[show abstract][hide abstract] ABSTRACT: Different parameters have been determined for prediction of treatment outcome in hepatitis c virus genotype 1 infected patients undergoing pegylated interferon, ribavirin combination therapy. Results on the importance of vitamin D levels are conflicting. In the present study, a comprehensive analysis of vitamin D levels before and during therapy together with single nucleotide polymorphisms involved in vitamin D metabolism in the context of other known treatment predictors has been performed.
In a well characterized prospective cohort of 398 genotype 1 infected patients treated with pegylated interferon-α and ribavirin for 24-72 weeks (INDIV-2 study) 25-OH-vitamin D levels and different single nucleotide polymorphisms were analyzed together with known biochemical parameters for a correlation with virologic treatment outcome.
Fluctuations of more than 5 (10) ng/ml in 25-OH-vitamin D-levels have been observed in 66 (39) % of patients during the course of antiviral therapy and neither pretreatment nor under treatment 25-OH-vitamin D-levels were associated with treatment outcome. The DHCR7-TT-polymorphism within the 7-dehydrocholesterol-reductase showed a significant association (P = 0.031) to sustained viral response in univariate analysis. Among numerous further parameters analyzed we found that age (OR = 1.028, CI = 1.002-1.056, P = 0.035), cholesterol (OR = 0.983, CI = 0.975-0.991, P<0.001), ferritin (OR = 1.002, CI = 1.000-1.004, P = 0.033), gGT (OR = 1.467, CI = 1.073-2.006, P = 0.016) and IL28B-genotype (OR = 2.442, CI = 1.271-4.695, P = 0.007) constituted the strongest predictors of treatment response.
While 25-OH-vitamin D-levels levels show considerable variations during the long-lasting course of antiviral therapy they do not show any significant association to treatment outcome in genotype 1 infected patients.
PLoS ONE 01/2014; 9(2):e87974. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cell therapy based on alloreactivity has completed clinical proof of concept against hematological malignancies. However, the efficacy of alloreactivity as a therapeutic approach to treat solid tumors is unknown. Using cell culture and animal models, we aimed to investigate the efficacy and safety of allogeneic suicide gene-modified killer cells as a cell-based therapy for hepatocellular carcinoma (HCC), for which treatment options are limited. Allogeneic killer cells from healthy donors were isolated, expanded, and phenotypically characterized. Antitumor cytotoxic activity and safety were studied using a panel of human or murine HCC cell lines engrafted in immunodeficient or immunocompetent mouse models. Human allogeneic suicide gene-modified killer cells (aSGMKCs) exhibit a high, rapid, interleukin-2-dependent, and non-major histocompatibility complex class I-restricted in vitro cytotoxicity toward human hepatoma cells, mainly mediated by natural killer (NK) and NK-like T cells. In vivo evaluation of this cell therapy product demonstrates a marked, rapid, and sustained regression of HCC. Preferential liver homing of effector cells contributed to its marked efficacy. Calcineurin inhibitors allowed preventing rejection of allogeneic lymphocytes by the host immune system without impairing their antitumor activity. Our results demonstrate proof of concept for aSGMKCs as immunotherapy for HCC and open perspectives for the clinical development of this approach.Molecular Therapy (2014); doi:10.1038/mt.2013.277.
[show abstract][hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide with a poor prognosis and limited therapeutic options. To aid the development of novel immunological interventions, we studied the breadth, frequency and tumor-infiltration of naturally occurring CD8(+) T-cell responses targeting several tumor-associated antigens (TAA). We used overlapping peptides spanning the entire AFP, GPC-3, MAGE-A1 and NY-ESO-1 proteins and major-histocompatibility-complex-class-I-tetramers specific for epitopes of MAGE-A1 and NY-ESO-1 to analyze TAA-specific CD8(+) T-cell responses in a large cohort of HCC patients. After non-specific expansion in vitro, we could detect interferon-γ (IFN-γ)-producing CD8(+) T-cells specific for all four TAA in the periphery as well as in liver and tumor tissue. These CD8(+) T-cell responses displayed clear immunodominance patterns within each TAA, but no consistent hierarchy was observed between different TAA. Importantly, the response breadth was highest in early stage HCC and associated with patient survival. After antigen-specific expansion, TAA-specific CD8(+) T-cells were detectable by tetramer staining but impaired in their ability to produce IFN-γ. Furthermore, regulatory T-cells (Treg ) were increased in HCC lesions. Depletion of Treg from cultures improved TAA-specific CD8(+) T-cell proliferation but did not restore IFN-γ-production. Conclusions: Naturally occurring TAA-specific CD8(+) T-cell responses are present in patients with HCC and therefore constitute part of the normal T-cell repertoire. Moreover, the presence of these responses correlates with patient survival. However, the observation of impaired IFN-γ-production suggests that the efficacy of such responses is functionally limited. These findings support the development of strategies that aim to enhance the total TAA-specific CD8(+) T-cell response by therapeutic boosting and/or specificity diversification. However, further research will be required to help unlock the full potential of TAA-specific CD8(+) T-cell responses. (Hepatology 2013;).
[show abstract][hide abstract] ABSTRACT: HLA-B∗27 is associated with spontaneous HCV genotype 1 clearance. HLA-B∗27-restricted CD8+ T-cells target three NS5B epitopes. Two of these epitopes are dominantly targeted in the majority of HLA-B∗27+ patients. In chronic infection, viral escape occurs consistently in these two epitopes. The third epitope (NS5B2820) was dominantly targeted in an acutely infected patient. This was in contrast, however, to the lack of recognition and viral escape in the large majority of HLA-B∗27+ patients. Here, we set out to determine the host factors contributing to selective targeting of this epitope.
Four-digit HLA class I typing and viral sequence analyses were performed in 78 HLA-B∗27+ patients with chronic HCV genotype 1 infection. CD8+ T-cell analyses were performed in a subset of patients. In addition, HLA/peptide affinity was compared for HLA-B∗27:02 and 05.
The NS5B2820 epitope is only restricted by the HLA-B∗27 subtype HLA-B∗27:02 (that is frequent in Mediterranean populations), but not by the prototype HLA-B∗27 subtype B∗27:05. Indeed, the epitope is very dominant in HLA-B∗27:02+ patients and is associated with viral escape mutations at the anchor position for HLA-binding in 12 out of 13 HLA-B∗27:02+ chronically infected patients.
The NS5B2820 epitope is immunodominant in the context of HLA-B∗27:02, but is not restricted by other HLA-B∗27 subtypes. This finding suggests an important role of HLA subtypes in the restriction of HCV-specific CD8+ responses. With minor HLA subtypes covering up to 39% of specific populations, these findings may have important implications for the selection of epitopes for global vaccines. (250/250).
Journal of Hepatology 08/2013; · 9.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC) is the fifth most common tumor worldwide with a high mortality. Available therapeutical options are limited, thus the development of new, innovative therapeutic strategies is crucial. Based on the immune system's antitumoral effect, immunotherapy is a promising new treatment option. Specific antitumoral T-cell responses can be detected in patients with HCC, however, their impact on tumor control seems to be rather weak. Various different immunosuppressive mechanisms seem to contribute to the failure of tumor-specific T-cell responses. Thus, the aim of immunotherapeutic strategies is to address these mechanisms of T-cell failure and to induce new or to boost existing antitumoral immune responses.
[show abstract][hide abstract] ABSTRACT: CD8+ T cell responses to hepatitis C virus (HCV) are important in generating a successful immune response and spontaneously clearing infection. HLA class I presents viral peptides to CD8+ T cells to permit detection of infected cells, and tapasin is an important component of the peptide loading complex for HLA class I. We sought to determine if tapasin polymorphisms affected the outcome of HCV infection. Patients with resolved or chronic HCV infection were genotyped for the known C/G coding polymorphism in exon 4 of the tapasin gene. In a European, but not a US, Caucasian population, the tapasin G allele was significantly associated with outcome of HCV infection, being found in 82.5% of resolvers versus 71.3% of persistently infected individuals (p=0.02, OR=1.90 95% CI=1.11-3.23). This was more marked at the HLA-B locus at which heterozygosity of both tapasin and HLA-B was protective (p<0.03). Individuals with an HLA-B allele with an aspartate at residue 114 and the tapasin G allele were more likely to spontaneously resolve HCV infection (p<0.00003, OR=3.2 95%CI=1.6-6.6). Additionally individuals with chronic HCV and the combination of an HLA-B allele with an aspartate at residue 114 and the tapasin G allele also had stronger CD8+ T cell responses (p=0.02, OR=2.58, 95% CI-1.05-6.5). Conclusion Tapasin alleles contribute to the outcome of HCV infection by synergising with polymorphisms at HLA-B in a population specific manner. This polymorphism may be relevant for peptide vaccination strategies against HCV infection. (HEPATOLOGY 2013.).
[show abstract][hide abstract] ABSTRACT: Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection are global health problems affecting 600 million people worldwide. Indeed, HBV and HCV are hepatotropic viruses that can cause acute and chronic liver disease progressing to liver cirrhosis and even hepatocellular carcinoma. Furthermore, co-infections of HBV and HCV with HIV are emerging worldwide. These co-infections are even more likely to develop persistent infection and are difficult to treat. There is growing evidence that virus-specific CD4+ and CD8+ T-cell responses play a central role in the outcome and pathogenesis of HBV and HCV infection. While virus-specific T-cell responses are able to successfully clear the virus in a subpopulation of patients, failure of these T-cell responses is associated with the development of viral persistence. In this review article, we will discuss similarities and differences in HBV- and HCV-specific T-cell responses that are central in determining viral clearance, persistence and liver disease.
[show abstract][hide abstract] ABSTRACT: The adaptive immune response plays a central role in the outcome of hepatitis C virus (HCV) infection. Indeed, spontaneous viral clearance is associated with an early neutralizing antibody response as well as vigorous and sustained HCV-specific CD4+ and CD8+ T cell responses. In persistent HCV infection, however, all three components of the antiviral adaptive immune response fail due to different viral evasion strategies. In this chapter, we will describe the components of a successful immune response against HCV and summarize the mechanisms of immune failure. We will also highlight characteristics of protective CD8+ T cell responses which is the key factor to the design of an efficacious vaccine.
Current topics in microbiology and immunology 01/2013; 369:243-62. · 4.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: Owing to the major limitations of current antiviral therapies in HBV (hepatitis B virus) infection, there is a strong need for novel therapeutic approaches to this major health burden. Stimulation of the host's innate and adaptive immune responses in a way that results in the resolution of viral infection is a promising approach. A better understanding of the virus-host interaction in acute and chronic HBV infection revealed several possible novel targets for antiviral immunotherapy. In the present review, we will discuss the current state of the art in HBV immunology and illustrate how control of infection could be achieved by immunotherapeutic interventions.
[show abstract][hide abstract] ABSTRACT: BACKGROUND & AIMS:: Dysfunctional CD8+ T cells are believed to contribute to the ability of hepatitis C virus (HCV) to evade the immune response. Most studies have focused on the effector functions of HCV-specific CD8+ T cells or their surface expression of inhibitory receptors. There is currently no information available about the ex vivo ability of HCV-specific CD8+ T cells to inhibit viral replication (antiviral efficacy). METHODS:: To analyze the antiviral efficacy of virus-specific CD8+ T cells ex vivo we utilized an immunological model based on a cell line that expresses HLA-A*02 and contains a stably replicating HCV reporter replicon. We isolated HCV-specific CD8+ T cells from 18 HLA-A*02-positive patients with chronic HCV infection and 15 subjects that resolved HCV infection (7 spontaneous, 8 after therapy). Replicon cells were labeled with virus-specific peptides; inhibition of HCV replication was determined by measuring luciferase activity after 72 hours co-culture with virus-specific CD8+ T cells. RESULTS:: HCV-specific CD8+ T cells from patients with chronic HCV infection had a significantly lower antiviral efficacy than influenza-, Epstein-Barr virus-, and cytomegalovirus-specific CD8+ T cells. Antiviral efficacy was associated with the ability of virus-specific CD8+ T cells to secrete interferon (IFN)-γ. Antiviral efficacy of HCV-specific CD8+ T cells was linked to surface expression of CD127 and PD-1. The cytokines interleukin (IL)-2, IL-7, and IL-15 increased the antiviral efficacy of CD127-positive, but not of CD127-negative, HCV-specific CD8+ T cells. Spontaneous, but not antiviral therapy-induced, viral clearance was associated with increased antiviral efficacy. CONCLUSION:: The ability of CD8+ T cells to inhibit HCV replication ex vivo is associated with their ability to secrete IFN-γ and their surface expression of CD127 and PD-1.
[show abstract][hide abstract] ABSTRACT: HLA-B*27 exerts protective effects in hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections. While the immunological and virological features of HLA-B*27-mediated protection are not fully understood, there is growing evidence that the presentation of specific immunodominant HLA-B*27-restricted CD8+ T-cell epitopes contributes to this phenomenon in both infections. Indeed, protection can be linked to single immunodominant CD8+ T-cell epitopes and functional constraints on escape mutations within these epitopes. To better define the immunological mechanisms underlying HLA-B*27-mediated protection in HCV infection, we analyzed the functional avidity, functional profile, antiviral efficacy and naïve precursor frequency of CD8+ T cells targeting the immunodominant HLA-B*27-restricted HCV-specific epitope as well as its antigen processing and presentation. For comparison, HLA-A*02-restricted HCV-specific epitopes were analyzed. The HLA-B*27-restricted CD8+ T-cell epitope was not superior to epitopes restricted by HLA-A*02 when considering the functional avidity, functional profile, antiviral efficacy or naïve precursor frequency. However, the peptide region containing the HLA-B*27-restricted epitope was degraded extremely fast by both the constitutive proteasome and the immunoproteasome. This efficient proteasomal processing that could be blocked by proteasome inhibitors was highly dependent on the hydrophobic regions flanking the epitope and led to rapid and abundant presentation of the epitope on the cell surface of antigen presenting cells. Our data suggest that rapid antigen processing may be a key immunological feature of this protective and immunodominant HLA-B*27-restricted HCV-specific epitope.
[show abstract][hide abstract] ABSTRACT: BACKGROUND & AIMS: CD40, a member of the tumor necrosis factor receptor family, and its ligand, CD40L (CD154), are important regulators of the antiviral immune response. CD40L is upregulated on lymphocytes and CD40 on hepatocytes during infection with hepatitis C virus (HCV); we investigated the role of CD40 signaling during HCV replication in hepatocytes. METHODS: Viral replication was studied in primary human hepatocytes (PHH) and Huh7.5 cells using the infectious HCV JFH1 culture system, and in co-culture with HCV antigen-specific CD8+ T cells. RESULTS: CD40L rapidly and transiently inhibits expression of the HCV non-structural proteins NS3 and NS5A as well as HCV structural proteins core and E2 in Huh7.5 cells. Similarly, CD40L prevented replication of HCV in PHH, in synergy with interferon (IFN)-alpha. In Huh7.5 cells with replicating HCV, CD40L prevented production of infectious viral particles. When HCV antigen-specific CD8+ T cells were co-cultured with HLA-A2-expressing Huh7 cells that had replicating virus, the T cells became activated, upregulated CD40L, and inhibited HCV replication. Inhibition of CD40L partially prevented the antiviral activity of the CD8+ T cells. The antiviral effect of CD40L required activation of JNK1/2, but not induction of apoptosis or the JAK/STAT pathway that is necessary for the antiviral effects of IFNs. CONCLUSIONS: CD40 inhibits HCV replication by a novel, innate immune mechanism. This pathway might mediate viral clearance, and disruptions might be involved in pathogenesis of HCV infection. (HEPATOLOGY 2012.).
[show abstract][hide abstract] ABSTRACT: The standard antiviral therapy for chronic hepatitis C is pegylated interferon-alfa (PegIFN) and ribavirin since about 10 years. This treatment regimen leads to a sustained virological response (SVR) in 40-50 % of patients infected with HCV genotype 1 and in approx. 80 % of those infected with HCV genotype 2 or 3. In recent years, many direct antiviral agents (DAA) have been developed and are being explored in clinical studies. These antiviral agents target different viral proteins that are central for HCV replication, incl. the NS3/4A protease, NS5B polymerase, and the NS5A protein. The protease inhibitors telaprevir and boceprevir have recently been approved for the treatment of chronic HCV genotype 1 infection in combination with PegIFN and ribavirin. These triple therapies increase the SVR rates in HCV genotype 1 patients from 40-50 % to approx. 70 %. Other DAAs will likely be approved in the near future and may result in an IFN-free antiviral therapy.
[show abstract][hide abstract] ABSTRACT: Dipeptidylpeptidase IV (CD26) is a multifunctional ectoenzyme involved in T cell activation that has been implicated in autoimmune pathophysiology. Because IL-17-producing CD4(+) T cells (Th17 cells) are important mediators of autoimmune disease, we analyzed the expression of CD26 and its enzymatic function on human Th17 cells. Analysis of CD26 expression on different CD4(+) T helper subsets showed that CD26 expression is highest on CD4(+) T cells producing type 17 cytokines (e.g., IL-22, IL-17, GM-CSF, or TNF) compared with Th1, Th2, and regulatory T cells. Phenotypic analysis revealed that CD26(++)CD4(+) T cells express the type 17 differentiation molecules CD161, CCR6, lL-23R, and retinoic acid-related orphan receptor-γt. Furthermore, sorted CD26(++)CD4(+) T cells contain >90-98% of Th17 cells, indicating that CD26(++) T cells harbor the Th17 lineage. A comparison with CD161 and CCR6 indicated that analysis of CD26 coexpression may improve the phenotypic characterization of Th17 cells. Of note, CD26(++) Th17 cells are enriched in the inflamed tissue of patients with hepatitis and inflammatory bowel disease. Functional analysis in migration assays revealed that CD26 expressed on Th17 cells is enzymatically active. Indeed, CD26 negatively regulates the chemotactic CD4(+) T cell response to the inflammatory chemokines CXCL9-12 that can be restored by pharmacological blockade of the enzymatic center of CD26. In summary, these results strongly suggest that CD26 may contribute to the orchestration of the immune response by Th17 cells in human inflammatory diseases. They also suggest that the phenotypic analysis of Th17 cells may be facilitated by determination of CD26 expression.
The Journal of Immunology 04/2012; 188(11):5438-47. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: A subset of CD8(+) T cells can secrete interleukin 17 (IL-17). However, very little information is currently available about their antigen specificity, tissue distribution, and biological relevance in chronic human viral infection. To address these issues, we comprehensively analyzed peripheral and intrahepatic CD8(+) T-cell responses in a cohort of patients with chronic hepatitis C virus (HCV) infection for the antigen-specific production of IL-17 and interferon (IFN) γ. We found that HCV-specific IL-17-producing and retinoic acid receptor related orphan receptorγt-expressing CD8(+) T cells are detectable in blood and liver and target different epitopes, compared with IFN-γ-producing CD8(+) T cells. Their highest frequency was found in patients with low inflammatory activity, suggesting a protective role in chronic HCV infection.
The Journal of Infectious Diseases 04/2012; 205(7):1142-6. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Perforin plays a central role in the immunopathogenesis of different viral infections. However, its role in hepatitis C virus (HCV) infection has not been fully understood. Here, we analyzed two closely related questions: first, is CD8+ T cell-mediated killing of HCV-replicating human hepatoma cells mediated by perforin? Second, if so, do HCV-specific CD8+ T cells obtained from chronically HCV infected patients express and upregulate perforin?
Susceptibility of HCV-replicating human hepatoma cells to the cytotoxic pathway was tested in vitro by addition of perforin substitute streptolysin O and granzyme B and by co-culture experiments with a perforin-expressing HCV-specific CD8+ T cell clone in the presence of perforin or caspase inhibitors. HCV-specific CD8+ T cells were obtained and analyzed for perforin expression and differentiation markers ex vivo from 12 chronically infected patients and 12 patients with resolved HCV infection.
HCV-replicating human hepatoma cells were susceptible to cytotoxic killing in vitro and a dominant role of perforin in HCV-specific CD8+ T cell-mediated cytolysis was observed. However, HCV-specific CD8+ T cells obtained ex vivo from chronically HCV infected patients expressed only low levels of perforin and showed an impaired ability to upregulate perforin. This was tightly linked to the distinct differentiation stage of HCV-specific CD8+ T cell differentiation ex vivo since early and intermediate differentiated HCV-specific CD8+ T cells only showed weak perforin expression in contrast to late differentiated CD8+ T cells that displayed strong perforin expression.
Our results suggest that perforin plays a dominant role in CD8+ T cell-mediated lysis of HCV-replicating human hepatoma cells but that lysis may be limited in human chronic viral infection by the low perforin expression of early/intermediate differentiated HCV-specific CD8+ T cells.
Journal of Hepatology 03/2012; 57(1):9-16. · 9.86 Impact Factor