HBsAg 阳性肝细胞的膜表面 HBsAg 抗原的检测

M1

Hep3B

HBsAg 阳性肝细胞

HepG2

91.3% 93.5

%

2.3%

Map of AAV Genome

E2A

Ori

V

AI

启动

子

Rep

Cap

E4

pHelper 质粒的改建

pHelper

HBsAb Fd

A series of human cells infected by tropic AAV/AFPp-GFP virus

（ 24 hours) HBsAg 阳性 HBsAg 阴性 Hep3B 肝细胞 肝癌细胞 HepG2 肝细胞

HBsAg 阴性

PBMC Hela LNcap Hs578T H2125 Lovo

A series of human cells infected by tropic AAV/ALBp-GFP virus

（ 24 hours) HBsAg 阳性 HBsAg 阴性 Hep3B 肝细胞 肝癌细胞 HepG2 肝细胞

HBsAg 阴性

PBMC Hela LNcap Hs578T H2125 Lovo

Tropic AAV/ALBp-HLA-A2 virus infection （ 24 hours)

未感染 感染

HBsAg 阳性肝细胞

5.6% 91.1%

HBsAg 阴性肝细胞

感染

1.4%

Tropic AAV/AFPp-HLA-A2 virus infection （ 24 hours)

未感染 感染

HBsAg 阳性 Hep3B 细胞 HBsAg 阴性 HepG2 细胞

感染

10.7% 87.8% 4.7%

HLA-A2-expressed Primary Hepatic Carcinoma Cells Killed by the rAAV/AFP-infected-DC Pulsed CTL

uninfected cells

AAV/AFPp-HLA-A2- infected cells

0.01% 90.1%

90.1%0.01%

50

40

30

20

10

0

Uninfected cells

AAV/HLA-2-infected cells

uninfected cells

AAV/HLA-2-infected cells

Uninfected cells

AAV/HLA-2-infected cells% killing

AAV-Rep78 function: interaction with HBV or AFP

Section II

Inhibit oncogene expression Inhibit cervical cancer

Inhibit Human papillomavirus p97 promoter

Inhibit E6 & E7 oncogene

Enhance recombinant AAV production

Can Rep78 inhibit HBV or AFP expression?

Expression of HBsAg inhibited by tropic AAV/AFPp-Rep78 virus infection

Expression of HBeAg inhibited by tropic AAV/AFPp-Rep78virus infection in HBV-positive PHC

84.6% 68.9% 53.9%32.9% 10.7%

感染前 24 小时 48 小时 72 小时 96 小时

Expression of AFP inhibited by tropic AAV/ALBp-Rep78 virus infection

Elevation of albumin by tropic AAV/ALBp-Rep78 virus infection in Hep3B cells

96.6

22.3%12.6%45.6% 50.8%

未感染前 48 小时 72 小时 96 小时

Rep78 (μg): 0 0.2 0.5 0 0.2 0.5 0 0.2 0.5 0 0.2 0.5

Enh I X 基因启动子 Enh II 前 C/C 启动子

AAV Rep78 与 HBV 基因的相互作用具有剂量依赖效应

Inhibition of HBV-C and –X Transcription by Rep78

C1.0ug1.0ug

C+Rep78

XX+GSTX+Rep78

Section III

Functions of expressed interferon in rAAV-transfeted hepatic cells

甲胎蛋白启动子

ALBp

TRTR Poly A

IFN-α 基因rAAV/Albp-IFN-α

ALBp

TRTR Poly A

IFN-γ 基因rAAV/Albp-IFN-γ

AFPp

TRTR Poly A

IFN-γ 基因rAAV/Albp-IFN-γ

构建携带干扰素基因的嗜肝性重组腺相关病毒

rAAV 在肝细胞中表达 IFN-α 抑制 HBsAg 和 HBeAg 的表达

77.2%12.1%52.9

52.9%25.7%

rAAV 在肝细胞中表达 IFN-a 对 HBeAg 表达的影响

0 小时 48 小时 72 小时 96 小时

rAAV 在感染的黑猩猩中表达人 IFN-a

AAV/Albp-IFN-αVirus, 1011 copies

ControlAAV

Expressed IFN-γ promote expression of MHC class I onrAAV-infected Hep3B cells

感染前

IFN-γ MHC class I

感染 AAV/AFPp

-IFN-γ 病毒后 12 hr

48 hr

96 hr

33.7%

48.5%

76.0%

52.3%

78.0%

85.2%

2.5%\\

%

rAAV 在肝细胞中表达的 IFN-γ 增强抗原特异性， MHC class I限制性 CTL 的杀伤活性70

60

50

40

30

20

10

0

CT

L杀

伤率

A B C D E F G H

HBsAg+ 肝细胞： B: HepG2

C: +anti-HLA I D:+INF-r

Hep3B: F: HBsAg+ 肝细胞

G:+anti-HLA I H:+INF-r

Conclusions

Generate rAAV with liver cell-specific promoters.

Gernerate liver-tropic rAAV.

Recover MHC class I expression by transfection of liver-tropic rAAV.

AAV Rep78 may become new anti-HBV infection or hepatic cancer drug.

Expressed INF-αcan inhibit HBV expression in the rAAV-transfected liver cells.

The liver-tropic AAV carrying IFN-Αcan express in animal models.

Expressed IFN-γcan promote expression of the HLA class I antigen in the rAAV-transfected liver cells.

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