐治疗
1) HCV-1b; 高病毒载量: PEG-IFN/RBV (48 周)
2) HCV-1b; 低病毒载量: PEG-IFN or IFN 单用, or
PEG-IFN/RBV (24 周)
3) HCV-2a, 2b; 高病毒载量: PEG-IFN/RBV (24 周)
4) HCV-2a, 2b;低病毒载量: PEG-IFN or IFN monotherapy,
PEG-IFN :聚乙二醇干扰素
RBV : 病毒唑(antiviral drug)
IFN治疗中丙肝病毒清除动力学
Feld & Hoofnagle, Nature 436:967-972, 2005
IFN
Feld & Hoofnagle, Nature 436:967-972, 2005
丙肝病毒对IFN / RBV联合治疗的反应
Sustained
Virological
Response
影响IFN疗效的因素
1. Stage : acute > chronic > cirrhosis
2. Age : young > elderly
3. Gender (?)
4. Race : non-black > black
5. Other genetic factors : HLA, SNPs, etc
6. Cell-mediated immunity
1. Viral load : low > high
2. Viral genotype/subtype :
HCV-2a > 2b > 1b
3. Viral mutations
NS5A sequence (ISDR, IRRDR)
Core (aa positions 71 and 90)
E2 sequence (PePHD)
宿主因素
病毒因素
Clinical response Ratio
Early Virological Response (EVR[12W])
End of Treatment Response (ETR)
Sustained Virological Response (SVR)
Null-Response
Relapse
54% (27/50)
72% (36/50)
46% (23/50)
22% (11/50)
32% (16/50)
PEG-IFN/RBV联合治疗的临床应答
Non-SVR 54% (27/50)
( Viral clearance at week 12 of therapy )
( Viral clearance at the end of 48-week therapy )
( Viral clearance at the end of 24-week follow-up )
El-Shamy et al. Hepatology 48:38-47, 2008
(IFN/RBV Resistance Determining Region)
IRRDR
V3
Pre
-V3
2`, 5`-OAS-BD
PKR-BD
ISDR
E1 E2 P7 NS2 NS3 NS4A NS4B NS5A NS5B5' UTR 3' UTR
1973 2120 2209 2248 2274 2334 2356 2379 2419
HCV核心蛋白及NS5A与IFN治疗应答的关系
R70 / L91 : IFN sensitive
Q70 / M91 : IFN resistant
Core
El-Shamy et al. Hepatology 48:38-47, 2008
Cons. VLTESTVSSALAELATKTFGSSGSSAVDSGTATAPPDQASDDGDKG 9 ......................E...A.........GLP.....A. 10 .................A.....P..A.G..TA.....T.....A. 12 I.........................A..........EPPG...T. 15 ..S...................E...........S...P..G..A. 18 I..............AT.....................S.SK..T. 21 ......................E...A..............G..P. 24 I...................N.....A........HT.P.....T. 28 ......................................P.....T. 32 I..D.N..T.............E...............P.....T. 35 ....................G.....A................... 37 ......................E..T.......G.L.......N.E 38 .................................G....P.....T. 43 ........T.............E...............P.....A. 44 I.........................A........H.HS..T..A. 61 ......................E.............YLL....GT. 65 ...G....TV............EPP.A..........RP.....A. 69 ......L...................A...M..........N..AR 72 I.....................R.......A.....GRP.....TE 73 ......L...............D.........S.....P.N...A. 88 I................A........A.....S...NLP.....A. 90 ...............N........P......T......V.N...N.92 ............................................RE 98 ......................................P..GE.TR
3 I...................................NL......A. 8 ....................................T.S.G.S..E 11 ..S.........................G.V.....G......... 13 I.....................................P.....A. 16 ................................S...G.......RE 17 ......................E.....................A. 27 ....................................N.L..E..A. 42 ......................E..............PP.....A. 59 ......................E...............P....GA. 91 ..............................I.....N.......A. 93 I...............................S..LN.......A.
14 .........V..........D.E...A...M.............R. 19 ..S...................E...........S...P.....A. 20 I....................................GP.....T. 22 ......................................P..F..T. 23 I.....................E.............NE......A. 33 .............................................. 45 ......................E............L..V...C... 47 ......................................T......E 49 ..........................A...........P.....A. 52 ......................E...............S.G...A. 57 I..................A...E............SRP.....T. 67 ......................E...A.......L.G......... 71 I..D................................V.P.....A. 85 ...................S....AT.......G....T......E 97 ..........................A.............G...T. 100 ......................E.............EE........
Cons. IPKARRPEGRTWAQPGYPWPLYGNEGLGWAGW 9 ................................ 10 ..........A..................... 12 ................................ 15 ................................ 18 ..........A..................... 21 ................................ 24 ................................ 28 ................................ 32 .....Q....A..................... 35 ..........A..................... 37 .....H.......................... 38 ................................ 43 ..........A...............M..... 44 ..........S..................... 61 ..........................M..... 65 ..........A..................... 69 ..........A..................... 72 ................................ 73 .....Q....A...............M..... 88 ..........A..................... 90 ......S................D........ 92 ................................ 98 .....Q....A.....................
3 .....Q....A..................... 8 ..........................M..... 11 .....Q....................M..... 13 ...V............................ 16 ..........A...............M..... 17 .....Q....A..................... 27 .....Q....A..................... 42 .........A................M..... 59 ..........A...............M..... 91 ................................ 93 .....Q....A.....................
14 .....Q.......................... 19 .....Q....A..................... 20 ..........................M..... 22 .....Q....S..................... 23 ................................ 33 .....Q....A..................... 45 ..........................M..... 47 ................................ 49 ......S...................M..... 52 .....Q....................M..... 57 ................................ 67 .....Q....................M..... 71 ..........A..................... 85 ...V......A..................... 97 ..........A..................... 100 ..........A.....................
Null-R
Relapse
SVR
IRRDR (NS5A)2334 237965 9670 91
SVR和Non-SVR的HCV序列比较
Core
敏感性 = 76%
特异性 = 92%
IRRDR 变异的数量预测SVR
Factor
No. of subjects / no. of subtotal*
SVR Non-SVR P value
A2360 10 / 23 (43%) 3 / 27 (11%) 0.02
IRRDR ≥6 和 R70/L91 与 SVR的相关性
IRRDR ≥6 16 / 23 (70%) 3 / 27 (11%) 0.0001NS5A
R70 / L91 17 / 23 (74%) 9 / 27 (33%) 0.01 Core
ISDR ≥4 3 / 23 (13%) 1 / 27 (4%) NS
Q70 / M91 1 / 23 (4%) 3 / 27 (11%) NS
Q70 3 / 23 (13%) 11 / 27 (41%) NS (0.06)
M91 3 / 23 (13%) 10 / 27 (37%) NS (0.11)
T2378 9 / 23 (39%) 4 / 27 (15%) NS
R70 19 / 23 (83%) 16 / 27 (59%) NS (0.14)
L91 20 / 23 (87%) 17 / 27 (63%) NS (0.11)
Q70 and/or M91 5 / 23 (22%) 18 / 27 (67%) 0.002
ISDR ≥2 10 / 23 (43%) 7 / 27 (26%) NS
Protein
IRRDR ≥6 及 R70/L91与IFN应答的关系
IRRDR ≥6
R70/L91
Non-R70/L91
IRRDR ≤5
H
C
V
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H
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[ Q70(and/or)M91 ]
SVR prediction Non-SVR prediction
SVR 及 Non-SVR的预测标志物
IRRDR ≥6 IRRDR ≤5
>
<
Core R70 / L91 Core Q70 (and/or) M91
El-Shamy et al. Hepatology 48:38-47, 2008
Akuta et al. J Hepatol 46:403-410, 2007
HCV抗病毒治疗前景
1. 干扰素(IFN), Peg- IFN
2. 利巴韦林
3. NS3 蛋白酶抑制剂
4. NS5B RNA依赖的RNA多聚酶抑制剂
5. NS3解螺旋酶抑制剂
6. 环孢霉素 A 诱导剂
C E1 E2
p7
NS2
NS4A
NS4B NS5A NS5BNS3
Thank you for your attention!!
Department of Microbiology
Kobe University Graduate School of Medicine
S. IshidoM. Nagano T. Toritani M. Itoh
S. OgataT. Taguchi
W.-Y. Tong T. Fujita
K. Hachida L. Deng
R.Hidajat
R.H Florese
T. Adachi
Y. Ide
M. I.Lusida
K. Oka
S. Muramatsu
J. Song
S. Inubushi
I. Shoji
M. Takamatsu
Y. Iwanaga
I. Yoshida
F. Wang
K. Sada
H. Hotta
謝 謝 !
日本神户大学医学研究院微生物学教授,神户大学医学院国际医学研究和治疗中心主任。�� 研究领域:微生物学�� 学术兼职:在日本病毒学会、日本肝病学会、日本细菌学会、日本免疫学会、日本癌症协会等多个团体任职。