T-cell Responses In Hepatitis Research Articles

Naturally occurring CD4 + T-cell epitope variants act as altered peptide ligands leading to impaired helper T-cell responses in hepatitis C virus infection

Hepatitis C virus (HCV) has a high rate of replication and lacks RNA-proofreading capabilities, thereby leading to variant or mutant viruses circulating within the host as a quasispecies. Previous work in our laboratory identified viral variants that emerged in a class-II immunodominant epitope NS3 358-375 of the non-structural-3 (NS3) protein region of HCV, the sequence of which is based on genotype 1A, the most prevalent genotype in the United States. Further work suggested that positive immune selection pressure was driving viral variation. Paradoxically, viral variants account for only a small percentage of the circulating virus in human beings and in chimpanzees, suggesting that passive evasion is not the only means of escape by HCV. This observation suggests a unique pathogenesis for HCV as it persists in the host. In the current study, we hypothesize that viral variants are acting as altered peptide ligands (APLs). To test this hypothesis, we used cloned T cells specific for NS3 358-375 peptide, which demonstrated attenuated T-cell and interferon-γ (IFN-γ) responses to individual variant peptides, when compared with the NS3 358-375 stimulated T-cell clones. Furthermore, such variants could act as APLs, based on their ability to antagonize the IFN-γ proliferative responses of clones specific for NS3 358-375. In addition, major histocompatibility complex (MHC) class II tetramer staining demonstrated that variant peptide–MHC complexes were able to specifically bind to NS3 358-375 T-cell clones and that both the variant and NS3 358-375 tetramers were able to bind to the same CD4 + T cells. Taken together, the results suggest that viral variants may act as APL to effectively blunt the T-cell response to an important HCV epitope.

Oral Nucleoside Analogs in HBV: Do They Really Improve Immune Responses?

© 2011, INASL 10 HBV Specific T-Cell Responses in Hepatitis B J Menachery*, D Rana**, Y Chawla*, A Duseja*, RK Dhiman*, S Arora** *Department of Hepatology, PGIMER, Chandigarh **Department of Immunopathology, PGIMER, Chandigarh Cellular immune responses seem to prevail in acute hepatitis, whereas chronically infected patients demonstrate generally suppressed cellular immune responses and significantly greater antibody responses. Aim: To study hepatitis B virus (HBV) specific T-cell proliferative responses in HBV-related liver diseases. Materials and Methods: We analyzed the T lymphocyte proliferative responses to the nonspecific mitogen phytohemagglutinin (PHA) and the HBV specific hepatitis B core antigen (HbcAg) by calculating T cell proliferation index in 10 acute viral hepatitis (AVH) patients, 19 chronic hepatitis B (CHB) patients, 10 HBV cirrhotics, 10 inactive carriers and 10 healthy controls using MTT assay. Results: The mean proliferation index (PI) to PHA was highest in healthy controls (133.2 ± 58.1) and lowest in cirrhotics (44.1 ± 46.9) with all other groups falling in between. On comparing the mean T cell responses to HBcAg, AVH patients had the highest mean response (186.48 ± 116.37) followed by CHB (137.9 ± 134.3), inactive carriers (63.2 ± 41.2) and cirrhotics (55.5 ± 42.7). Conclusion: Patients with AVH had the highest T cell response to HBcAg which probably explains the clearance of virus infection in these patients, in contrast to patients with cirrhosis who had the least. Conflict of Interest: None Oral Nucleoside Analogs in HBV: Do They Really Improve Immune Responses? J Menachery*, D Rana**, Y Chawla*, A Duseja*, RK Dhiman*, S Arora** Departments of *Hepatology and **Immunopathology, PGIMER, Chandigarh, India The fact that chronic hepatitis B (CHB) patients have T cell hyporesponsiveness to hepatitis B virus (HBV) antigens is well known and restoration of immune responses has been described with successful antiviral therapy. Aim: We undertook this study to evaluate the changes in T-cell proliferative response during treatment of CHB patients with entecavir. Materials and Methods: Nine treatment naive CHB patients were given entecavir 0.5 mg/d. T cell responses to phytohemagglutinin (PHA) and hepatitis B core antigen (HBcAg) in CHB patients were compared before starting treatment and after 3 months of therapy by calculating the proliferation index (PI) using an MTT assay. Results: Proliferation index (PI) to HBcAg on treatment with entecavir increased in 7 out of 9 patients and the median PI increased from 36.4 to 105.8. Conclusion: T cell responses to hepatitis B antigens improve on successful antiviral treatment and the restoration of specific immune response may be important in the clearance of virus. Conflict of Interest: None Prevalence of Positive Serology for Hepatitis B and Hepatitis C in Community and Hospital Setting R Balal, P Kumar, M Jain, D Gupta Jagjivanram Hospital, Mumbai Aim: To analyze the prevalence of positive serology for hepatitis B and C in general population and in hospital setting. Methods: One thousand two hundred people from general railway population residing at two railway colonies: Bandra and Matunga were screened for hepatitis B and C to determine the prevalence in community. All patients attending the IPD and OPD services between 2007 and 2008 (12546) were screened based on the following inclusion criteria to determine the hospital prevalence: 1. Abnormal liver function tests, 2. Acute hepatitis, 3. Chronic liver disease, 4. History of high-risk behavior, 5. Positive family history, 6. HIV infection and 7. Renal failure patients on hemodialysis. A total of 2645 patients were screened for HBsAg and anti-HCV. Results: The community prevalence of HBsAg and anti-HCV positivity were 1.47% and 0.09%, respectively. The prevalence in high-risk patients attending hospital was 6.78% for hepatitis B and 1.48% for hepatitis C. 03_JCEH-Abstract.indd 10 3/18/2011 11:13:03 AM

Prevalence of Positive Serology for Hepatitis B and Hepatitis C in Community and Hospital Setting

© 2011, INASL 10 HBV Specific T-Cell Responses in Hepatitis B J Menachery*, D Rana**, Y Chawla*, A Duseja*, RK Dhiman*, S Arora** *Department of Hepatology, PGIMER, Chandigarh **Department of Immunopathology, PGIMER, Chandigarh Cellular immune responses seem to prevail in acute hepatitis, whereas chronically infected patients demonstrate generally suppressed cellular immune responses and significantly greater antibody responses. Aim: To study hepatitis B virus (HBV) specific T-cell proliferative responses in HBV-related liver diseases. Materials and Methods: We analyzed the T lymphocyte proliferative responses to the nonspecific mitogen phytohemagglutinin (PHA) and the HBV specific hepatitis B core antigen (HbcAg) by calculating T cell proliferation index in 10 acute viral hepatitis (AVH) patients, 19 chronic hepatitis B (CHB) patients, 10 HBV cirrhotics, 10 inactive carriers and 10 healthy controls using MTT assay. Results: The mean proliferation index (PI) to PHA was highest in healthy controls (133.2 ± 58.1) and lowest in cirrhotics (44.1 ± 46.9) with all other groups falling in between. On comparing the mean T cell responses to HBcAg, AVH patients had the highest mean response (186.48 ± 116.37) followed by CHB (137.9 ± 134.3), inactive carriers (63.2 ± 41.2) and cirrhotics (55.5 ± 42.7). Conclusion: Patients with AVH had the highest T cell response to HBcAg which probably explains the clearance of virus infection in these patients, in contrast to patients with cirrhosis who had the least. Conflict of Interest: None Oral Nucleoside Analogs in HBV: Do They Really Improve Immune Responses? J Menachery*, D Rana**, Y Chawla*, A Duseja*, RK Dhiman*, S Arora** Departments of *Hepatology and **Immunopathology, PGIMER, Chandigarh, India The fact that chronic hepatitis B (CHB) patients have T cell hyporesponsiveness to hepatitis B virus (HBV) antigens is well known and restoration of immune responses has been described with successful antiviral therapy. Aim: We undertook this study to evaluate the changes in T-cell proliferative response during treatment of CHB patients with entecavir. Materials and Methods: Nine treatment naive CHB patients were given entecavir 0.5 mg/d. T cell responses to phytohemagglutinin (PHA) and hepatitis B core antigen (HBcAg) in CHB patients were compared before starting treatment and after 3 months of therapy by calculating the proliferation index (PI) using an MTT assay. Results: Proliferation index (PI) to HBcAg on treatment with entecavir increased in 7 out of 9 patients and the median PI increased from 36.4 to 105.8. Conclusion: T cell responses to hepatitis B antigens improve on successful antiviral treatment and the restoration of specific immune response may be important in the clearance of virus. Conflict of Interest: None Prevalence of Positive Serology for Hepatitis B and Hepatitis C in Community and Hospital Setting R Balal, P Kumar, M Jain, D Gupta Jagjivanram Hospital, Mumbai Aim: To analyze the prevalence of positive serology for hepatitis B and C in general population and in hospital setting. Methods: One thousand two hundred people from general railway population residing at two railway colonies: Bandra and Matunga were screened for hepatitis B and C to determine the prevalence in community. All patients attending the IPD and OPD services between 2007 and 2008 (12546) were screened based on the following inclusion criteria to determine the hospital prevalence: 1. Abnormal liver function tests, 2. Acute hepatitis, 3. Chronic liver disease, 4. History of high-risk behavior, 5. Positive family history, 6. HIV infection and 7. Renal failure patients on hemodialysis. A total of 2645 patients were screened for HBsAg and anti-HCV. Results: The community prevalence of HBsAg and anti-HCV positivity were 1.47% and 0.09%, respectively. The prevalence in high-risk patients attending hospital was 6.78% for hepatitis B and 1.48% for hepatitis C. 03_JCEH-Abstract.indd 10 3/18/2011 11:13:03 AM

Characterization of CD8+ T-cell response in acute and resolved hepatitis A virus infection

In contrast to the infection with other hepatotropic viruses, hepatitis A virus (HAV) always causes acute self-limited hepatitis, although the role for virus-specific CD8 T cells in viral containment is unclear. Herein, we analyzed the T cell response in patients with acute hepatitis by utilizing a set of overlapping peptides and predicted HLA-A2 binders from the polyprotein. A set of 11 predicted peptides from the HAV polyprotein, identified as potential binders, were synthesized. Peripheral blood mononuclear cells (PBMCs) from patients were tested for IFNγ secretion after stimulation with these peptides and ex vivo with HLA-A2 tetramers. Phenotyping was carried out by staining with the activation marker CD38 and the memory marker CD127. Eight out of 11 predicted HLA-A2 binders showed a high binding affinity and five of them were recognized by CD8+ T cells from patients with hepatitis A. There were significant differences in the magnitude of the responses to these five peptides. One was reproducibly immunodominant and the only one detectable ex vivo by tetramer staining of CD8+ T cells. These cells have an activated phenotype (CD38hi CD127lo) during acute infection. Three additional epitopes were identified in HLA-A2 negative patients, most likely representing epitopes restricted by other HLA-class I-alleles (HLA-A11, B35, B40). Patients with acute hepatitis A have a strong multi-specific T cell response detected by ICS. With the tetramer carrying the dominant HLA-A2 epitope, HAV-specific and activated CD8+ T cells could be detected ex vivo. This first description of the HAV specific CTL-epitopes will allow future studies on strength, breadth, and kinetics of the T-cell response in hepatitis A.

Hepatitis B Virus Splice-Generated Protein Induces T-Cell Responses in HLA-Transgenic Mice and Hepatitis B Virus-Infected Patients

Hepatitis B virus splice-generated protein (HBSP), encoded by a spliced hepatitis B virus RNA, was recently identified in liver biopsy specimens from patients with chronic active hepatitis B. We investigated the possible generation of immunogenic peptides by the processing of this protein in vivo. We identified a panel of potential epitopes in HBSP by using predictive computational algorithms for peptide binding to HLA molecules. We used transgenic mice devoid of murine major histocompatibility complex (MHC) class I molecules and positive for human MHC class I molecules to characterize immune responses specific for HBSP. Two HLA-A2-restricted peptides and one immunodominant HLA-B7-restricted epitope were identified following the immunization of mice with DNA vectors encoding HBSP. Most importantly, a set of overlapping peptides covering the HBSP sequence induced significant HBSP-specific T-cell responses in peripheral blood mononuclear cells from patients with chronic hepatitis B. The response was multispecific, as several epitopes were recognized by CD8(+) and CD4(+) human T cells. This study provides the first evidence that this protein generated in vivo from an alternative reading frame of the hepatitis B virus genome activates T-cell responses in hepatitis B virus-infected patients. Given that hepatitis B is an immune response-mediated disease, the detection of T-cell responses directed against HBSP in patients with chronic hepatitis B suggests a potential role for this protein in liver disease progression.

Impact of the genetic restriction of virus-specific T-cell responses in hepatitis C virus infection

The immunobiology of hepatitis C virus (HCV) is significantly influenced by the host immune response to the virus, especially by virus-specific T-cell responses. Virus-specific T cells are restricted by human leucocyte antigen class I and II molecules. Of note, associations between these polymorphic loci and outcome and course of HCV infection have been reported in large and well-documented cohorts. This review will briefly summarize these studies and focus especially on the immunological and virological basis for the reported associations. The outcome and course of HCV infection is most likely determined by a complex interplay of genetic, immunological and virological factors. A better understanding of these host-virus interactions is essential not only to gain better insights into the mechanisms of viral clearance and persistence but also for the development of new antiviral vaccine strategies.

Interferon-$alpha; for hepatitis C: antiviral or immunotherapy?

Interferon-$alpha; for hepatitis C: antiviral or immunotherapy?

Effects of interferon treatment on the antiviral T-cell response in hepatitis C virus genotype 1b- and genotype 2c-infected patients

The viral genotype may influence the response to interferon (IFN) treatment in chronic hepatitis C virus (HCV) infection. To characterize potential mechanisms responsible for this effect, we assessed whether IFN modulation of HCV-specific T-cell responses differs in patients infected by different genotypes. The T-cell response to HCV core protein was sequentially analyzed before and during IFN treatment in two groups of patients chronically infected with HCV genotype 1b (eight patients) or 2c (eight patients). Overlapping 20 mer peptides corresponding to the amino acid sequence of the prevalent viral population identified in the serum of each patient were used for the analysis of the T-cell proliferative response to avoid possible problems caused by amino acid differences between infecting virus and HCV proteins used in vitro. Recombinant HCV core antigen was used in parallel. The level of viremia was monitored by competitive polymerase chain reaction (PCR). The T-cell response to HCV peptides and recombinant core protein detected throughout the follow-up was significantly more vigorous in genotype 2c- than in genotype 1b-infected patients. This difference was the result of a greater enhancement of the T-cell response caused by IFN treatment in genotype 2c-compared with genotype 1b-infected patients. The different IFN modulatory effect on T cells from genotype 1b- and genotype 2c-infected patients illustrates an aspect of the virus-host interaction, which may contribute toward the explanation of why different genotypes differ in responsiveness to IFN treatment.

Effects of interferon treatment on the antiviral T-cell response in hepatitis C virus genotype 1b- and genotype 2c-infected patients

The viral genotype may influence the response to interferon (IFN) treatment in chronic hepatitis C virus (HCV) infection. To characterize potential mechanisms responsible for this effect, we assessed whether IFN modulation of HCV-specific T-cell responses differs in patients infected by different genotypes. The T-cell response to HCV core protein was sequentially analyzed before and during IFN treatment in two groups of patients chronically infected with HCV genotype 1b (eight patients) or 2c (eight patients). Overlapping 20 mer peptides corresponding to the amino acid sequence of the prevalent viral population identified in the serum of each patient were used for the analysis of the T-cell proliferative response to avoid possible problems caused by amino acid differences between infecting virus and HCV proteins used in vitro. Recombinant HCV core antigen was used in parallel. The level of viremia was monitored by competitive polymerase chain reaction (PCR). The T-cell response to HCV peptides and recombinant core protein detected throughout the follow-up was significantly more vigorous in genotype 2c- than in genotype 1b-infected patients. This difference was the result of a greater enhancement of the T-cell response caused by IFN treatment in genotype 2c-compared with genotype 1b-infected patients. The different IFN modulatory effect on T cells from genotype 1b- and genotype 2c-infected patients illustrates an aspect of the virus-host interaction, which may contribute toward the explanation of why different genotypes differ in responsiveness to IFN treatment. (Hepatology 1997 Sep;26(3):792-7)