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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
Background
Hepatitis C
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.
Experimental models of hepatitis C virus
infection, replication, and pathogenesis.
Hepatology 2001;33:489–95.
Background
HCV genome structure
Hepatitis C virus proteins World J Gastroenterol
2007 May 7; 13(17): 2406-2415
Background
HCV protein and its function
Hepatitis C virus proteins World J Gastroenterol
2007 May 7; 13(17): 2406-2415
Background
Core Protein
Description:Structural Protein, part of
the viral nucleocapsid
Position:N-terminal of the polyprotein
Formation: cleavage at ER by signal
peptidases
Background
Structure
N-terminus:
Hydrophilic, highly basic and contains RNA
and DNA binding domains, nuclear
localization signals.
C-terminus:
Hydrophobic, mediates anchorage to the
ER.
Background
Form
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.
Background
Function
Steatosis
cell
growth
carcinogenesis
Core Protein
apoptosis
signaling
Background
Function
Hepatic steatosis:
Function
Chronic hepatitis C
Intrahepatic core protein
Hepatic
steatosis
In human hepatoma cells:
upregulate transcription of most genes
involved in fat/lipid metabolism.
Background
Function
CORE PROTEIN
Interact with
and maintain
an activated
form of (PKR)
the RNAdependent
protein kinase
mitogen-activated
protein kinase
/extracellular signalrelated kinase
Activate
MAPK/ERK
pathway, Wnt1 signaling
HCC
Background
Challenges
Lack of a preventive vaccine
Lack of effective antiviral therapies
New solution in need!
Background
scFv---single-chain variable fragments
Background
Advantages of scFv
Small Molecular Weight,high
permiability!
Low immunogenicity----can be used
repeatedly!
With easy gene process----can be
obtain in large quantity!
Background
Fuction of scFv
Folding
Interaction
alter target
Protein
Modification
Subcellular
localization
Potential
therapeutics
against
cancer and
HIV
Background
How do we get it?
Phage Display Technology
Background
Why do we use scFv?
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
Background
Research purposes
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.
Background
Research Method
Technology Routine
Extract Bone
marrow
Human scFv
Library
Affinity
Selection
EIA
Transfect
Huh7 cells
Construct
plasmids of
scFV42C and
Core Protein
Map the
Epitope
Recognized
by scFv42C
Immunofluorescence
Confocal
Laser
Scanning
and FRET
Immunoblotting
DNA
Sequenc
ing
Expression
and
Purificationof
scFv42C
Cell
Proliferation
Assay.
Method
Extract Bone marrow
Patients:
Serologically documented HCV infection (HCV
antibody by third-generation enzyme-linked
immunoassay)
Serum HCV-RNA by reverse transcription
polymerase chain reaction(RT-PCR)
Method
Human scFv Library
1. PCR human VH and VL fragments from bone
marrow aspirate extracts
2. Cloned into the phagemid vector pAK100.25
3. Expression of an scFv as a gene III fusion
protein on the surface of filamentous phages
Method
Affinity Selection EIA
Extract Bone
marrow
Core protein
Phage Display Technology
Method
Map the Epitope Recognized by scFv42C.
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.
Method
Molecular cloning
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
Method
Molecular cloning
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 Nterminal fusion of the enhanced cyan
fluorescence protein (CFP)
Method
Transfection Experiments
 CFP-C191/Core-pRc/CMV
scFv42C-YFP/scFvIR-YFP
Transfect Huh7 for
 pZeo/core and pEF/sc42
Transfect Huh7 for
colocalization assay
Western blot
 JFH1(replicon RNA)
and pEF/sc42
Transfect Huh7.5 for
Western and Northern
blot
Method
Colocalization Experiments
Immunofluorescence
Confocal Laser Scanning Microscope
Fluorescence Resonance Energy
Transfer Technique(FRET).
Method
Cell Proliferation Assay
Cell proliferation:day 1 or 2 after
transfection
Add tetrazolium to medium
Measure absorbence at 450 nm
after 1-4 hours
Method
Results
Identification of High-Affinity Single-Chain
Antibodies Against HCV Core Protein.
Results
Amino Acid Sequence
Results
Purification of scFv42C
Figure 3
Results
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
Results
Mapping of the scFv42C Binding
Motif on the Core Protein.
Phage Display
Peptide Library
scFv42C
Phage Display Technology
Results
Mapping of the scFv42C Binding
Motif on the Core Protein.
Results
Intracellular Colocalization of scFv42C
with HCV Core Protein.
 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).
Results
Localization of Core Protein and
scFv42c individually
Figure 5
Results
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.
Results
FRET
Fluorescence Resonance Energy Transfer Technique
Core protein
scFv-IR
Core protein
scFv-42C
Results
FRET Analysis
Results
scFv42C’s reduce Core protein
The scFv42C Antibody Reduced Core
Protein Level but not Viral Replication
per se in Transient Transfection.
Results
Cotransfect with core
Results
Cotransfect with HCV
Results
Transfect with HCV
Results
Effect of scFv42C on cell proliferation
Results
Discussion
Advantages of scFv over intact antibody
Smaller size
Ease of production
Improved tissue penetration
Discussion
Applications
 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
Discussion
Applications
 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
Discussion
The case of HCV
 HCV causes serious diseases:
steatosis
HCC
fibrosis
cirrhosis
 Single-chain antibodies may prove useful in the
therapy of chronic HCV infection.
Discussion
The case of HCV
 scFvs against NS3 (helicase) and NS5B (RNAdependent RNA polymerase):
Effective in inhibiting their enzymatic activities
both in vitro and in transfected cells.
 scFvs represent potential candidates for
intracellular immunization against HCV infection.
Discussion
scFv against core protein
Core protein:a major viral factor
contributing to HCV pathogenesis.
Targeting the viral core protein may
alleviate HCV-related pathological
changes.
Discussion
Verification
High affinity
scFv against
core protein
Features:recognize
motif PXPLXG
Interact with core
protein:deplete
core protein and
its pathogenesis
Interrupt viral
life cycle?
Discussion
Interrupt viral life cycle?
In this study:
scFv42C antibody did not directly affect
HCV replication in transient transfection.
Reason:
Core protein:
1.Not required for viral genome replication
2.Necessary for virus particle assembly.
Discussion
Interrupt viral life cycle?
Mechanism:
Interfere with nucleocapsid formation or RNA
encapsidation.
Unpublished data:
scFv42C antibody reduce viral particle
formation.
Discussion
scFv
HCV