Heterogeneity of hepatitis C-virus in genotype 1 patients treated with the combination therapy of pegylated interferon and ribavirin [Elektronische Ressource] / vorgelegt von Hua Cao
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Aus der Medizinische Klinik und Poliklinik Abteilung Innere Medizin II der Albert-Ludwigs-Universität Freiburg i. Br. Heterogeneity of hepatitis C-virus in genotype 1 patients treated with the combination therapy of pegylated interferon and ribavirin INAUGURAL-DISSERTATION zur Erlangung des Medizinischen Doktorgrades Der Medizinischen Fakultät Der Albert-Ludwigs-Universität Freiburg i. Br. Vorgelegt 2004 von Hua Cao geboren in Wuhan, China Dekan: Prof. Dr. med. Josef Zentner 1. Gutachter: Prof. Dr. med. Jens W. F. Rasenack 2. Gutachter: Prof. Dr. med. Frank Hufert Jahr der Promotion: 2004 To my father Daode Cao, my mother Nailing Yun, and my sister Zhen Cao I 1. Introduction 01 1.1 Epidemiology of hepatitisC01 1.2 Hepatitis C Virus 01 1.2.1 Genome structure ofHCV02 1.2.1.1 The structural proteins 02 1.2.1.2 The non-structural proteins 03 1.2.2 Heterogeneity of HCV 04 1.2.2.1 Genotypes and subtypes 04 1.2.2.2 Quasispecies 04 1.3 Diagnosis of Hepatitis C 06 1.3.1 Laboratory Diagnosis 06 1.3.2 Clinical Diagnosis 06 1.3.3 Course of the disease 06 1.4 Therapy 07 1.4.1 Treatment of hepatitis C 07 1.4.1.1 Interferon 07 1.4.1.1.1 Classification and antiviral activity 07 1.4.1.1.2 Mechanisms of action 08 1.4.1.
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| Publié par | albert-ludwigs-universitat_freiburg |
| Publié le | 01 janvier 2004 |
| Nombre de lectures | 25 |
| Langue | English |
Extrait
Aus der Medizinische Klinik und Poliklinik Abteilung Innere Medizin II der Albert-Ludwigs-Universität Freiburg i. Br.
Heterogeneity of hepatitis C-virus in genotype 1 patients
treated with the combination therapy of pegylated
interferon and ribavirin
INAUGURAL-DISSERTATION zur Erlangung des Medizinischen Doktorgrades Der Medizinischen Fakultät Der Albert-Ludwigs-Universität Freiburg i. Br.
Vorgelegt 2004 von Hua Cao geboren in Wuhan, China
Dekan:
1. Gutachter:
2. Gutachter:
Jahr der Promotion:
Prof. Dr. med. Josef Zentner
Prof. Dr. med. Jens W. F. Rasenack
Prof. Dr. med. Frank Hufert
2004
To my father Daode Cao, my mother Nailing Yun, and my sister Zhen Cao
Hepatitis C Virus
Genome structure of HCV
1.2.1
1.2.1.1
The structural proteins
1.4.1.3
1.4.1.3.1
1.4.1.2.1
1.4.1.1.2
1.4.1.2.2
1.4.1.2
Introduction Epidemiology of hepatitis C
1.2
1. 1.1
1.4.1.3.2
Aim of the study
Oligonucleotide Primers
Enzymes
2. Materials and methods 2.1 Materials
Nucleotide
1.4.1
1.4.1.1.1
1.4.1.1
10
10
09
1.7
1.6
1.4.2
1.5
09
09
13
2.1.1
2.1.3
2.1.2
01 01
01
02
02
Mechanisms of action and resistance
Chemistry and antiviral activity
Combination therapy
Therapeutic Uses
Ribavirin
Pegylated interferon
Pharmacology
Mechanisms of Escape
Pathomechanism of hepatitis C
Interferon
Classification and antiviral activity
Mechanisms of action
Therapy
Course of the disease
Treatment of hepatitis C
Laboratory Diagnosis
Diagnosis of Hepatitis C
Heterogeneity of HCV
The non-structural proteins
Quasispecies
Genotypes and subtypes
Clinical Diagnosis
1.2.1.2
07
06
06
06
06
07
07
07
13
13
13
1.4
1.3.3
1.3.2
1.3.1
04
03
04
04
1.3
1.2.2.2
1.2.2.1
1.2.2
I
13
09
08
09
11
12
11
2.1.4 2.1.5
2.1.6 2.1.7 2.1.8 2.1.9
2.1.10 2.1.11 2.2 2.2.1
2.2.1.1 2.2.1.1.1 2.2.1.1.2 2.2.1.1.3
2.2.1.4 2.2.1.2 2.2.2 2.2.2.1 2.2.2.2 2.2.2.3
2.2.3 2.2.3.1 2.2.3.2
2.2.3.2.1 2.2.3.2.2
2.2.3.3 2.2.4 2.2.4.1
2.2.4.2 2.2.5 2.2.6 2.2.7 2.2.8 2.2.9
II
RNase Inhibitor Kits Electrophoresis buffers and solutions Stop Solution of SSCP and Clone Medium and Plate Gel Chemicals Devices Methods Extraction of viral RNA from serum The QIAamp Viral RNA Mini Kits Principle Virus lysis Adsorption to the QIAamp membrane Removal of residual contaminants Elution with Buffer AVE Instruction of HCV RNA extracting Methods for cDNA synthesis Reverse transcription (RT) of HCV HVR1 Polymerase chain reaction(PCR) of HVR1 Agarose gel electrophoresis Clone The TOPO TA Cloning®Kit principle Instruction of cloning Setting up the TOPO®cloning reaction Transforming One Shot®TOP10 competent cells Analysis of positive Clones Isolation of plasmid DNA The QIAprep Miniprep Kits principle Instruction of QIAprep®Miniprep kit Digestion eluted plasmid DNA PCR of eluted plasmid DNA Elute DNA from Agrose Gel for SSCP f-SSCP Statistical analysis
14 14 14
15 15 15 16
17 17 17 17
17 17 18
18 18 19 19 20 21 22 22 22
22 23
23 23 24 24 24 25 26
26 27
Summary Zusammenfassung
Abbreviations
Curriculum Vitae
Reference
6. 7. 8. 9.
3. Results 3.1 Patients 3.2 SSCP analysis in patients treated with PEG-IFN and Ribavirin 3.2.1 HCV genomic complexity in the HVR1 and response to treatment 3.2.2 The SSCP analysis of E2-HVR1 quasispecies evolution during therapy 3.2.3 The SSCP analysis of pre-therapy and post-therapy 3.3 Comparison of SSCP and clones analysis of HVR1 3.4 Relationship of titer of baseline serum and response to therapy 4. Discussion 4.1 Relationship between HCV herogeneity and response to treatment 4.1.1 Quasispeceis and treatment response 4.1.2 Low quasispeceis and response to treatment 4.2 Change of HCV quasispecies 4.2.1 Change of HCV quasispecies 4.2.2 Effect of IFN on HCV quasispecies 4.2.3 Ribavirin and quasispecies 4.3 Influence of HCV titre on response to the therapy 4.4 Conclusion 5.
Acknowledgments
64 65
80
60 62 62
65 66
69
67 68
58 59
56
III
82
28 28 28 32 33 35 39
28
83
1. Introduction
1.1 Epidemiology of hepatitis C
1
Hepatitis C is a global health problem. The estimated mean prevalence of hepatitis
C virus infection worldwide is about 1 to 2% (Roudot-Thoraval, 1997). According to
most recent estimates, around 170-200 million individuals have chronic HCV
infection worldwide, including 400-500 thousand Germans (Alberti, 2003; Alter,
1999). The HCV prevalence shows significant geographical variations and significant
demographical variations within the same geographic region (Alberti, 2003). The
prevalence of the HCV infection increases with age (Kim, 2002).
The primary mode of HCV transmission is exposure to infected human blood via
intravenous drug use or unscreened transfusions (Lauer, 2001). Persons who received
blood transfusions or an organ transplant before 1992 and hemophiliacs who received
clotting factor concentrates produced before 1987 are also at risk for HCV (CDC
Report, 1998). The incidence of new infections with HCV is decreasing in all western
countries while it is still high in the underdeveloped world, mainly because of
consequence of the use of unscreened blood transfusions and unsafe parenteral
exposure use (Alter, 2002).
Injecting drug use accounts for 60% of all new HCV infections in the US, through
sharing of syringes directly, or possibly through sharing of drug preparation
equipments (CDC Report, 1998). Mother to child transmission of HCV is about 0.5 to
5 % and dependent on virus titre. Only in mothers co-infected with HIV the
transversal transmission rate may exceed 15 % or more (Roberts, 2002). Healthcare
workers exposed to needle sticks contaminated with HCV positive blood have a risk
of HCV infection between 0.5 and 3 % (Alter, 2002). In about 20% of cases the cause
of infection remains unknown (Roudot-Thoraval, 1997).
1.2 Hepatitis C Virus
Hepatitis C virus (HCV) was first described in 1989 by Choo (Choo, 1989). The
structure of the virus has been deduced largely from molecular analysis. It belongs to
the family of flaviviruses and has similarities to flaviviruses and pestiviruses (Zakim,
2003).
Filtration studies suggest that the virus is 30-60 nm in diameter (He, 1987).
Infectivity is abolished by chloroform, suggesting that the virus has a lipid envelope
(Bradley, 1985).
1.2.1 Genome structure of HCV
2
The genome is a single-stranded RNA of positive-sense with about 9.6 kilo bases
(kB) in length. It contains a single, large open reading frame (ORF) that encodes a
single, large polyprotein of approximately 3000 amino acids which is cleaved post-
translationally into multiple structural and non-structural (NS) peptides (Major, 1997).
The structural proteins are encoded at the 5’ end and the non-structural proteins at the
3’ end of the ORF. The HCV genome also has important and highly conserved 5’ and
3’ untranslated regions (Major, 1997; Honda 1999; Kolykhalov, 2000). The 5’
untranslated region has an internal ribosomal entry site essential for initiation of viral
protein translation, and the 3’ untranslated region has structured RNA elements
essential for both viral replication and translation (Hoofnagle, 2002) (Figure 1)
Figure 1 Genome structure of HCV
(wwwna.et.luu/edms~ddeanWWr/
1.2.1.1 The structural proteins
W/335/HCV.html).
The structural proteins consist of a single nucleocapsid core protein (C) and two
envelope glycoproteins (E1 and E2). The core protein has a molecular weight of 21
kD and is the putative nucleocapsid of the virus. The nucleotide sequence of the core
region is highly conserved (Hijikata, 1991; Bukh, 1994). The core protein localizes to
the cytoplasm and its N terminal portion binds non-specifically to RNA (Santolini,
1994; Shimoike, 1999). The core protein provides the protective nucleocapsid
structure for HCV.
The putative viral envelope glycoprotein, E1 (31kb) and E2 (70kb), provide the
coat proteins of the virus. They are separated co-translationally and are targeted to the
endoplasmic reticulum where they undergo N-linked glycosylation (Matsuura, 1992;
Grakoui, 1993; Ralston, 1993; Cocquerel, 2000).
The E2 protein has two highly variable regions (HVR1 and HVR2), which are
3
candidates for the major neutralizing epitopes of the virus (Weiner, 1991; Kato, 1992).
HVR1 (nt 1150-1230) consists of the first 27 amino acids of E2 at the N terminus.
Antibodies specific to HVR1 have been reported to protect against reinfection
(Habersetzer, 1998; Farci, 1996). Spontaneous mutations in this region are believed to
play a role in immune escape (Kato, 1993; Manzin, 1998; McAllister, 1998; Farci,
2000). E2 also contains a conserved region known as the PePHD (PKR (IFN-induced
RNA-dependent protein kinase)-eIF2alpha phosphorylation homology domain),
which may interact with intracellular protein kinase R, an interferon-induced enzyme.
These findings suggest that the PePHD region may confer interferon resistance
(Taylor, 1999).
1.2.1.2 The non-structural proteins
The various non-structural proteins of HCV mediate the enzymatic activity
necessary for viral replication, including a viral protease and RNA polymerase
(Figure 1). The NS proteins consist of NS2, NS3, NS4A, NS4B, NS5A, and NS5B.
HCV does not have a NS1 region, a domain in the flaviviruses on which the
nomenclature is based. The specific functions and structures of most, but not all, of
the individual HCV non-structural proteins have been defined (Zakim, 2003).
The NS5 region contains the RNA-dependent RNA polymerase activity essential
for RNA viral replication. These enzymatic activities are potential targets for antiviral
compounds (Major, 1997; Honda, 1999; Kolykhalov, 2000). The NS5 protein is
composed of two products known as NS5A and NS5B. The function of NS5A is still
unclear, although mutations in this region appear to enhance replication (Blight, 2000;
Krieger, 2001). NS5A exists in phosphorylated and hyperphosphorylated forms, the
degree of which varies among genotypes (Koch, 1999; Hirota, 1999). NS5A contains
a putative interferon sensitivity-determining region (ISDR), a small sequence in the
carboxyl-terminal region of NS5A gene (Enomoto, 1995), that interactsin vitrowith
the interferon-induced protein kinase R, a cellular protein induced by IFN (Gale,
1999). Patients carrying HCV 1b with wild-type ISDR or with 1-3 amino acid
substitutions (intermediate type) usually failed to develop a sustained response to
therapy, whereas multiple substitutions in this amino acid sequence were associated
with sustained clearance of the virus (Enomoto, 1996). Thus NS5A is probably a
requisite part of the replication complex of HCV.
4
1.2.2 Heterogeneity of HCV HCV can be classified into genotypes, subtypes, and isolates based on sequence
diversity of the genome, so called quasispecies. Six major genotypes have been
identified. Greater than 30 percent sequence divergence indicates different genotypes,
designated 1 to 6 (Bukh, 1995; Simmonds, 1995). Variation between 10 percent and
30 percent is characteristic for different subtypes of HCV, which are designated by
lower case letters after the genotype, such as 1a or 3b. More than 70 subtypes of HCV
have been described (Simmonds, 1994). Variability of 1 percent to 5 percent is typical
of the quasispecies diversity found in a single infected patient (Zakim, 2003).
1.2.2.1 Genotypes and subtypes
The classification system of HCV into genotypes and subtypes is based on
phylogenetic analysis (Bukh, 1995). Genotypes are stable in a particular patient,
although infection with multiple genotypes or subtypes can occur. There are distinct
geographic variations in frequencies of different genotypes. Genotypes 1 and 2 are
found worldwide; genotype 3 is common in the Indian subcontinent and Southeast
Asia; genotype 4 is the major genotype in Africa and the Middle East; genotype 5 has
been found largely in South Africa; and 6 in Hong Kong and Viet Nam. As population
shifts occur and modes of transmission change, changes in HCV genotype
distributions occur. Genotypes 1a and 3a were probably rather recently introduced in
the United States and European populations, perhaps as a result of the spread of
injection drug use in the 1960s and 1970s (Zakim, 2003).
1.2.2.2 Quasispecies
As a typical RNA virus, the HCV genome has a high mutation rate, which is reported to be 1.44 X 10-3 perper site per year (Okamoto, 1992). This is genome
partially explained by the high rate of synthesis of virions in chronic hepatitis C, ranging from 1010to 1012virions per day. In addition, there is also rapid turnover of
virus, at least in the serum. The half-life of HCV is short, averaging only 2 to 3 hours
(Neumann, 1998).
The term “quasispecies refers to the genetic diversity of a virus population with
individual viral genomes differing by 1%-5% in nucleotide sequence that can be
observed in a single infected individual due to changes taking place during the course
of the infection (Martell, 1992; Bukh, 1995). Within an individual patient, such
5
variation can be characterized in terms of diversity or complexity. Diversity is the mean genetic distance calculated for all pairs of sequences, where genetic distance is directly proportional to the number of nucleotide differences between two avirna. ts Complexity refers to the population distribution of variants and is calculated from
sequence data (Navas, 1998). The sequence variability can occur in any region of the
viral genome, although some regions are more highly conserved (the 5’ and 3’ UTRs
and core regions) and some more variable (particularly the HVR1 and HVR2 regions
of E2) than others (Kanazawa, 1994; Farci, 1997).
Replicative fitness and immune pressure probably dictate which variations in
sequence are more likely to persist (Kanazawa, 1994; Farci, 1997). Selection of HCV
quasispecies is related to many factors, such as viral replication efficacy,
immunomodulation of the host, therapeutic interventions and co-infection with others
viruses (Domingo, 1985). The high rate of viral replication, the lack of proofreading
of the viral polymerase and the immune pressure of the host probably account for the
fact that the HCV RNA genome mutates frequently. It may also account for other
biological behaviours of RNA viruses, such as persistent infection, escape mutants,
defective interfering particles, cell tropism, and drug resistance (Holland, 1992;
Steinhauer, 1987). The quasispecies diversity of HCV may contribute to the
development of chronicity and immune escape during infection because the envelope
protein changes rapidly in response to immune pressure (Hoofnagle, 2002).
Hot-spots for mutation are described within the genome encoding two portions of
E2 termed the hypervariable regions (HVRI and II), which encode neutralizing
epitopes (Weiner, 1991; Weiner, 1992). Elevated tolerance for amino acid substitution
and sensitivity to the selective pressure exerted by the host’s immune system makes
HVR1 particularly suitable for quasispecies heterogeneity studies (Weiner, 1992;
Farci, 1994; Pawlotsky, 1999).
Most mutant viral particles cannot replicate, but the remainder can transmit new
genetic information to their progeny. The fittest infectious particles are selected on the
basis of their replication capacities and especially by the selective pressure exerted by
their interactions with host cells proteins and the immune response (the so-called
“genetic bottleneck), which targets regions encoding both cytotoxic and neutralizing
epitopes (Duarte, 1994). Immune selective pressures act on regions encoding
cytotoxic and neutralizing epitopes. For example, The HVR1 is a target for the anti-
HCV neutralizing response (Weiner, 1992; Farci, 1994). It is commonly used to
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