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Gastroenterology ; 156(6): 1820-1833, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30768983


BACKGROUND & AIMS: Hepatitis D virus (HDV) superinfection in patients with hepatitis B virus (HBV) is associated with rapid progression to liver cirrhosis and hepatocellular carcinoma. Treatment options are limited, and no vaccine is available. Although HDV-specific CD8+ T cells are thought to control the virus, little is known about which HDV epitopes are targeted by virus-specific CD8+ T cells or why these cells ultimately fail to control the infection. We aimed to define how HDV escapes the CD8+ T-cell-mediated response. METHODS: We collected plasma and DNA samples from 104 patients with chronic HDV and HBV infection at medical centers in Europe and the Middle East, sequenced HDV, typed human leukocyte antigen (HLA) class I alleles from patients, and searched for polymorphisms in HDV RNA associated with specific HLA class I alleles. We predicted epitopes in HDV that would be recognized by CD8+ T cells and corresponded with the identified virus polymorphisms in patients with resolved (n = 12) or chronic (n = 13) HDV infection. RESULTS: We identified 21 polymorphisms in HDV that were significantly associated with specific HLA class I alleles (P < .005). Five of these polymorphisms were found to correspond to epitopes in HDV that are recognized by CD8+ T cells; we confirmed that CD8+ T cells in culture targeted these HDV epitopes. HDV variant peptides were only partially cross-recognized by CD8+ T cells isolated from patients, indicating that the virus had escaped detection by these cells. These newly identified HDV epitopes were restricted by relatively infrequent HLA class I alleles, and they bound most frequently to HLA-B. In contrast, frequent HLA class I alleles were not associated with HDV sequence polymorphisms. CONCLUSIONS: We analyzed sequences of HDV RNA and HLA class I alleles that present epitope peptides to CD8+ T cells in patients with persistent HDV infection. We identified polymorphisms in the HDV proteome that associate with HLA class I alleles. Some variant peptides in epitopes from HDV were only partially recognized by CD8+ T cells isolated from patients; these could be mutations that allow HDV to escape the immune response, resulting in persistent infection. HDV escape from the immune response was associated with uncommon HLA class I alleles, indicating that HDV evolves, at the population level, to evade recognition by common HLA class I alleles.

Linfócitos T CD8-Positivos/imunologia , Antígenos HLA-A/genética , Antígenos HLA-B/genética , Hepatite B Crônica/genética , Hepatite D Crônica/genética , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/imunologia , Vigilância Imunológica/imunologia , Superinfecção/genética , Alelos , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Epitopos de Linfócito T/imunologia , Evolução Molecular , Antígenos HLA-A/imunologia , Antígenos HLA-B/imunologia , Humanos , Tolerância Imunológica , Interferon gama/metabolismo , Mutação , Polimorfismo Genético
Gut ; 68(5): 905-915, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30622109


OBJECTIVE: A hallmark of chronic HBV (cHBV) infection is the presence of impaired HBV-specific CD8+ T cell responses. Functional T cell exhaustion induced by persistent antigen stimulation is considered a major mechanism underlying this impairment. However, due to their low frequencies in chronic infection, it is currently unknown whether HBV-specific CD8+ T cells targeting different epitopes are similarly impaired and share molecular profiles indicative of T cell exhaustion. DESIGN: By applying peptide-loaded MHC I tetramer-based enrichment, we could detect HBV-specific CD8+ T cells targeting epitopes in the HBV core and the polymerase proteins in the majority of 85 tested cHBV patients with low viral loads. Lower detection rates were obtained for envelope-specific CD8+ T cells. Subsequently, we performed phenotypic and functional in-depth analyses. RESULTS: HBV-specific CD8+ T cells are not terminally exhausted but rather exhibit a memory-like phenotype in patients with low viral load possibly reflecting weak ongoing cognate antigen recognition. Moreover, HBV-specific CD8+ T cells targeting core versus polymerase epitopes significantly differed in frequency, phenotype and function. In particular, in comparison with core-specific CD8+ T cells, a higher frequency of polymerase-specific CD8+ T cells expressed CD38, KLRG1 and Eomes accompanied by low T-bet expression and downregulated CD127 indicative of a more severe T cell exhaustion. In addition, polymerase-specific CD8+ T cells exhibited a reduced expansion capacity that was linked to a dysbalanced TCF1/BCL2 expression. CONCLUSIONS: Overall, the molecular mechanisms underlying impaired T cell responses differ with respect to the targeted HBV antigens. These results have potential implications for immunotherapeutic approaches in HBV cure.

Linfócitos T CD8-Positivos/fisiologia , Produtos do Gene pol/metabolismo , Vírus da Hepatite B/imunologia , Hepatite B Crônica/metabolismo , Proteínas do Core Viral/metabolismo , Carga Viral , Adulto , Idoso , Estudos de Coortes , Feminino , Hepatite B Crônica/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo
J Virol ; 92(13)2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29669837


Virus-specific CD8 T cell response seems to play a significant role in the outcome of hepatitis delta virus (HDV) infection. However, the HDV-specific T cell epitope repertoire and mechanisms of CD8 T cell failure in HDV infection have been poorly characterized. We therefore aimed to characterize HDV-specific CD8 T cell epitopes and the impacts of viral mutations on immune escape. In this study, we predicted peptide epitopes binding the most frequent human leukocyte antigen (HLA) types and assessed their HLA binding capacities. These epitopes were characterized in HDV-infected patients by intracellular gamma interferon (IFN-γ) staining. Sequence analysis of large hepatitis delta antigen (L-HDAg) and HLA typing were performed in 104 patients. The impacts of substitutions within epitopes on the CD8 T cell response were evaluated experimentally and by in silico studies. We identified two HLA-B*27-restricted CD8 T cell epitopes within L-HDAg. These novel epitopes are located in a relatively conserved region of L-HDAg. However, we detected molecular footprints within the epitopes in HLA-B*27-positive patients with chronic HDV infections. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infections, indicating that the substitutions represent viral escape mutations. Molecular modeling of HLA-B*27 complexes with the L-HDAg epitope and its potential viral escape mutations indicated that the structural and electrostatic properties of the bound peptides differ considerably at the T cell receptor interface, which provides a possible molecular explanation for the escape mechanism. This viral escape from the HLA-B*27-restricted CD8 T cell response correlates with a chronic outcome of hepatitis D infection. T cell failure resulting from immune escape may contribute to the high chronicity rate in HDV infection.IMPORTANCE Hepatitis delta virus (HDV) causes severe chronic hepatitis, which affects 20 million people worldwide. Only a small number of patients are able to clear the virus, possibly mediated by a virus-specific T cell response. Here, we performed a systematic screen to define CD8 epitopes and investigated the role of CD8 T cells in the outcome of hepatitis delta and how they fail to eliminate HDV. Overall the number of epitopes identified was very low compared to other hepatotropic viruses. We identified, two HLA-B*27-restricted epitopes in patients with resolved infections. In HLA-B*27-positive patients with chronic HDV infections, however, we detected escape mutations within these identified epitopes that could lead to viral evasion of immune responses. These findings support evidence showing that HLA-B*27 is important for virus-specific CD8 T cell responses, similar to other viral infections. These results have implications for the clinical prognosis of HDV infection and for vaccine development.

Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Antígenos HLA-B/imunologia , Hepatite D/imunologia , Vírus Delta da Hepatite/imunologia , Antígenos da Hepatite delta/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Linfócitos T CD8-Positivos/metabolismo , Epitopos de Linfócito T/metabolismo , Antígenos HLA-B/genética , Antígenos HLA-B/metabolismo , Hepatite D/genética , Hepatite D/virologia , Vírus Delta da Hepatite/genética , Antígenos da Hepatite delta/metabolismo , Humanos , Mutação , Homologia de Sequência