Juliane Karthe, Kathi Tessmann, et al. HEPATOLOGY, Vol. 48, No. 3, 2008

1. Specific Targeting of Hepatitis C Virus Core Protein by an Intracellular Single-Chain Antibody of Human Origin Juliane Karthe, Kathi Tessmann,et al. HEPATOLOGY, Vol. 48, No. 3, 2008 -----Presented by Gan Weiqiang

2. Background

3. Hepatitis C virus (HCV): discovered in 1989, infects approximately 200 million people worldwide. Persistent infection:chronic hepatitis, hepatic steatosis, cirrhosis, HCC. HCV:small, enveloped RNA virus, Hepacivirus genus of the Flaviviridae family. Hepatitis C Experimental models of hepatitis C virus infection, replication, and pathogenesis. Hepatology 2001;33:489–95.

4. HCV genome structure Hepatitis C virus proteins World J Gastroenterol 2007 May 7; 13(17): 2406-2415

5. HCV protein and its function Hepatitis C virus proteins World J Gastroenterol 2007 May 7; 13(17): 2406-2415

6. Description:Structural Protein, part of the viral nucleocapsid Position:N-terminal of the polyprotein Formation: cleavage at ER by signal peptidases Core Protein

7. N-terminus: Hydrophilic, highly basic and contains RNA and DNA binding domains, nuclear localization signals. C-terminus: Hydrophobic, mediates anchorage to the ER. Structure

8. Longer form (p21):residues 1-191,localizes to the cytoplasm related to ER membrane. Shorter form (p19): residues 1-173,derived from p21. Translocated to the nucleus. Form

9. Function Steatosis carcinogenesis cell growth Core Protein apoptosis signaling

10. Hepatic steatosis: Chronic hepatitis C Intrahepatic core protein Hepatic steatosis In human hepatoma cells: upregulate transcription of most genes involved in fat/lipid metabolism. Function Function

11. HCC CORE PROTEIN Function mitogen-activated protein kinase /extracellular signal-related kinase Interact with and maintain an activated form of (PKR) Activate MAPK/ERK pathway, Wnt-1 signaling the RNA-dependent protein kinase

12. Lack of a preventive vaccine Lack of effective antiviral therapies New solution in need! Challenges

13. scFv---single-chain variable fragments

14. Small Molecular Weight,high permiability! Low immunogenicity----can be used repeatedly! With easy gene process----can be obtain in large quantity! Advantages of scFv

15. alter target Protein Fuction of scFv Folding Potential therapeutics against cancer and HIV Interaction Modification Subcellular localization

16. How do we get it? Phage Display Technology

17. Core protein:most conserved HCV protein ---- have a broad effect on different genotypes Viral life cycle and pathogenesis---- 1.Abrogate core particle assembly and RNA packaging 2.Affect its direct effect on cell proliferation and lipid metabolism Why do we use scFv?

18. Develop a human antibody fragment scFv against HCV core protein ; Investigate its intracellular distribution; Find out its association with the core protein; Look into its impact on cell proliferation triggered by the core protein. Research purposes

19. Research Method

20. DNA Sequencing DNA Sequencing Affinity Selection EIA Extract Bone marrow Human scFv Library Construct plasmids of scFV42C and Core Protein Construct plasmids of scFV42C and Core Protein Map the Epitope Recognized by scFv42C Expression and Purificationof scFv42C Expression and Purificationof scFv42C Transfect Huh7 cells Confocal Laser Scanning and FRET Cell Proliferation Assay. Immunoflu-orescence Immuno-blotting Technology Routine

21. Patients: Serologically documented HCV infection (HCV antibody by third-generation enzyme-linked immunoassay) Serum HCV-RNA by reverse transcription polymerase chain reaction(RT-PCR) Extract Bone marrow

22. PCR human VH and VL fragments from bone marrow aspirate extracts Cloned into the phagemid vector pAK100.25 Expression of an scFv as a gene III fusion protein on the surface of filamentous phages Human scFv Library

23. Affinity Selection EIA Extract Bone marrow Core protein Phage Display Technology

24. Identify peptide sequences recognized by the scFv42C antibody: 1.The Ph.D.-7 phage display library peptides kit containing linear peptides(randomly presenting 7-12 amino acids) 2.Selected clones were sequenced and the amino acid sequence of the presented peptides was deduced to determine the consensus binding motif. Map the Epitope Recognized by scFv42C.

25. 1. scFv42C-YFP and scFvIR-YFP: cDNA encoding scFv42C and scFvIR (irrelevant scFv) antibody fragments were cloned into pEYFP-N1 with its C-terminus fused to enhanced yellow fluorescence protein(YFP) 2.pEF/sc42:scFv42C cDNA was cloned into EF/myc/cyto vector Molecular cloning

26. 3.pZeo/core: cDNA encoding the full-length core protein (aa 1-191 of 1b genotype)was cloned into pcDNA3.1/Zeo(+) vector 4. CFP-C191:cDNA encoding the full-length core protein (aa 1-191 of 1b genotype)was subcloned into pRc/CMV and pECFP-C1 vector with N-terminal fusion of the enhanced cyan fluorescence protein (CFP) Molecular cloning

27. CFP-C191/Core-pRc/CMV scFv42C-YFP/scFvIR-YFP pZeo/core and pEF/sc42 JFH1(replicon RNA) and pEF/sc42 Transfection Experiments Transfect Huh7 for colocalization assay Transfect Huh7 for Western blot Transfect Huh7.5 for Western and Northern blot

28. Immunofluorescence Confocal Laser Scanning Microscope Fluorescence Resonance Energy Transfer Technique(FRET). Colocalization Experiments

29. Cell proliferation:day 1 or 2 after transfection Add tetrazolium to medium Measure absorbence at 450 nm after 1-4 hours Cell Proliferation Assay

30. Results

31. Identification of High-Affinity Single-Chain Antibodies Against HCV Core Protein.

32. Amino Acid Sequence

33. Purification of scFv42C Figure 3

34. Verification The purified antibody could recognize the core protein (aa 1-115) in Western blot, albeit the signal was much weaker than that of mouse anti-core antibody (Fig.3B). Figure 3

35. Mapping of the scFv42C BindingMotif on the Core Protein. Phage Display Peptide Library scFv42C Phage Display Technology

36. Mapping of the scFv42C BindingMotif on the Core Protein.

37. Confocal laser scanning microscope was used to investigate intracellular interaction between scFv42C and core protein. To facilitate their detection,CFP was attached to the N-terminus of core protein(CFP-191),whereas YFP was tagged to the C-terminus of scFv42C (scFv42C-YFP) or scFvIR (scFvIR-YFP). Intracellular Colocalization of scFv42C with HCV Core Protein.

38. Localization of Core Protein and scFv42c individually Figure 5

39. Coexpression of scFv42C with the core protein Figure 5 Coexpression of scFv42C with the core protein shifted the scFv42C expression pattern to globular-like, which colocalized with HCV core.

40. FRET Fluorescence Resonance Energy Transfer Technique Core protein scFv-42C scFv-IR Core protein

41. FRET Analysis

42. The scFv42C Antibody Reduced Core Protein Level but not Viral Replication per se in Transient Transfection. scFv42C’s reduce Core protein

43. Cotransfect with core

44. Cotransfect with HCV

45. Transfect with HCV

46. Effect of scFv42C on cell proliferation

47. Discussion

48. Smaller size Ease of production Improved tissue penetration Advantages of scFv over intact antibody

49. In the study of HIV 1.scFvs targeting the tat or VIF protein: Inhibite HIV replication; 2.scFvs against the integrase: Confer cellular resistance to HIV infection Applications

50. As for cancer therapy 1.scFvs targeting vascular endothelial growth factor receptor 2: Reduce the malignant potential of melanoma cells 2.scFvs recognizing lung cancer–associated common antigens: Reduce the growth of lung cancer cells Applications