RESUMO
We compared the kinetics and magnitude of hepatitis B virus (HBV) infection in hepatitis C virus (HCV)-naive and chronically HCV-infected chimpanzees in whose livers type I interferon-stimulated gene (ISG) expression is strongly induced. HBV infection was delayed and attenuated in the HCV-infected animals, and the number of HBV-infected hepatocytes was drastically reduced. These results suggest that establishment of HBV infection and its replication space is limited by the antiviral effects of type I interferon in the chronically HCV-infected liver.
Assuntos
Coinfecção/imunologia , Hepacivirus/fisiologia , Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Hepatite C Crônica/imunologia , Interferon Tipo I/imunologia , Replicação Viral , Animais , Coinfecção/virologia , Modelos Animais de Doenças , Hepacivirus/crescimento & desenvolvimento , Hepacivirus/imunologia , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/imunologia , Hepatócitos/virologia , Interferon Tipo I/biossíntese , Fígado/virologia , Pan troglodytesRESUMO
We previously reported that exosomal transfer of hepatitis C virus (HCV) positive-strand RNA from human Huh-7 hepatoma cells to human plasmacytoid dendritic cells (pDCs) triggers pDC alpha/beta interferon (IFN-α/ß) production in a Toll-like receptor 7 (TLR7)-dependent, virus-independent manner. Here we show that human pDCs are also activated by a TLR7-dependent, virus-independent, exosomal RNA transfer mechanism by human and mouse hepatoma and nonhepatoma cells that replicate the negative-strand lymphocytic choriomeningitis virus (LCMV).
Assuntos
Células Dendríticas/virologia , Vírus da Coriomeningite Linfocítica/isolamento & purificação , Células Dendríticas/citologia , Humanos , Técnicas In VitroRESUMO
It is widely believed that the hepatitis B virus (HBV) is completely cleared by antiviral antibodies and specific cytotoxic T lymphocytes (CTLs) during acute viral hepatitis. We now demonstrate that traces of HBV are often detectable in the blood for many years after clinical recovery from acute hepatitis, despite the presence of serum antibodies and HBV-specific CTLs, which can be present at acute-stage levels. The strength of the CTL response to HBV following clinical recovery correlates with persistence of HBV DNA. It is of particular interest that HBV-specific CTLs from patients studied up to 23 years after clinical and serological recovery expressed activation markers (HLA-DR, CD69) indicating recent contact with antigen. These results suggest that sterilizing immunity to HBV frequently fails to occur after recovery from acute hepatitis and that traces of virus can maintain the CTL response for decades following clinical recovery, apparently creating a negative feedback loop that keeps the virus under control, perhaps for life.
Assuntos
Vírus da Hepatite B/isolamento & purificação , Hepatite B/virologia , Linfócitos T Citotóxicos/imunologia , Doença Aguda , Sequência de Aminoácidos , Convalescença , Seguimentos , Antígeno HLA-A2/análise , Hepatite B/imunologia , Anticorpos Anti-Hepatite B/sangue , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Humanos , Imunidade Celular/efeitos dos fármacos , Interleucina-2/farmacologia , Dados de Sequência Molecular , Proteínas Recombinantes/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacosRESUMO
Rosette inhibitory factor, RIF, previously described in serum from patients with hepatitis B virus infection, has been isolated and identified as a minor species of beta-lipoprotein of the low-density (LDL) class. It is unrelated to hepatitis B virus proteins or particles. Although discrete by reference to charge and density (1.050 +/- 0.004 g/cm3), RIF appears to be a complex macromolecular structure containing apolipoproteins A, B, and C. Greater than 400% recovery is achieved upon 300,000-fold purification from RIF+ sera suggesting activation of a precursor form that is not present in normal serum. RIF inhibits E rosette function of T lymphocytes in vitro with a lag period of approximately 4 h and maximal effect at 24 h consistent with a metabolically-induced event. RIF is functionally active at concentrations of 1 X 10(-12) M or greater, rapidly binds to lymphocytes, and has a functionally effective half-life of approximately 1.5 h. Approximately 2,900 receptors for RIF appear to be present per cell and a high mutual affinity is apparent (k approximately to 9 X 10(10) liters/mol). RIF has no detectable effect on mitogen (PHA) responsiveness of lymphocytes, but inhibits the capacity of lymphocytes to respond to histoincompatible cells in vitro at concentrations greater than 10(-8) M. Equivalent RIF- lipoprotein fractions from normal serum are equally inhibitory in the mixed lymphocyte reaction suggesting that this effect is not directly attributable to RIF activity. These data indicate that RIF is a unique and functionally specific species of LDL that represents either an association complex of lipoproteins or a hybrid molecule of unusual composition. The association of this factor with viral-induced hepatocellular injury underscores the need to elucidate its structure and function in greater detail.
Assuntos
Lipoproteínas LDL/sangue , Linfócitos T/imunologia , Adulto , Cromatografia , DNA/biossíntese , Eletroforese , Eritrócitos/imunologia , Hepatite A/sangue , Humanos , Reação de Imunoaderência , Imunoeletroforese , Lipoproteínas LDL/análise , Lipoproteínas LDL/farmacologia , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , MasculinoRESUMO
The capacity of normal human T lymphocytes to form rosettes with sheep red blood cells can be inhibited by drugs or agents which induce elevations in intracellular levels of cyclic AMP. The effect is early in the presence of agents which elicit rapid elevations in intracellular cyclic AMP (isoproterenol, aminophylline) and occurs later in the presence of cholera toxin which induces a dalayed increase in endogenous cyclic AMP. Dibutyryl cyclic AMP is inhibitory, and the effects of dibutyryl cyclic AMP and the adenyl cyclase stimulators are potentiated by inhibition of phosphodiesterase. These data provide substantial evidence that elevation of intracellular cyclic AMP diminishes E rosette function of lymphocytes.
Assuntos
Eritrócitos/imunologia , Linfócitos T/imunologia , Aminofilina/farmacologia , Animais , Bucladesina/farmacologia , Enterotoxinas/farmacologia , Humanos , Reação de Imunoaderência , Isoproterenol/farmacologia , Ovinos/imunologia , Linfócitos T/efeitos dos fármacos , Fatores de TempoRESUMO
We have examined T cell recognition of a hepatitis B surface antigen (HBsAg), pre-S(2)-region synthetic peptide, p120-145, in terms of fine specificity, H-2-linked genetic influences, comparison to antibody binding, and relevance to T cell recognition of the native protein. We showed that the immune response to the synthetic peptide is regulated by H-2-linked genes, but that the pattern of H-2 restriction differed from that observed for the native anti-pre-S(2) response. Dominant and nonoverlapping T cell and B cell recognition sites were identified on the synthetic peptide p120-145. T cell recognition is focussed on the NH2-terminal sequence, and antibody (B cell) recognition is focussed on the COOH-terminal sequence. The fine specificity of T cell recognition of p120-145 was defined by a single, subtype-dependent amino acid substitution. With respect to the immunogenicity of p120-145, the synthetic peptide containing both T and B cell determinants is highly immunogenic in responder strains, whereas separate T or B cell peptide determinants are minimally immunogenic. Furthermore, the synthetic T cell recognition site can prime T cell help for antibody production to the synthetic B cell site, which is crossreactive with the native pre-S(2) region of HBsAg/p33 particles. This system provides evidence that totally synthetic T cell and B cell recognition sites can be combined to yield a functional immunogen.
Assuntos
Linfócitos B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/biossíntese , Reações Cruzadas , Epitopos/imunologia , Feminino , Antígenos H-2/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BLRESUMO
We have previously reported that hepatitis B virus (HBV)-specific CD8(+) cytotoxic T lymphocytes and CD4(+) helper T lymphocytes can inhibit HBV replication in the liver of HBV transgenic mice by secreting interferon (IFN)-gamma when they recognize viral antigen. To determine whether an activated innate immune system can also inhibit HBV replication, in this study we activated natural killer T (NKT) cells in the liver of HBV transgenic mice by a single injection of alpha-galactosylceramide (alpha-GalCer), a glycolipid antigen presented to Valpha14(+)NK1.1(+) T cells by the nonclassical major histocompatibility complex class I-like molecule CD1d. Within 24 h of alpha-GalCer injection, IFN-gamma and IFN-alpha/beta were detected in the liver of HBV transgenic mice and HBV replication was abolished. Both of these events were temporally associated with the rapid disappearance of NKT cells from the liver, presumably reflecting activation-induced cell death, and by the recruitment of activated NK cells into the organ. In addition, prior antibody-mediated depletion of CD4(+) and CD8(+) T cells from the mice did not diminish the ability of alpha-GalCer to trigger the disappearance of HBV from the liver, indicating that conventional T cells were not downstream mediators of this effect. Finally, the antiviral effect of alpha-GalCer was inhibited in mice that are genetically deficient for either IFN-gamma or the IFN-alpha/beta receptor, indicating that most of the antiviral activity of alpha-GalCer is mediated by these cytokines. Based on these results, we conclude that alpha-GalCer inhibits HBV replication by directly activating NKT cells and by secondarily activating NK cells to secrete antiviral cytokines in the liver. In view of these findings, we suggest that, if activated, the innate immune response, like the adaptive immune response, has the potential to control viral replication during natural HBV infection. In addition, the data suggest that therapeutic activation of NKT cells may represent a new strategy for the treatment of chronic HBV infection.
Assuntos
Antivirais/imunologia , Galactosilceramidas/imunologia , Vírus da Hepatite B/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Replicação Viral/imunologia , Animais , Antivirais/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Relação Dose-Resposta a Droga , Feminino , Galactosilceramidas/administração & dosagem , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Tempo , Replicação Viral/efeitos dos fármacosRESUMO
Although coinfection of hepatitis B virus (HBV) and Schistosoma mansoni is a frequent event in humans, little is known about the interactions between these two pathogens. S. mansoni infection induces T helper cell type 2 (Th2)-type cytokines in the liver of humans and mice. The intrahepatic induction of nitric oxide (NO) and Th1-type cytokines, such as interferon (IFN)-gamma and IFN-alpha/beta, inhibits HBV replication noncytopathically in the liver of transgenic mice. To examine whether S. mansoni infection and the accompanying induction of Th2-type cytokines could interfere with HBV replication in the liver, HBV transgenic mice were infected with S. mansoni. By 5 wk after infection, HBV replication disappeared concomitant with the intrahepatic induction of NO and Th1-type cytokines, and in the absence of Th2-type cytokines. By 6-8 wk after infection, HBV replication remained undetectable and this was associated with further induction of NO and Th1-type cytokines together with the appearance of Th2-type cytokines. The S. mansoni-dependent antiviral effect was partially blocked by genetically deleting IFN-gamma, although it was unaffected by deletion of IFN-alpha/beta. These results indicate that IFN-gamma (probably via NO) mediates most of this antiviral activity and that Th2-type cytokines do not counteract the antiviral effect of IFN-gamma. Similar events may suppress HBV replication during human S. mansoni infection.
Assuntos
Vírus da Hepatite B/fisiologia , Esquistossomose mansoni/virologia , Replicação Viral , Animais , Interferon gama/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico/fisiologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo IIRESUMO
We have previously identified two antiviral cytokines (interferon [IFN]-gamma and IFN-alpha/beta) that downregulate hepatitis B virus (HBV) replication in the liver of transgenic mice. The cytokine-inducible downstream events that inhibit HBV replication have not been identified. One possible factor is nitric oxide (NO), a pleiotropic free radical with antiviral activity that is produced in the liver by the inducible NO synthase (iNOS). To examine the role of NO in our model, we crossed transgenic mice that replicate HBV with mice that lack a functional iNOS. Importantly, iNOS-deficient mice were almost completely resistant to the noncytopathic inhibitory effect of HBV-specific cytotoxic T lymphocytes on viral replication, an effect that we have shown previously to depend on the intrahepatic induction of IFN-gamma. Conversely, iNOS-deficient mice were not resistant to the antiviral effect of IFN-alpha/beta induced by either polyinosinic-polycytidylic acid complex or by lymphocytic choriomeningitis virus (LCMV) infection. These results indicate that NO mediates the antiviral activity of IFN-gamma, whereas the antiviral activity of IFN-alpha/beta is NO independent. We also compared the relative sensitivity of LCMV to control by NO in these animals. Interestingly, LCMV replicated to higher levels in the liver of iNOS-deficient mice than control mice, indicating that NO controls LCMV replication in the liver, as well as HBV.
Assuntos
Vírus da Hepatite B/imunologia , Fígado/virologia , Óxido Nítrico/imunologia , Replicação Viral/imunologia , Animais , Feminino , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/fisiologia , Indutores de Interferon/imunologia , Interferon-alfa/imunologia , Interferon beta/imunologia , Interferon gama/imunologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/imunologia , Óxido Nítrico Sintase Tipo II , Poli I-C/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
We have previously shown that hepatitis B virus (HBV) replication is abolished in the liver of HBV transgenic mice by inflammatory cytokines induced by HBV-specific cytotoxic T cells and during unrelated viral infections of the liver. We now report that intrahepatic HBV replication is also inhibited in mice infected by the malaria species Plasmodium yoelii 17X NL. P. yoelii infection triggers an intrahepatic inflammatory response characterized by the influx of natural killer cells, macrophages, and T cells. During this process, interferon (IFN)-gamma and IFN-alpha/beta suppress HBV gene expression and replication in the liver. Collectively, the data suggest that malaria infection might influence the course and pathogenesis of HBV infection in coinfected humans.
Assuntos
Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Interferons/imunologia , Fígado/imunologia , Malária/imunologia , Plasmodium yoelii/fisiologia , Alanina Transaminase/sangue , Animais , Northern Blotting , Citocinas/biossíntese , Citocinas/genética , Citocinas/imunologia , Hepatite B/complicações , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Humanos , Interferons/biossíntese , Interferons/genética , Fígado/parasitologia , Fígado/patologia , Fígado/virologia , Macrófagos/imunologia , Malária/complicações , Malária/parasitologia , Camundongos , Camundongos Transgênicos , Linfócitos T/imunologia , Replicação ViralRESUMO
The virological and immunological features of hepatitis C virus (HCV) infection were studied weekly for 6 months after accidental needlestick exposure in five health care workers, four of whom developed acute hepatitis that progressed to chronicity while one subject cleared the virus. In all subjects, viremia was first detectable within 1-2 weeks of inoculation, 1 month or more before the appearance of virus-specific T cells. The subject who cleared the virus experienced a prolonged episode of acute hepatitis that coincided with a CD38+ IFN-gamma- CD8+ T cell response to HCV and a small reduction in viremia. Subsequently, a strong CD4+ T cell response emerged and the CD8+ T cells became CD38- and started producing IFN-gamma in response to HCV, coinciding with a rapid 100,000-fold decrease in viremia that occurred without a corresponding surge of disease activity. Chronic infection developed in two subjects who failed to produce a significant T cell response and in two other subjects who initially mounted strong CD4+ T cell responses that ultimately waned. In all subjects, viremia was higher at the peak of acute hepatitis than it was when the disease began, and the disease improved during the viremia. These results provide the first insight into the host-virus relationship in humans during the incubation phase of acute HCV infection, and they provide the only insight to date into the virological and immunological characteristics of clinically asymptomatic acute HCV infection, the commonest manifestation of this disease. In addition, the results suggest that the vigor and quality of the antiviral T cell response determines the outcome of acute HCV infection, that the ability of HCV to outpace the T cell response may contribute to its tendency to persist; that the onset of hepatitis coincides with the onset of the CD8+ T cell response, that disease pathogenesis and viral clearance are mediated by different CD8+ T cell populations that control HCV by both cytolytic and noncytolytic mechanisms, and that there are different pathways to viral persistence in asymptomatic and symptomatic acute HCV infection.
Assuntos
Hepatite C/imunologia , Doença Aguda , Adulto , Alanina Transaminase/sangue , Linfócitos T CD4-Positivos/imunologia , Feminino , Hepatite C/virologia , Humanos , Interferon gama/fisiologia , Masculino , Pessoa de Meia-Idade , RNA Viral/análiseRESUMO
We have previously shown that interferon and tumor necrosis factor noncytopathically abolish hepatitis B virus (HBV) replication from the hepatocyte and kidney tubular epithelial cells in vivo. Here we show that a persistent lymphocytic choriomeningitis virus (LCMV) infection is cleared from the hepatocyte noncytopathically when the same cytokines are induced in the liver by antigen-nonspecific stimuli. These results indicate that, like HBV, LCMV is also susceptible to intracellular inactivation by cytokine-induced antiviral mechanisms that are operative in the hepatocyte. In contrast, LCMV is not cleared from intrahepatic nonparenchymal cells or splenocytes, indicating that, unlike the hepatocyte, these cells do not produce the factors required to inactivate LCMV. Antiviral mechanisms like these may have evolved to maintain the functional integrity of vital organs in the face of massive infection.
Assuntos
Citocinas/metabolismo , Fígado/virologia , Coriomeningite Linfocítica/metabolismo , Vírus da Coriomeningite Linfocítica , Adenoviridae , Alanina Transaminase/sangue , Animais , Antígenos CD/análise , Expressão Gênica , Interferons/metabolismo , Interleucinas/metabolismo , Túbulos Renais/virologia , Fígado/patologia , Coriomeningite Linfocítica/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , RNA Viral/análise , Baço/virologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Virais/análiseRESUMO
Hepatocellular carcinoma (HCC) is a common complication of chronic hepatitis B virus (HBV) infection. The pathogenetic mechanisms potentially responsible for HCC during chronic HBV infection are not well defined. This study demonstrates that chronic immune-mediated liver cell injury triggers the development of HCC in the absence of viral transactivation, insertional mutagenesis, and genotoxic chemicals. These results strongly suggest that the immune response to HBV is both necessary and sufficient to cause liver cancer during chronic HBV infection, and that all other procarcinogenic events associated with HCC are probably dependent on this process.
Assuntos
Carcinoma Hepatocelular/imunologia , Vírus da Hepatite B , Hepatite B Crônica/imunologia , Neoplasias Hepáticas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Hepatite B Crônica/patologia , Fígado/imunologia , Fígado/patologia , Masculino , Camundongos , Camundongos Transgênicos , TimectomiaRESUMO
We have previously demonstrated that the murine humoral immune responses to the group-specific a and subtype-specific d/y determinants of hepatitis B surface antigen (HBsAg) are controlled by H-2-linked immune response (Ir) genes. High responder (H-2d,q), intermediate responder (H-2a greater than b greater than k) and nonresponder (H-2f,s) haplotypes have been identified (8, 9). The kinetics and specificity of in vivo antibody production after HBsAg immunization in congeneic, H-2-recombinant strains was analyzed to further define relevant Ir genes and their influence on the immune response to distinct antigenic determinants. These studies indicate that the humoral anti-HBs response is regulated by at least two Ir genes, one in the I-A subregion (Ir-HBs-1) and one in the I-C subregion (Ir-HBs-2) of the murine H-2 complex. Ir-HBs-1 regulates the primary responses to all HBsAg determinants, whereas the influence of Ir-HBs-2 is determinant specific, affecting the responses to the d or y determinants. The anti-a response is regulated exclusively by Ir-HBs-1. Strains possessing only the Ir-HBs-2 gene [B10.S(9R) and B10.HTT] produce no anti-a response and a subtype-specific antibody response is detected only after secondary or tertiary immunization. In contrast, the influence of Ir-HBs-2 in the presence of Ir-HBs-1 is detected upon primary immunization and is additive rather than exclusive. There is also suggestive evidence that the presence of the Ek molecule, at least in the context of I-Ak, may have a suppressive influence on the anti-HBs response. Additionally, HBsAg-specific, T cell proliferative responses were H-2 restricted and the kinetics and specificity of T cell proliferative responses paralleled in vivo antibody production. These data indicate that, although the I-A subregion exerts a dominant influence, distinct Ir-HBs genes, mapping in separate I subregions, control immune responses to alternate HBsAg determinants on the same protein molecule.
Assuntos
Genes MHC da Classe II , Antígenos H-2/genética , Anticorpos Anti-Hepatite B/biossíntese , Antígenos de Superfície da Hepatite B/genética , Animais , Especificidade de Anticorpos , Mapeamento Cromossômico , Epitopos/genética , Feminino , Antígenos H-2/imunologia , Anticorpos Anti-Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Cinética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Recombinação Genética , Especificidade da Espécie , Linfócitos T/imunologiaRESUMO
The absence of readily manipulable experimental systems to study the cytotoxic T lymphocyte (CTL) response against hepatitis B virus (HBV) antigens has thus far precluded a definitive demonstration of the role played by this response in the pathogenesis of liver cell injury and viral clearance during HBV infection. To circumvent the problem that HBV infection of human cells in vitro for production of stimulator/target systems for CTL analysis is not feasible, a panel of 22 overlapping synthetic peptides covering the entire amino acid sequence of the HBV core (HBcAg) and e (HBeAg) antigens were used to induce and to analyze the HBV nucleocapsid-specific CTL response in nine patients with acute hepatitis B, six patients with chronic active hepatitis B, and eight normal controls. By using this approach, we have identified an HLA-A2-restricted CTL epitope, located within the NH2-terminal region of the HBV core molecule, which is shared with the e antigen and is readily recognized by peripheral blood mononuclear cells from patients with self-limited acute hepatitis B but less efficiently in chronic HBV infection. Our study provides the first direct evidence of HLA class I-restricted T cell cytotoxicity against HBV in humans. Furthermore, the different response in HBV-infected subjects who successfully clear the virus (acute patients) in comparison with patients who do not succeed (chronic patients) suggests a pathogenetic role for this CTL activity in the clearance of HBV infection.
Assuntos
Epitopos/análise , Antígeno HLA-A2/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos E da Hepatite B/imunologia , Linfócitos T Citotóxicos/imunologia , Alanina Transaminase/análise , Hepatite B/imunologia , HumanosRESUMO
The molecular and cellular mechanisms responsible for cytotoxic T lymphocyte (CTL)-induced immunopathology are not well defined. Using a model in which hepatitis B surface antigen (HBsAg)-specific CTL cause an acute necroinflammatory liver disease in HBsAg transgenic mice, we demonstrate that class I-restricted disease pathogenesis is an orderly, multistep process that involves direct as well as indirect consequences of CTL activation. It begins (step 1) almost immediately as a direct antigen-specific CTL-target cell interaction that triggers the HBsAg-positive hepatocyte to undergo programmed cell death (apoptosis). It progresses (step 2) within hours to a focal inflammatory response in which antigen-nonspecific lymphocytes and neutrophils amplify the local cytopathic effect of the CTL. The most destructive pathogenetic function of the CTL, however, is to secrete interferon gamma when they encounter antigen in vivo, thereby activating the intrahepatic macrophage and inducing a delayed-type hypersensitivity response (step 3) that destroys the liver and kills the mouse. We propose that the principles illustrated in this study are generally applicable to other models of class I-restricted, CTL-induced immunopathology, and we suggest that they contribute to the immunopathogenesis of viral hepatitis during hepatitis B virus infection in humans.
Assuntos
Encefalopatia Hepática/imunologia , Encefalopatia Hepática/patologia , Antígenos de Superfície da Hepatite B/biossíntese , Antígenos de Histocompatibilidade Classe I/imunologia , Fígado/patologia , Linfócitos T Citotóxicos/imunologia , Animais , Complexo CD3/análise , Antígenos CD4/análise , Antígenos CD8/análise , Antígenos de Superfície da Hepatite B/análise , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/genética , Técnicas Imunoenzimáticas , Imunoterapia Adotiva , Inflamação , Interferon gama/fisiologia , Fígado/imunologia , Masculino , Metalotioneína/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Necrose , Regiões Promotoras Genéticas , Subpopulações de Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
We have recently developed the technology to identify and characterize the human histocompatibility leukocyte antigen (HLA) class I-restricted, CD8+ cytotoxic T lymphocyte (CTL) response to hepatitis B virus (HBV)-encoded antigens in patients with acute viral hepatitis. CTL are expanded in vitro by stimulation with HBV-derived synthetic peptides and selected by restimulation with a panel of HLA-matched stable transfectants that express the corresponding HBV protein. We have recently reported the existence of an HLA-A2-restricted, CD8+ CTL response to an epitope located between residues 18 and 27 of the HBV nucleocapsid core antigen (HBcAg). We now report the discovery of a CTL epitope located between HBcAg residues 141 and 151 that completely overlaps a critical domain in the viral nucleocapsid protein that is essential for its nuclear localization and genome packaging functions as well as processing of the precore protein. The CTL response to this epitope is dually restricted by the HLA-A31 and HLA-Aw68 alleles, which, unexpectedly, appear to use a common binding motif based on the results of alanine substitution and competition analysis, and the binding properties of these two alleles predicted from their known primary sequence, and from the three-dimensional structure of HLA-Aw68. We have also demonstrated that the HBV-specific CTL response to this epitope is polyclonal during acute viral hepatitis, since these two restriction elements can present the HBcAg 141-151 epitope to independent CTL clones derived from a single patient; and that the CTL response is multispecific, since HLA-A2-restricted and HLA-Aw68-restricted CTL responses to HBcAg 18-27 and HBcAg 141-151, respectively, have been identified to coexist in another patient. The foregoing argue against the emergence of CTL escape mutants as a significant problem during HBV infection, especially at this locus, where mutations might be incompatible with viral replication. Finally, our data suggest an association between the HBV-specific CTL response and viral clearance, and they have implications for the design of immunotherapeutic strategies to terminate HBV infection in chronically infected patients.
Assuntos
Capsídeo/imunologia , Epitopos/imunologia , Antígenos HLA-A/análise , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas do Core Viral/imunologia , Doença Aguda , Alelos , Sequência de Aminoácidos , Linhagem Celular , Feminino , Antígenos HLA-A/genética , Antígenos da Hepatite B/imunologia , Antígenos da Hepatite B/farmacologia , Antígenos do Núcleo do Vírus da Hepatite B/análise , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/farmacologia , Humanos , Masculino , Dados de Sequência Molecular , Linfócitos T Citotóxicos/patologia , TransfecçãoRESUMO
Mutations that abrogate recognition of a viral epitope by class I-restricted cytotoxic T lymphocyte (CTL) can lead to viral escape if the CTL response against that epitope is crucial for viral clearance. The likelihood of this type of event is low when the CTL response is simultaneously directed against multiple viral epitopes, as has been recently reported for patients with acute self-limited hepatitis B virus (HBV) infection. The CTL response to HBV is usually quite weak, however, during chronic HBV infection, and it is generally acknowledged that this is a major determinant of viral persistence in this disease. If such individuals were to produce a mono- or oligospecific CTL response, however, negative selection of the corresponding mutant viruses might occur. We have recently studied two HLA-A2-positive patients with chronic hepatitis B who, atypically, developed a strong HLA-A2-restricted CTL response against an epitope (FLPSDFFPSV) that contains an HLA-A2-binding motif located between residues 18-27 of the viral nucleocapsid protein, hepatitis B core antigen (HBcAg). These patients failed, however, to respond to any of other HLA-A2-restricted HBV-derived peptides that are generally immunogenic in acutely infected patients who successfully clear the virus. Interestingly, DNA sequence analysis of HBV isolates from these two patients demonstrated alternative residues at position 27 (V --> A and V --> I) and position 21 (S --> N, S --> A, and S --> V) that reduced the HLA and T cell receptor-binding capacities of the variant sequences, respectively. Synthetic peptides containing these alternative sequences were poorly immunogenic compared to the prototype HBc18-27 sequence, and they could not be recognized by CTL clones specific for the prototype peptide. While we do not know if the two patients were originally infected by these variant viruses or if the variants emerged subsequent to infection because of immune selection, the results are most consistent with the latter hypothesis. If this is correct, the data suggest that negative selection of mutant viral genomes might contribute to viral persistence in a subset of patients with chronic HBV infection who express a narrow repertoire of anti-HBV CTL responses.
Assuntos
Epitopos , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Linfócitos T Citotóxicos/imunologia , Doença Aguda , Sequência de Aminoácidos , Sequência de Bases , Doença Crônica , Antígeno HLA-A2/imunologia , Humanos , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
Using transgenic mice that replicate hepatitis B virus (HBV) at high levels in the liver as recipients of HBV-specific cytotoxic T lymphocytes (CTLs), we showed that the chemokines responsive to gamma-2/IFN-gamma inducible protein ([Crg2]IP-10) and monokine induced by interferon-gamma (Mig) are rapidly and strongly induced in the liver after CTL transfer. The transferred CTLs produce neither chemokine; rather, they activate (via the secretion of IFN-gamma) hepatocytes and nonparenchymal cells of the liver to produce (Crg2)IP-10 and Mig. Importantly, blocking these chemokines in vivo reduces the recruitment of host-derived lymphomononuclear cells into the liver and the severity of the liver disease without affecting the IFN-gamma-dependent antiviral potential of the CTLs. The finding that neutralization of these chemokines is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.
Assuntos
Citotoxicidade Imunológica , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Interferon gama/imunologia , Monocinas/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Quimiocina CXCL10 , Hepatite B/genética , Hepatite B/patologia , Interferon gama/genética , Fígado/imunologia , Fígado/patologia , Fígado/virologia , Camundongos , Camundongos Transgênicos , Monocinas/genéticaRESUMO
Cytotoxic T lymphocytes (CTL) are thought to contribute to viral clearance and liver cell injury during hepatitis B virus (HBV) infection. Using a strategy involving the in vitro stimulation of peripheral blood mononuclear cells (PBMC) with HBV-derived synthetic peptides containing HLA-A2.1, -A31, and -Aw68 binding motifs, we have previously described CTL responses to several epitopes within the HBV nucleocapsid and envelope antigens in patients with acute hepatitis. In this study we define six HLA-A2-restricted CTL epitopes located in the highly conserved reverse transcriptase and RNase H domains of the viral polymerase protein, and we show that the CTL response to polymerase is polyclonal, multispecific, and mediated by CD8+ T cells in patients with acute viral hepatitis, but that it is not detectable in patients with chronic HBV infection or uninfected healthy blood donors. Importantly, the peptide-activated CTL recognize target cells that express endogenously synthesized polymerase protein, suggesting that these peptides represent naturally processed viral epitopes. DNA sequence analysis of the viruses in patients who did not respond to peptide stimulation indicated that CTL nonresponsiveness was not due to infection by viral variants that differed in sequences from the synthetic peptides. CTL specific for one of the epitopes were unable to recognize several naturally occurring viral variants, except at high peptide concentration, underlining the HBV subtype specificity of this response. Furthermore, CTL responses against polymerase, core, and envelope epitopes were detectable for more than a year after complete clinical recovery and seroconversion, reflecting either the persistence of trace amounts of virus or the presence of long lived memory CTL in the absence of viral antigen. Finally, we demonstrated that wild type viral DNA and RNA can persist indefinitely, in trace quantities, in the serum and PBMC after complete clinical and serological recovery, despite a concomitant, vigorous, and sustained polyclonal CTL response. Since viral persistence is not due to escape from CTL recognition under these conditions, the data suggest that HBV may retreat into immunologically privileged sites from which it can seed the circulation and reach CTL-inaccessible tissues, thereby maintaining the CTL response in apparently cured individuals and, perhaps, prolonging the liver disease in patients with chronic hepatitis.