Parvo-Virus B19 infection in hepatitis C

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Medizinische Hochschule Hannover Zentrum Innere Medizin

Klinik für Gastroenterologie, Hepatologie und Endokrinologie

(Direktor: Prof. Dr. med. Michael P. Manns)

Role of Parvovirus B19 Infection in hepatitis C

Dissertation

zur Erlangung des Doktorgrades der Medizin an der Medizinischen Hochschule Hannover

vorgelegt von Chun Wang aus Shanghai China

Hannover 2008

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Role of Parvovirus B19 infection in hepatitis C

Angenommen vom Senat der Medizinschen Hochschule Hannover am ：

Gedruckt mit Genehmigung der Medizinschen Hochschule Hannover Präsident: Prof. Dr. Dieter Bitter-Suermann

Betreuer: Priv. Doz. Dr. med. Heiner Wedemeyer Referent: Prof. Dr. Thomas F. Schulz

Korreferent: Prof. Dr. Stefan Poehlmann

Tag der mündlichen Prüfung:

20. Mai 2008

Promotionsausschussmitglieder:

Prof. Dr. med. Alexander Kapp

Prof. Dr. med. Burkhard Wippermann

Prof. Dr. med. Stefan Kubicka

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Role of Parvovirus B19 infection in hepatitis C

Hepatitis C virus is a major causative agent of end-stage liver damage (cirrhosis) and hepatocellular carcinoma. Alcohol consumption, virus co-infection (e.g. HBV, HIV), genetics, and other liver disease such as NASH, are the major factors, which can influence the natural history of HCV and will contribute to high risk of developing liver cirrhosis in the range from 1-40% after twenty or thirty years [3].

The detailed mechanisms how these factors contribute to progression of chronic liver disease are largely undefined. In particular, the role of coinfections with other pathogens than HIV and HBV has rarely been studied.

1.1.2 Virology

The hepatitis C virus is an enveloped noncytopathic single-stranded RNA of ~10, 000 nucleotides, 30-60nm diameter, belonging to the Flaviviridae family, which acts as an mRNA in the cytoplasm to translate a polyprotein. Cellular and viral proteases are involved in the generation of structural proteins: core, envelope protein 1 (E1) and 2 (E2), p7 and nonstructural proteins (NS2, -3, -4A, -4B, -5A and -5B) [4]. Binding of the HCV E2 protein to the second extracellular loop of CD81 and endocytosis of HCV via the LDL (low density lipoprotein) receptor may play an important role in proceeding infection. Recently, Claudin-1 has been reported as a co-receptor in the late period of viral entry [5]. However, the detailed mechanism how HCV is infecting the hepatocyte is still unknown.

Genetic heterogeneity is the most notable feature of HCV. Six major genotypes and over 100 subtypes of HCV have been described. HCV genotypes show some different patterns in natural history. While genotype 3 infection is associated with steatosis [6, 7], more frequent flares of hepatitis were observed in genotype 2 infected persons [8].

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Recently it was suggested that the risk for the development of hepatocellular carcinoma might be higher in genotype 1b infection [9]. Moreover, an association of HCV genotypes with responses to antiviral therapy is well established as individuals with HCV genotype 2 infection show the highest response rates followed by genotype 3 infected individuals. The lowest response rate are observed in genotype 1 infection with no significant differences between genotype 1b and genotype 1a [3].

1.1.3 Immunology of HCV infection

The immune response against HCV is a crucial factor for viral replication, elimination, as well as the outcome of virus infection. The immune response can target all HCV structural and non-structural protein. However there is no specific antibody pattern associated with recovery or replication.

It has been frequently demonstrated that predominant multi-epitope specific CD4⁺ and CD8⁺ T-cell responses are correlated with spontaneous recovery from HCV infection. By contrast, delayed, transient or narrowly focused T-cell responses lead to chronic hepatitis C [10-13] . Simultaneously, an early IFN-gamma response by CD8⁺ T cells induces resolution of HCV, whereas persistence of function impaired CD8⁺ T cells leads to chronic infection. HCV-specific T cell responses have rarely been studied in the context of viral co-infections. In individuals infected with HCV and HIV, HCV-specific T cell responses are impaired along with the depletion of total CD4⁺ T cells following the progression of HIV infection. Moreover, HIV infected patients with spontaneous control of HCV show a high risk for HCV viremia re- occurrence due to CD4⁺ T cell depletion [14] . HCV and Schistosomiasis coinfections were studied on Egyptian individuals. This coinfection leads to high HCV viral load, more frequently chronic HCV infection and more severe liver disease due to impaired HCV-specific T cell responses cause by Schistosomiasis [15]. No study yet has investigated HCV specific immunity in the context of B19-infections.

However, this may have implications as heterologous immunity has been shown to significantly influence the outcome of various viral infections in rodents and humans [16, 17].

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1.1.4 Therapy

Even though the acute infection phase is usually asymptomatic, chronic HCV could be prevented by early treatment of acute HCV infection with interferon alpha monotherapy for just 6 months [18, 19]. Approximately 50% of genotype 1 patients and 80-90% of genotype 2 and 3 infected patients can reach sustained virologic response with the combination therapy of pegylated interferons and ribavirin. Patients with high risk factor for progressive liver disease (e.g. ISHAK fibrosis score >= 2) should be treated early [3]. Other factors being associated with a better treatment response are younger age, female sex, low viral load before therapy, a rapid response with HCV-RNA negativity until week 4 of therapy, a low baseline gamma GT level, absence of hepatic steatosis and high adherence to therapy [3].

1.2 Parvovirus B19

1.2.1 Epidemiology and natural history

Parvovirus B19 (B19) was occasionally discovered by Cossart et al. during testing for hepatitis B virus surface antigen in 1974 [20]. The infection with this virus is worldwide especially in childhood. The prevalence of anti-B19 IgG antibody is 2- 15% in young children (1-5 years), 15-60% in school age children (6-19 years), 30- 60% in adults, and more than 85% in the senior population [21-24]. Pathways of transmission are respiratory route, blood-derived products and vertically from mother to fetus. B19 is a pathogen with a variety of clinical manifestations, ranging from asymptomatic courses in immunocompetent individuals to lethal cytopenias in immunocompromised patients [25]. The most common disease associated with B19 infection is erythema infectiosum, also termed fifth disease or “slapped-cheek” disease.

The duration of viremia has been reported from weeks to months in immunocompetent hosts following the development of virus specific antibodies.

However, Lindblom etal [26] reported that viremia was detectable during the whole follow-up period (128 weeks) in 4 of 5 immunocompetent individuals after acute B19 infection in spite of the resolution of clinical symptoms. Thus, the clearance of B19 viremia may be slower than previously thought. Bone marrow is the primary site of replication [25].

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1.2.2 Virology

B19 is a single-stranded nonenveloped DNA virus, 22-24 nm diameters, containing 5,596 nucleotides (nt), and is member of the family Parvoviridae known to be pathogenic in humans.

The B19 genome has two large open reading frames, encoding for the nonstructural protein (NS1) and for structural capsid proteins (VP1 and VP2). NS1 manifests site-specific DNA-binding, subserves multiple replicative functions and is cytotoxic to host cells [27, 28]. VP2 is the major structural protein to make up 96% of the total capsid protein [29]. VP1 accounts for the remaining 4% and is identical to VP2 with the addition of 227 amino acids (termed the VP1 unique region, VP1u) at the amino terminus [29, 30]. Importantly, the VP1 protein comprises a viral phospholipase A2 activity [31] which was suggested to be critical for efficient transfer of the viral genome to initiate replication. The Erythrocyte P antigen has been identified as the B19 receptor for entering into cell to initiate infection [32]. In addition, a5b1-integrin and the Ku80 autoantigen have been described as co-receptors [33-35].

Unlike HCV, the nucleotide sequence of B19 is rather conserved, containing only 6% divergence. So far three genotypes: B19-, LaLi-, and V9-related viruses have been identified [36]. However, the variation of B19 sequence has no correlation with specific disease symptoms and persistent infection [25, 37, 38].

1.2.3 Immunology of B19 infection

Humoral immune response against B19 is correlated with the eradication of the virus and has a long lasting protection against re-infection [39]. B19-specific cellular immune responses have been studied only in the recent decade. NS1, VP1 and VP2 can be targets of the host’s cellular immune responses. B19 specific CD8⁺ and CD4⁺ T cell responses can be found in acute infection, persistent infection and remote infection [40-47]. The maintenance of B19-specific T cell responses may indicate persistence of low loads of residual virus as it has been shown in the case of recovery after HBV infection [48]. Importantly, specific T cell epitopes have been characterized in recent years. One group [47] has described two particularly frequently detected epitopes in healthy B19 IgG-positive but IgM/DNA-negative individuals. Although one epitope (LASEESAFYVLEHSSFQLLG) was DRB1*1501 restricted, positive T cell responses were detectable in DRB1*1501 negative

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individuals too. In 6 serologically recovered B19 infected individuals with positive responses against this epitope only 2 were DRB1*15 positive. The lack of an efficient T cell response against B19 may lead to persistent infection [49].

B19-specific T cell responses have not been studied yet in the context of hepatitis virus infections. There is no information on frequency, specificity and strength of B19 specific T cell immunity in hepatitis C versus responses in healthy controls. Thus, one aim of this thesis project was to address this question accordingly.

1.2.4 Therapy

Normally, B19 infection requires no specific antiviral treatment. In immunocompetent individuals, some patients may need symptomatic treatment (NSAIDs)because of B19-induced arthralgia. Erythrocyte transfusion is required in cases of B19-induced transient aplastic crisis [50]. In immunocompromised patients, infusion of immunoglobulin is an effective therapy to against chronic anemia or cytopenia due to persistent B19 infection. Fetal blood transfusions is a curative treatment in severe B19-related hydrops fetalis [25, 51, 52]. In patients with B19- related myocarditis, interferon beta is a kind of successful treatment contributes to virus clearance and protects left ventricular function [53]. Currently, larger trials are exploring the efficacy of interferon beta treatment of B19-related mycoarditits.

1.2.5 B19 infection and liver disease

In the past decade, a growing number of studies have been published to reveal an association between B19 and liver disease especially in the etiology of fulminant liver failure and the persistence of B19 in liver [54-63]. The summary of these studies is shown in Table 1. Recently, an investigation aimed to evaluate the effect of B19- infection on the course of HBV-associated liver disease by Toan and colleague demonstrated that B19-infection was not only frequent in HBV-infected Vietnamese patients but also obviously associated with severe hepatitis B-associated liver disease.

In this study, total of 463 Vietnamese individuals was recruited. Of these, 399 were HBV-infected patients (311 symptomatic HBV-infected patients with well- characterized clinical profiles, 88 asymptomatic chronic HBV carriers with no liver disease) and 64 were healthy individuals. Sera of these individuals were tested for the presence of B19-DNA by nPCR and quantity real-time PCR and DNA-sequencing.

Paralleled liver biochemical and serological testing were also assayed. The prevalence

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of B19 DNA was significantly higher in HBV-infected patients compared to the healthy control group (24.1% vs 4.7%, p<0.001). Moreover, it showed a significantly higher prevalence of B19 DNA in the HCC subgroup compared with no-HCC subgroup (38.6% vs 18.5%, p<0.001). The prevalence of B19 DNA in “severe (LC and HCC)” patients group and “mild (AHB, CHB, and ASYM)” patients group were 29.9% and 18.5%, respectively (p=0.008). By using multivariate analysis they also demonstrated the serum B19 viral load is correlated with the HBV viral load and serum ALT levels. Based on these findings the authors concluded that B19 may be persistent in HBV patients and B19 may play an important role in the pathogenesis of HBV in Vietnamese.

1.3 Aims

Based on the recent findings of B19 being possibly involved in disease progression in Asian hepatitis B patients, we here aimed to investigate whether B19 infection may be a co-factor for disease progression in European patients with chronic hepatitis C. Therefore, the following questions were addressed:

(i) What is the prevalence of B19 infection in German patients with chronic HCV and HBV infection versus healthy individuals?

(ii) If B19 can be detected in hepatitis C patients – how does antiviral therapy with interferon alpha and ribavirin influence B19 viremia?

(iii) Does B19 persist in blood and/or liver of immunocompetent hosts after apparent recovery from B19 infection?

(iv) Is any virological or serological marker of B19 infection associated with the outcome of chronic hepatitis C infection?

(v) What is frequency and strength of B19-specific T cell responses in serologically recovered healthy controls vs. patients with chronic hepatitis virus infection?

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Table 1: Summary of studies on B19 and fulminant hepatitis or non-A-E hepatitis

Study group Subjects Conclusion

Eis-Huebinger (Germany)[54]

Explanted liver tissue, liver and bone marrow from autopsied adults and serum samples

B19 DNA was present frequently in livers of adults with severe liver damage

indicating the persistence of B19 in the liver

Abe (Japan) [55] Explanted liver tissue and serum samples

B19 may act as an causative agent of fulminant hepatitis

He (China) [56] Serum samples B19 is not associated with non-A-E hepatitis. The prevalence of B19 infection in patients with non-A-E hepatitis is similar to that in patients with A-E hepatitis. B19 and HBV coinfection does not lead to more severe liver damage Wong

(USA)[57]

Explanted liver tissue

The prevalence of B19 DNA in livers from patients with fulminant hepatitis (FH) or hepatitis-associated aplastic anemia (HAA) is similar to livers from patients with HBV or HCV infection. Thus, B19 is not

associated with FH or HAA.

Karetnyi (USA)[60]

Explanted liver tissues

B19 may play a role in liver damage in patients with non-A-E fulminant liver failure.

Sokal (Belgium) [61]

Serum samples The evidence of acute B19 infection in children younger than 5 years with FH of unknown etiology showed an eusemia Yoto (Japan)[62] Serum samples B19 may be the causative agent of acute

hepatitis So. K (Australia)

[63]

Drago (Italy) [58]

Hillingso (Denmark)[59]

Case reports Acute B19 infection leaded to abnormal liver parameters. B19 may be a causative agent of fulminant liver failure or

fulminant hepatitis.

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Role of Parvovirus B19 infection in hepatitis C -- Subjects and Methods

2. Subjects and methods 2.1 Subjects

2.1.1 Hepatitis C patients. Stored serum samples were studied from 75 patients with chronic hepatitis C infection who have been treated with interferon alpha and ribavirin combination therapy. All sera were taken before treatment was started. All patients were treated in the outpatient clinic of the Department of Gastroenterology, Hepatology and Endocrinology of Medical School Hannover, Germany. Another 16 sera and paired peripheral blood mononuclear cells (PBMCs) were collected from patients with chronic hepatitis C infection without antiviral therapy for evaluating cellular immune responses. These 91 patients had a mean age of 46 years ranging from 19-73 years (male: 49; female: 42). Characteristics are summarized in Table 4.

2.1.2 Hepatitis B patients. Serum samples were taken from randomly selected 50 patients with chronic hepatitis B infection (mean age: 47 years, range: 15-72, male 33;

female: 17). The presence of hepatitis B surface antigen (HBsAg) for more than 6 months was required for the diagnosis of chronic HBV infection.

2.1.3 Liver tissue samples

Explanted liver tissues were obtained at the time of liver transplantation from 50 patients with end-stage liver damage undergoing orthotopic liver transplantation for various reasons from 1993 to 2000 at Medical School Hanover (mean age: 47 years, range: 8-69). Of these, 19 had HCV related end-stage liver disease, while 31 had non- HCV liver cirrhosis (e.g. primary biliary cirrhosis, primary sclerosing cholangitis, Morbus Wilson and Polycystic liver disease).

Routine liver biopsy samples were obtained from 32 patients (mean age: 46 years, range: 33-75). Of these, 13 had chronic hepatitis C. 19 biopsies were taken from patients with no evidence of HCV infection.

All of these samples were stored at -20°C until used.

All patients were recruited at Medical School Hannover, Germany.

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2.1.4 Healthy individuals

Sera and paired PBMCs were collected from 30 laboratory and clinic healthy volunteers (mean age: 37.5 years, range: 25-65, male: 16, female: 14). Among them, 19 were B19-IgG antibody positive while 11 were negative.

2.2 Methods

2.2.1 Serological tests.

Anti-B19 IgM and anti-B19 IgG antibodies were tested using the Parvovirus B19 IgM/IgG Enzyme Immunoassay (Biotrin, Ireland) according to the manufacture’s instruction. Anti-HCV antibodies were tested using ARCHITECT Anti-HCV assay (Abbott, USA).

2.2.2 Extraction of nucleic acids from liver tissue

DNAs were extracted from 20-30mg frozen liver tissue using the QIAamp DNA mini kit (Qiagen, Germany) according to the manufacture’s instruction.

Contamination was excluded by re-extraction of these same samples in another laboratory by a different person.

DNAs from liver biopsy samples were extracted by a person who had never contacted with B19 virus in another laboratory.

2.2.3 Polymerase chain reaction (PCR).

Nested PCR (nPCR) and quantitative real time PCR (qPCR) were performed to detect B19 DNA. Primers specific for the VP1/VP2 structural sequence were employed in nPCR as described elsewhere [64]. Briefly, the primer pairs for the first round of PCR were as follows: sense 5’-AGCATGTGGAGTGAGGGGGC-3’ and anti-sense 5’-AAAGCATCAGGAGCTATACTTCC-3’. The primers for second round were sense 5’-GCTAACTCTGTAACTTGTAC-3’ and anti-sense 5’- AAATATCTCCATG GGGTTGAG (NCBI GenBank accession No. U38509). The whole reaction consisted of two 35-cycle programs (1 cycle= 94°C for 30 seconds, 50°C for 30 seconds and 72°C for 45 seconds)

Quantitative real-time PCR (qPCR) was performed using the Parvo B19 artus LC-PCR kit (Qiagen, Germany) according to the manufacture’s instruction. A qPCR of genomic C-reactive protein (CRP) DNA was performed with same samples in

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order to determine the amount of human CRP DNA representing the actual amount of amplifiable cellular DNA in each sample [65, 66]. B19 copy numbers per cell were calculated from the amplification of B19 divided by the amount of human CRP DNA.

Samples were defined as a “serologically recovered” cohort by the presence of anti-B19 IgG with the absence of IgM and DNA.

2.2.4 Isolation of PBMCs and carboxy fluorescein succinimidyl ester (CFSE)- based T cell proliferation assay.

PBMCs were separated from heparinized blood samples by gradient centrifugation on Ficoll-Paque and stored in liquid nitrogen until used.

CFSE-based T cell proliferation assay was performed as described previously [11]. Briefly, frozen PBMCs were thawed and suspended at 10⁷/ml in PBS plus 0.2%

BSA and incubated at 37°C for 7 min with 2.5ųM CFSE (Sigma, USA). An equal volume of FCS was added thereafter and cells were incubated on ice for 5 min to stop reaction, followed by 3 times washing. Then, labelled cells were resuspended in medium (RPMI 1640 supplemented with 10% inactive AB serum and penicillin), plated at 0.3*10⁶ cells per 200ul per well in round-bottom 96-well microtiter plates and cultured with DMSO alone (background), synthetic peptides (VP1/2 7.2 and VP1/2 4.7 [47], ProImmune, UK, Table 2) at a final concentration of 1ųg/ml and 5ųg/ml, tetanus toxoid (TT, 3ųg/ml) and PHA (6ųg/ml) as positive controls at 37°C with 5% CO2 for 7 days. Each condition was duplicated.

PBMCs from patients with chronic HCV infection were stimulated also with recombinant genotype 1a-derived HCV core, NS3 and NS4 protein (Microgen, Germany) at a final concentration of 1ųg/ml.

Flow cytometric analysis: at the time of harvest, CFSE-labelled PBMCs were washed in PBS containing 2% FCS and 0.05% sodium azide, and stained with the following antibodies: anti-human CD4-APC, CD3-PE (Becton Dickson, Germany) at 4°C. Flow cytometric data (100,000 nongated events) were acquired on a BD FACSCalibur 4-color flow cytometer using BD Cellquest software (both from BD Biosciences). For analysis, BD FlowJo 6.1.1 (Treestar, Ashland, OR, USA) was used to gate on CD4⁺CD3⁺ T cell populations. The number of cells that had proliferated was determined by gating on the lineage-positive CFSE^low and CFSE^high subset. The background of CD4⁺ T cells proliferative frequency (%) was calculated as the number of CFSE^low CD4⁺ T cells /(numbers of CFSE^low CD4⁺ T cells + numbers of CFSE^high

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CD4⁺ T cells) *100 in the absence of antigen. The PF was calculated by subtracting the mean background proliferation from the proliferating fraction in response to specific antigen. The SI was calculated by dividing the antigen-induced PF by the background PF. Both a PF of 1.0% or more and an SI of 2.0 or more are considered as a positive response, as previously defined [67, 68] .

Table 2:Synthetic peptide specification

B19 region CD4+ T-cell-restricted peptide sequence peptide no

VP1/2 FLIPYDPEHHYKVFSPAASS 4.7

VP1/2 LASEESAFYVLEHSSFQLLG 7.2

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Role of Parvovirus B19 infection in hepatitis C -- Results

3. Results

3.1 Prevalence of B19 serologically recovered infection and the presence of B19 DNA in serum samples from patients with chronic hepatitis C and B infection.

Anti-B19 IgG antibodies were found in 67/91 (74%) of the chronic HCV infected patients and in 19/30 (63%) healthy individuals, only one HCV sample tested positive for B19 IgM antibody. The percentage of patients with serological evidence for a previous B19 infection was similar in these two groups (Fig. 1).

Figure 1

Figure 1, Prevalence of anti-B19 IgG antibody positive in European individuals Legend: Anti-B19 IgG antibodies were tested in 30 healthy individuals and 91 chronic HCV infected patients. The frequency of positive samples is similar in these two groups.

63

74

37

26

0 10 20 30 40 50 60 70 80

he al thy con trol (n=30) chroni c HC V pati e nts (n =91)

frequency(%) ^IgG_pos

IgG neg

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In contrast to previous studies on Asian HCV infected patients [69, 70], all but one HCV serum were negative for B19 DNA by qPCR. This unique B19 DNA positive serum sample was also positive for both anti-B19 IgM and IgG. Consecutive serum samples of the same patient were collected for up to 48 months. The B19 viremia lasted at least one year even under combination antiviral therapy of daily injections with recombinant interferon alpha-2b plus ribavirin and was not associated with ALT level or HCV viremia (Fig. 2). However, the patient became B19-negative during a second course of pegylated interferon alpha 2b plus ribavirin therapy.

Importantly, B19 DNA was also undetectable in all 50 sera collected from HBsAg positive patients (Table 3). All sera were collected in Germany. Those data are in contrast to recent data from Vietnamese patients [69]. Thus, B19 viremia is an extremely rare finding in German patients with viral hepatitis B and C.

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Figure 2

Figure 2: Fluctuation of ALT, HCV-RNA and B19 viremia in the single HCV- infected patient with B19 viremia.

The patient was followed for 48 months and treated twice with interferon alpha and ribavirin. B19 viremia lasted at least one year even during the first combination therapy.

The first combination therapy (from follow-up month 0 to 5) consisted of interferon alpha-2b plus ribavirin for 22 weeks. The dose of interferon alpha-2b was 10 MU per day in the first two weeks followed by 3 MU per day for 8 weeks followed by 3 MU every 2^nd day. The second combination therapy (from follow-up month 13 to 19) consisted of PEG-interferon alpha-2b (100 µg qw) plus ribavirin for 20 weeks.

Table 3: Frequency of B19 viremia in patients with chronic hepatitis C or B infection

Number of patients B19 viremia

chronic hepatitis C 91 1

chronic hepatitis B 50 0

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

0 1 2 3 4 5 6 13 15 16 17 18 19 20 27 48

follow-up month

ALT x ULN

0.1 1 10 100 1000 10000

HCV RNA (log IE/ml)

HCV RNA 1^sttherapy 2^ndtherapy

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

0 1 2 3 4 5 6 13 15 16 17 18 19 20 27 48

0.1 1 10 100 1000 10000

ALT

ALT HCV RNA

pos pos pos pos neg neg neg

1^sttherapy 2^ndtherapy B19

DNA

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

0 1 2 3 4 5 6 13 15 16 17 18 19 20 27 48

follow-up month

ALT x ULN

0.1 1 10 100 1000 10000

HCV RNA (log IE/ml)

HCV RNA 1^sttherapy 2^ndtherapy

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

0 1 2 3 4 5 6 13 15 16 17 18 19 20 27 48

0.1 1 10 100 1000 10000

ALT ALT ALT

ALT HCV RNA

pos pos pos pos neg neg neg

1^sttherapy 2^ndtherapy B19

DNA

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3.2 Retrospective analysis of Clinical characteristics in relation to B19 serology Clinical characteristics, histological staging and grading of hepatitis C patients are shown in Table 4. These 91 chronic HCV infected patients were divided into two groups based on the serological testing for B19. In addition, the histological staging and inflammation score of these two groups are shown in Figure 3. Importantly, there was no significant difference in any of the clinic investigated parameters. Also histological staging did not differ between the two groups. Thus, past B19 infection had no apparent effect on the course of HCV infection in this cross-sectional study.

Table 4: Characteristics of HCV-infected patients with or without anti-B19 IgG antibodies

anti-B19 IgG pos (n=67)

anti-B19 IgG neg (n=24)

p value Age(years, mean±SD,

range:19-73)

48±10 44±8.7 0.26

Male: 38 Male: 12

Gender

Female: 29 Female: 12

0.5 F score(mean±SD,

range:0-6)

2.6±2.1 2.2±1.5 0.34

Percentage of live cirrhosis^a(%)

23 8

Activity score(mean±SD, range:0-18)

5.2±2.0 5.2±2.2 1

ALT ULN(times) 3.1±2.3 3.2±2.6 0.91

AST ULN(times) 2.4±1.8 2.3±1.5 0.71

gGT ULN(times) 2.1±2.2 2.2±2.8 0.85

Total bilirubin(umol/l) 12±7.9 13±5.6 0.74

Albumin(g/l) 44±3.4 45±3.1 0.2

Prothrombin time (Quick value %)

98±23 96±8.6 0.43

Platelets(Tsd/ul) 187±56 187±93 0.97

Presence of B19 viremia 1 0 0.55

Presence of anti-B19 IgM 1 0 0.55

Legend: a, F score >=5. There were no significant differences of clinic characteristics in these two groups.

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Figure 3: Histological grading and staging chronic hepatitis C patients with and without anti-B19 IgG antibodies.

Histological staging was available for 85/91 chronic HCV infected patients.

Inflammation scores was available for 82/91 patients. There were no significant differences between the two groups.

0 1 2 3 4 5 6

Anti-B19 IgG pos Anti-B19 IgG neg

F score (ISHAK)

0 1 2 3 4 5 6

F score (ISHAK)

0 2 4 6 8 10 12 14 16 18

Inflammation score

0 2 4 6 8 10 12 14 16 18

Inflammation score

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3.3 Presence of B19 DNA in explanted livers and routine liver biopsy samples The presence of B19 DNA in liver by qPCR is shown in Figure 4. Surprisingly, B19 DNA was amplified from more than half of the liver tissues studied and more frequently detectable in explanted end-stage liver tissues (74%) than in biopsy samples (44%)(p<0.05). This held also true for non-HCV subgroups (non-HCV explanted liver vs. non-HCV liver biopsy samples, p<0.05). Importantly, there was no difference in frequency of B19-DNA detection between HCV-positive and HCV- negative samples for both explanted livers and routine liver biopsy samples.

In biopsy samples, the presence of B19 DNA was similar in severe liver disease (F score >=5) and moderate liver disease (F score <5). Moreover, in the same group, there was no difference of grading (inflammatory activity) in B19 DNA positive samples versus negative samples.

There was no significant difference of virus copy number per cell both between explanted liver and routine biopsy samples and HCV-positive compared with HCV- negative liver samples (Fig. 5). The level of viral load was relatively low.

In the same samples, none was B19 DNA positive by nPCR.

The result of qPCR was reliable as similar results were obtained using re- extracted DNA from the same samples in different lab by a different person (Part of data shown in Table 5). Values depicted are the average of two independent qPCR tests.

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Figure 4

Figure 4: Presence of B19 DNA in end-stage liver disease (explanted livers) and routine liver biopsy samples.

B19 DNA was investigated in 50 explanted livers and 32 routine liver biopsy samples by qPCR. B19 DNA was amplified more frequently in explanted end-stage liver tissues (74%) than in biopsy samples (44%)(p<0.05).

74

44

68

46

77

42

59 64

0 10 20 30 40 50 60 70 80 90

explanted livers(n=50) routine liver biopsy samples(n=32) explanted livers- HCV subgroup(n=19) biopsy samples- HCV subgroup(n=13) explanted livers- non-HCV subgroup(n=31) biopsy samples- non-HCV subgroup(n=19) HCV subgroup (n=32) non-HCV subgroup(n=50)

Presence of B19 DNA

% ^p<0.05 ^p<0.05

74

44

68

46

77

42

59 64

0 10 20 30 40 50 60 70 80 90

explanted livers(n=50) routine liver biopsy samples(n=32) explanted livers- HCV subgroup(n=19) biopsy samples- HCV subgroup(n=13) explanted livers- non-HCV subgroup(n=31) biopsy samples- non-HCV subgroup(n=19) HCV subgroup (n=32) non-HCV subgroup(n=50)

Presence of B19 DNA

% ^p<0.05 ^p<0.05

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Figure 5

Figure 5: B19 DNA copy numbers per cell in routine liver biopsy samples and end-stage liver disease (explanted livers)

B19 DNA copy numbers/cell of 14 B19 positive routine liver biopsy samples and 37 explanted livers was calculated from the amplification of B19 divided by the amount of human CRP DNA representing the actual amount of amplifiable cellular DNA in each sample. The median of copy numbers/cell in routine liver biopsy samples was 8.29E-05 (in chronic hepatitis C subgroup was 1.38E-04, while in non-HCV liver disease subgroup was 7.18E-05). The median of copy numbers/cell in explanted livers was 9.63E-05 (in end-stage hepatitis C subgroup was 6.95E-05, while in non-HCV end-stage liver disease subgroup was 2.42E-04). There was no significant difference of virus load in these groups.

The result of qPCR was reliable as similar results were obtained using re-extracted DNA from the same samples in different lab by a different person. Values depicted are the average of two independent qPCR tests.

1.00E-06 1.00E-05 1.00E-04 1.00E-03 1.00E-02 1.00E-01 1.00E+00

Copy numbers/cell

HCV (n=6) Non-HCV (n=8) HCV (n=13) Non-HCV (n=24) routine liver biopsy samples explanted livers

(23)

Table 5: Virus copy numbers of B19 DNA in explanted liver tissues using qPCR with DNA templates extracted in 2 different labs

patient copy numbers/cell(1^st qPCR) copy numbers/cell(2^nd qPCR)

PED 0 0

DJN 2.14E-05 2.88E-05

BHN 0 0

MHA 0 0

REH 1.39E-05 2.74E-04

BAD 1.48E-04 1.06E-04

IFA 1.10E-04 1.04E-03

SDR 3.14E-05 8.87E-05

CGO 1.97E-01 2.89E+00

SD 5.33E-01 3.94E-05

BAA 1.81E-05 8.43E-05

EKH 0 0

MVR 7.49E-06 2.09E-04

KTT 0 0

LAE 4.43E-03 2.19E-03

MKT 0 0

KVR 3.33E-05 1.09E-05

BRH 6.38E-05 8.09E-05

MGD 1.24E-03 1.67E-04

DHS 5.48E-05 6.39E-05

GMS 0 2.55E-05

GMA 0 3.15E-05

POA 1.71E-05 0

MKZ 4.61E-05 3.16E-05

WKS 3.35E-03 1.88E-03

FDH 4.39E-04 2.13E-03

SRA 5.29E-05 1.13E-04

SBD 2.64E-04 5.23E-04

BOR 0 0

SAE 8.26E-05 0

FWR 0 0

FAA 3.19E-04 1.16E-04

BMN 0 0

RMS 2.14E-03 5.64E-04

RHD 2.90E-05 1.03E-04

SCA 0 0

LHA 0 0

(24)

3.4 Proliferation of B19 specific CD4⁺ T cells in chronic HCV infected patients and healthy individuals with B19 serologically recovered infection

A flow cytometry-CFSE assay was performed to evaluate the proliferation of B19 specific CD4⁺ T cell after stimulation of PBMCs with synthetic peptides. In B19 serologically recovered healthy individuals, 3/19 (16%) previous showed a positive CD4⁺ T cell responses to the peptide VP1/2 7.2. In chronic HCV infected patients with B19 serologically recovered 3/13 (23%) patients had positive responses to peptide VP1/2 7.2 and/or peptide 4.7. Thus the frequency of positive responses was similar in these two groups. Representative examples of three individuals are shown in figure 6.

In addition, the positive responses of HCV-specific CD4⁺ T cells against NS3, NS4 and core were detectable in 2/13, 5/13 and 0/13 chronic HCV infected patients, respectively. The summary of CD4⁺ T cells responses after stimulating with specific antigen is shown in Table 6.

Parvo-Virus B19 infection in hepatitis C

Medizinische Hochschule Hannover Zentrum Innere Medizin

Klinik für Gastroenterologie, Hepatologie und Endokrinologie

Role of Parvovirus B19 Infection in hepatitis C

Dissertation

zur Erlangung des Doktorgrades der Medizin an der Medizinischen Hochschule Hannover

vorgelegt von Chun Wang aus Shanghai China

Hannover 2008

Angenommen vom Senat der Medizinschen Hochschule Hannover am ：

Gedruckt mit Genehmigung der Medizinschen Hochschule Hannover Präsident: Prof. Dr. Dieter Bitter-Suermann

Betreuer: Priv. Doz. Dr. med. Heiner Wedemeyer Referent: Prof. Dr. Thomas F. Schulz

Korreferent: Prof. Dr. Stefan Poehlmann

Tag der mündlichen Prüfung:

20. Mai 2008

Promotionsausschussmitglieder:

Prof. Dr. med. Alexander Kapp

Prof. Dr. med. Burkhard Wippermann

Prof. Dr. med. Stefan Kubicka

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