Manfred Wiese

Cellular immune responses persist and humoral responses decrease twodecades after recovery from a single-source outbreak of hepatitis C

As acute hepatitis C virus (HCV) infection is clinically inapparent in most cases, the immunologic correlates of recovery are not well defined. The cellular immune response is thought to contribute to the elimination of HCV-infected cells and a strong HCV-specific T-helper-cell (Th) response is associated with recovery from acute hepatitis C (ref. 2). However, diagnosis of resolved hepatitis C is based at present on the detection of HCV-specific antibodies and the absence of detectable HCV RNA, and detailed comparison of the humoral and cellular immune response has been hampered by the fact that patient cohorts as well as HCV strains are usually heterogeneous and that clinical data from acute-phase and long-term follow-up after infection generally are not available. We studied a cohort of women accidentally exposed to the same HCV strain of known sequence and found that circulating HCV-specific antibodies were undetectable in many patients 18–20 years after recovery, whereas HCV-specific helper and cytotoxic T-cell responses with an interferon (IFN)-γ-producing (Tc1) phenotype persisted. The data indicate these HCV-specific CD4+ and CD8+ T cells are biomarkers for a prior HCV exposure and recovery. Because of undetectable antibodies against HCV, the incidence of self-limited HCV infections and recovery may be underestimated in the general population.

A Polymorphism Near IL28B Is Associated With Spontaneous Clearance of Acute Hepatitis C Virus and Jaundice

BACKGROUND & AIMS A single nucleotide polymorphism (SNP) upstream of the IL28B gene has been associated with response of patients with chronic hepatitis C to therapy with pegylated interferon and ribavirin and also with spontaneous clearance of acute hepatitis C in a heterogeneous population. We analyzed the association between IL28B and the clinical presentation of acute hepatitis C virus (HCV) infection in a homogeneous population. METHODS We analyzed the SNP rs12979860 in 190 women from the German anti-D cohort (infected with HCV genotype 1b via contaminated rhesus prophylaxis) and its association with spontaneous clearance. Clinical data were available in 136 women with acute infection who were also evaluated for IL28B genotype. Based on results of a TaqMan polymerase chain reaction assay, the rs12979860 SNP genotypes studied were C/C, C/T, or T/T. RESULTS Spontaneous clearance was more common in patients with the C/C genotype (43/67; 64%) compared with C/T (22/90; 24%) or T/T (2/33; 6%) (P < .001). Jaundice during acute infection was more common among patients with C/C genotype (32.7%) than non-C/C patients (with C/T or T/T) (16.1%; P = .032). In C/C patients, jaundice during acute infection was not associated with an increased chance of spontaneous clearance (56.3%) compared with those without jaundice (60.6%). In contrast, in non-C/C patients, jaundice was associated with a higher likelihood of spontaneous clearance (42.9%) compared with those without jaundice (13.7%). CONCLUSIONS The SNP rs12979860 upstream of IL28B is associated with spontaneous clearance of HCV. Women with the C/T or T/T genotype who did not develop jaundice had a lower chance of spontaneous clearance of HCV infection.

Hepatitis C virus mutation affects proteasomal epitope processing

The high incidence of hepatitis C virus (HCV) persistence raises the question of how HCV interferes with host immune responses. Studying a single-source HCV outbreak, we identified an HCV mutation that impaired correct carboxyterminal cleavage of an immunodominant HLA-A2-restricted CD8 cell epitope that is frequently recognized by recovered patients. The mutation, a conservative HCV nonstructural protein 3 (NS3) tyrosine to phenylalanine substitution, was absent in 54 clones of the infectious source, but present in 15/21 (71%) HLA-A2-positive and in 11/24 (46%) HLA-A2-negative patients with chronic hepatitis C. In order to analyze whether the mutation affected the processing of the HLA-A2-restricted CD8 cell epitope, mutant and wild-type NS3 polypeptides were digested in vitro with 20S constitutive proteasomes and with immunoproteasomes. The presence of the mutation resulted in impaired carboxyterminal cleavage of the epitope. In order to analyze whether impaired epitope processing affected T cell priming in vivo, HLA-A2-transgenic mice were infected with vaccinia viruses encoding either wild-type or mutant HCV NS3. The mutant induced fewer epitope-specific, IFN-gamma;-producing and fewer tetramer(+) cells than the wild type. These data demonstrate how a conservative mutation in the flanking region of an HCV epitope impairs the induction of epitope-specific CD8(+) T cells and reveal a mechanism that may contribute to viral sequence evolution in infected patients.

Non-Invasive Assessment of Hepatic Steatosis in Patients with NAFLD Using Controlled Attenuation Parameter and 1H-MR Spectroscopy

Introduction Non-invasive assessment of steatosis and fibrosis is of growing relevance in non-alcoholic fatty liver disease (NAFLD). 1H-Magnetic resonance spectroscopy (1H-MRS) and the ultrasound-based controlled attenuation parameter (CAP) correlate with biopsy proven steatosis, but have not been correlated with each other so far. We therefore performed a head-to-head comparison between both methods. Methods Fifty patients with biopsy-proven NAFLD and 15 healthy volunteers were evaluated with 1H-MRS and transient elastography (TE) including CAP. Steatosis was defined according to the percentage of affected hepatocytes: S1 5-33%, S2 34–66%, S3 ≥67%. Results Steatosis grade in patients with NAFLD was S1 36%, S2 40% and S3 24%. CAP and 1H-MRS significantly correlated with histopathology and showed comparable accuracy for the detection of hepatic steatosis: areas under the receiver-operating characteristics curves were 0.93 vs. 0.88 for steatosis ≥S1 and 0.94 vs. 0.88 for ≥S2, respectively. Boot-strapping analysis revealed a CAP cut-off of 300 dB/m for detection of S2-3 steatosis, while retaining the lower cut-off of 215 dB/m for the definition of healthy individuals. Direct comparison between CAP and 1H-MRS revealed only modest correlation (total cohort: r = 0.63 [0.44, 0.76]; NAFLD cases: r = 0.56 [0.32, 0.74]). For detection of F2–4 fibrosis TE had sensitivity and specificity of 100% and 98.1% at a cut-off value of 8.85 kPa. Conclusion Our data suggest a comparable diagnostic value of CAP and 1H-MRS for hepatic steatosis quantification. Combined with the simultaneous TE fibrosis assessment, CAP represents an efficient method for non-invasive characterization of NAFLD. Limited correlation between CAP and 1H-MRS may be explained by different technical aspects, anthropometry, and presence of advanced liver fibrosis.

Evaluation of liver disease progression in the German hepatitis C virus (1b)‐contaminated anti‐D cohort at 35 years after infection

The natural course of HCV infection remains controversial. The German HCV (1b)‐contaminated anti‐D cohort provides an ideal population to investigate the natural course of HCV infection in a large, homogenous cohort of young women from the date of HCV inoculation. Our previous follow‐up studies at 20 and 25 years after infection suggested slow fibrosis progression rates in this unique cohort. The aim of our prospective, community‐based, multicenter study was to reevaluate the liver disease progression in 718 patients of the original anti‐D cohort at 35 years after infection. Patients with self‐limited HCV infection (n = 189) were compared to those who failed to eliminate the virus spontaneously (n = 529), comprising patients who were treatment naïve (n = 197) or achieved a sustained virological response (SVR; n = 149), respectively, failed to clear the virus (non‐SVR; n = 183) after antiviral therapy. In the overall cohort, 9.3% of patients showed clinical signs of liver cirrhosis at 35 years after infection. Liver disease progression largely depended on HCV infection status. The highest proportion of patients with clinical signs of end‐stage liver disease was observed in the non‐SVR group (15.3%), whereas decreased cirrhosis rates were detected in the SVR group (6%) and in patients with self‐limited HCV infection (1.1%; P = 6.2 × 10−6). Overall survival was significantly enhanced after SVR, compared to treatment‐naïve patients or non‐SVR (P = 0.027). Conclusion: The present study provides further evidence for a mild, but significant, disease progression at 35 years after infection in the German HCV (1b)‐contaminated anti‐D cohort. Patients with self‐limited HCV infection or SVR after antiviral treatment were protected from progressive liver disease and showed the best clinical long‐term outcome. (Hepatology 2014;58:49–57)

Quality of life in patients with various liver diseases: patients with HCV show greater mental impairment, while patients with PBC have greater physical impairment.

Summary.  Little is known comparing and contrasting quality of life (QoL) in patients with hepatitis C, compared to patients with other liver diseases. We performed two independent prospective cross‐sectional studies including 511 and 284 patients with different forms of liver diseases. SF‐36 was used in both studies. Fatigue Impact Score, WHO‐BREF and Hospital Anxiety and Depression Scale (HADS) were used in either study only. In both studies, HCV‐positive patients scored worse in the mental aspects of health‐related QoL compared to other liver diseases, except for HBV in one study. Surprisingly, in both studies, quality of life was also significantly impaired in patients with viral clearance after interferon therapy but not after spontaneous clearance. Furthermore, patients with primary biliary cirrhosis showed significantly better mental health but significantly worse physical well‐being. Liver diseases differ in their form of impaired QoL. In HCV, this impairment might not always return to normal after treatment‐induced viral clearance. This may suggest that HCV either may not be involved in QoL impairment or may induce a process which persists after viral clearance in some patients.

Escape from HLA-B*08-Restricted CD8 T Cells by Hepatitis C Virus Is Associated with Fitness Costs

ABSTRACT The inherent sequence diversity of the hepatitis C virus (HCV) represents a major hurdle for the adaptive immune system to control viral replication. Mutational escape within targeted CD8 epitopes during acute HCV infection has been well documented and is one possible mechanism for T-cell failure. HLA-B*08 was recently identified as one HLA class I allele associated with spontaneous clearance of HCV replication. Selection of escape mutations in the immunodominant HLA-B*08-restricted epitope HSKKKCDEL1395-1403 was observed during acute infection. However, little is known about the impact of escape mutations in this epitope on viral replication capacity. Their previously reported reversion back toward the consensus residue in patients who do not possess the B*08 allele suggests that the consensus sequence in this epitope is advantageous for viral replication in the absence of immune pressure. The aim of this study was to determine the impact of mutational escape from this immunodominant epitope on viral replication. We analyzed it with a patient cohort with chronic HCV genotype 1b infection and in a single-source outbreak (genotype 1b). Sequence changes in this highly conserved region are rare and selected almost exclusively in the presence of the HLA-B*08 allele. When tested in the subgenomic replicon (Con1), the observed mutations reduce viral replication compared with the prototype sequence. The results provide direct evidence that escape mutations in this epitope are associated with fitness costs and that the antiviral effect of HLA-B*08-restricted T cells is sufficiently strong to force the virus to adopt a relatively unfavorable sequence.

CD8+ T-Cell Response Promotes Evolution of Hepatitis C Virus Nonstructural Proteins

BACKGROUND & AIMS Hepatitis C virus (HCV) acquires mutations that allow it to escape the CD8+ T-cell response, although the extent to which this process contributes to viral evolution at the population level is not clear. We studied viral adaptation using data from a large outbreak of HCV genotype 1b infection that occurred among women immunized with contaminated immunoglobulin from 1977 to 1978. METHODS The HCV nonstructural protein coding regions NS3-NS5B were sequenced from 78 patients, and mutations were mapped according to their location inside or outside previously described CD8+ T-cell epitopes. A statistical approach was developed to identify sites/regions under reproducible selection pressure associated with HLA class I. RESULTS The frequency of nonsynonymous mutations was significantly higher inside previously described CD8+ T-cell epitopes than outside-particularly in NS3/4A and NS5B. We identified new regions that are under selection pressure, indicating that not all CD8+ T-cell epitopes have been identified; 6 new epitopes that interact with CD8+ T cells were identified and confirmed in vitro. In some CD8+ T-cell epitopes mutations were reproducibly identified in patients that shared the relevant HLA allele, indicating immune pressure at the population level. There was statistical support for selection of mutations in 18 individual epitopes. Interestingly, 14 of these were restricted by HLA-B allele. CONCLUSIONS HLA class I-associated selection pressure on the nonstructural proteins and here predominantly on NS3/4A and NS5B promotes evolution of HCV. HLA-B alleles have a dominant effect in this selection process. Adaptation of HCV to the CD8+ T-cell response at the population level creates challenges for vaccine design.

The “interferon sensitivity determining region” of hepatitis C virus is a stable sequence element

BACKGROUND/AIMS A sequence of 40 amino acids within the nonstructural protein 5A of hepatitis C virus (HCV) has been suggested to be an interferon sensitivity determining region (ISDR). The variations in the ISDR after 12-14 years of chronic infection and the correlation between ISDR and interferon response were studied in patients who were infected by the same HCV isolate. METHODS We determined the HCV-ISDRs of 13 chronically infected patients by direct sequencing of polymerase chain reaction products. All patients were infected by isolate HCV-AD78, but differed with respect to their sensitivity to interferon. Four patients were complete responders, two patients were non-responders, and seven showed a partial response. RESULTS The ISDR of HCV-AD78 differed from a prototypical HCV-1b sequence in one amino acid and was therefore classified as an intermediate type. Direct sequencing of the HCV-ISDRs of the patients 12-14 years after infection, but before interferon therapy, revealed a rate of 2.2x10(-3) nucleotide substitutions per site per year, resulting in only single intermediate type amino acid exchanges. All sequences ranked with the intermediate type. Moreover, during interferon treatment no selection to a wild type ISDR was observed in five partial responders. CONCLUSIONS Within the homogeneous patient group examined here, no correlation was found between the ISDR and the interferon response. Recent studies found only a small number of mutant type ISDRs in Europe. Additionally, our results indicate that the ISDR is a stable sequence element. This provides an explanation for the divergent data relating to the importance of the ISDR in different geographical regions.

The predictive value of IL28B gene polymorphism for spontaneous clearance in a single source outbreak cohort is limited in patients carrying the CCR5Δ32 mutation

BACKGROUND & AIMS The CCR5Δ32 mutation has been suspected to adversely affect outcomes of HCV infection, although reports have remained controversial. Here, we investigated the relative genetic contributions of the CCR5Δ32 deletion and the IL28B rs12979860 polymorphisms to spontaneous clearance of hepatitis C in a single-source outbreak. METHODS We retrieved 396 Caucasian women (119 women with spontaneous HCV clearance) who had been infected with HCV genotype 1-contaminated anti-D immunoglobulin in 1978, and determined their IL28B and CCR5 alleles. RESULTS IL28B CC, CT, and TT genotypes were found in 35.4%, 50%, and 14.6% of patients and corresponded to spontaneous clearance rates of 50%, 21.2%, and 12.1% (Chi(2)=38.7, p=5.0×10(-10)), respectively. CCR5 WT/WT, WT/Δ32, and Δ32/Δ32 genotypes were observed in 76%, 22.7%, and 1.3% of patients and corresponded to clearance rates of 33.2%, 21.2%, and 0% (Chi(2)=6.9, p=0.009), respectively. In a stepwise forward-conditional multivariate regression model both CCR5 (OR 2.1, p=0.01 for WT/WT) and IL28B genetic variants (OR 4.3, p=4.6×10(-10) for the C/C genotype) were identified as independent predictors of spontaneous HCV clearance. Importantly, favorable response rates were associated with the IL28B CC genotype only in CCR5 wild-type homozygous women, while HCV clearance in CCR5Δ32 carriers remained poor even in patients with the rs12979860 CC genotype. CONCLUSIONS Both IL28B rs1297860 and CCR5Δ32 allelic variants are independent genetic determinants of spontaneous HCV clearance. The variable relative distribution between IL28B rs1297860 and CCR5Δ32 allelic variants in different populations may have masked the role of the CCR5Δ32 mutation in some studies.