Comparative Study of Anti-hepatitis B Virus RNA Interference by Double-stranded Adeno-associated Virus Serotypes 7, 8, and 9

Using a hepatitis B virus (HBV) transgenic mouse model, we previously showed that a single dose of double-stranded adeno-associated virus (dsAAV) vector serotype 8 carrying a small hairpin RNA (shRNA) effectively reduces HBV replication and gene expression, but the effect gradually decreases with time. In this report, we compared the anti-HBV RNA interference (RNAi) effect of dsAAV8 with those of dsAAV7 and dsAAV9, two other hepatotropic AAV vectors, and examined whether the sequential use of these heterologous AAV vectors could prolong the anti-HBV effect. Our results showed that shRNA delivered by each of the three dsAAV vectors profoundly reduced the serum HBV titer and liver HBV mRNA and DNA levels in the transgenic mice for up to 22 weeks, with dsAAV8 having the greatest inhibitory effect, followed by dsAAV9 and dsAAV7. The potency of dsAAV8 correlated with the presence of higher levels of vector DNA and anti-HBV shRNA in the liver. An in vivo cross-administration experiment showed that preexisting anti-AAV8 antibody completely blocked the anti-HBV RNAi effect of dsAAV8, but had no effect on the potency of dsAAV7 and dsAAV9. Moreover, we demonstrated that a longer anti-HBV effect could be achieved by the sequential use of dsAAV8 and dsAAV9. These results indicate that effective and persistent HBV suppression might be achieved by a combination of the power of RNAi silencing effect and multiple treatments with different AAV serotypes.     

Hepatorenal Correction in Murine Glycogen Storage Disease Type I With a Double-stranded Adeno-associated Virus Vector

Glycogen storage disease type Ia (GSD-Ia) is caused by the deficiency of glucose-6-phosphatase (G6Pase). Long-term complications of GSD-Ia include life-threatening hypoglycemia and proteinuria progressing to renal failure. A double-stranded (ds) adeno-associated virus serotype 2 (AAV2) vector encoding human G6Pase was pseudotyped with four serotypes, AAV2, AAV7, AAV8, and AAV9, and we evaluated efficacy in 12-day-old G6pase (−/−) mice. Hypoglycemia during fasting (plasma glucose <100 mg/dl) was prevented for >6 months by the dsAAV2/7, dsAAV2/8, and dsAAV2/9 vectors. Prolonged fasting for 8 hours revealed normalization of blood glucose following dsAAV2/9 vector administration at the higher dose. The glycogen content of kidney was reduced by >65% with both the dsAAV2/7 and dsAAV2/9 vectors, and renal glycogen content was stably reduced between 7 and 12 months of age for the dsAAV2/9 vector-treated mice. Every vector-treated group had significantly reduced glycogen content in the liver, in comparison with untreated G6pase (−/−) mice. G6Pase was expressed in many renal epithelial cells of with the dsAAV2/9 vector for up to 12 months. Albuminuria and renal fibrosis were reduced by the dsAAV2/9 vector. Hepatorenal correction in G6pase (−/−) mice demonstrates the potential of AAV vectors for the correction of inherited diseases of metabolism.     

Use of RNA interference to modulate liver adenoma development in a murine model transgenic for hepatitis B virus

Chronic hepatitis B virus (HBV) infection is closely related to the development of severe liver complications, including hepatocellular carcinoma. In previous studies, we reported that in vivo long-term HBV suppression in transgenic mice can be achieved without apparent toxicity by short hairpin RNA sequentially delivered using adeno-associated viral (AAV) vectors of different serotypes. Our goal herein was to address the clinical utility of this delivery system and, in particular, to determine whether RNA interference (RNAi) and its ability to induce long-term HBV suppression will modulate the development of HBV-associated liver pathology. As a model system, we used a unique HBV transgenic mouse model, containing a 1.3 times over length of the HBV genome, on the ICR mouse background. These transgenic mice produce high serum HBV titers comparable with human chronic HBV patients, and, importantly, manifest characteristic HBV-associated pathology, including progressive hepatocellular injury and the development of hepatocellular adenoma. Using this system, we injected animals with AAV vectors expressing either HBV-specific or a control luciferase-specific short hairpin RNA and followed animals for a total of 18 months. We report herein that AAV-mediated RNAi therapy profoundly inhibits HBV replication and gene expression, with a significant reduction in hepatic regeneration, liver enzymes and, importantly, the appearance of liver adenomas. Indeed, the therapeutic effect of RNAi correlated with the reduction in HBV titers. Our data demonstrate that appropriately designed RNAi therapy has the potential to prevent formation of HBV-associated hepatocellular adenoma.     

In vitro characterization of the activity of PF-05095808, a novel biological agent for hepatitis C virus therapy

PF-05095808 is a novel biological agent for chronic hepatitis C virus (HCV) therapy. It comprises a recombinant adeno-associated virus (AAV) DNA vector packaged into an AAV serotype 8 capsid. The vector directs expression of three short hairpin RNAs (shRNAs) targeted to conserved regions of the HCV genome. These shRNAs are processed by the host cell into the small interfering RNAs which mediate sequence-specific cleavage of target regions. For small-molecule inhibitors the key screens needed to assess in vitro activity are well defined; we developed new assays to assess this RNA interference agent and so to understand its therapeutic potential. Following administration of PF-05095808 or corresponding synthetic shRNAs, sequence-specific antiviral activity was observed in HCV replicon and infectious virus systems. To quantify the numbers of shRNA molecules required for antiviral activity in vitro and potentially also in vivo, a universal quantitative PCR (qPCR) assay was developed. The number of shRNA molecules needed to drive antiviral activity proved to be independent of the vector delivery system used for PF-05095808 administration. The emergence of resistant variants at the target site of one shRNA was characterized. A novel RNA cleavage assay was developed to confirm the spectrum of activity of PF-05095808 against common HCV clinical isolates. In summary, our data both support antiviral activity consistent with an RNA interference mechanism and demonstrate the potential of PF-05095808 as a therapeutic agent for chronic HCV infection.     

Natural killer T cell activation inhibits hepatitis B virus replication in vivo

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.     

The short hairpin RNA driven by polymerase II suppresses both wild-type and lamivudine-resistant hepatitis B virus strains

BACKGROUND: Chronic infection with hepatitis B virus (HBV) is widespread because of the limited availability of therapeutic treatments. Although previous reports have suggested that RNA interference has promise as a treatment for HBV infection, further studies of long-term and off-target drug effects on HBV, especially on drug-resistant strains of HBV, are needed. Therefore, seven vectors that express short hairpin RNAs (shRNAs), driven by the polymerase II promoter, pSilencer4.1/HBV, were constructed to target open reading frames (ORFs) of the HBV C and S genes from wild-type and drug-resistant strains. Treatment efficiency was also assessed. METHODS: The pSilencer4.1/HBV vectors were investigated in HepG2.2.15 cells and transgenic mice that consistently produce wild-type HBV. Additionally, vectors that produce a lamivudine-resistant strain of HBV were developed and cotransfected, along with pSilencer/HBV, into both HepG2 cells and mice. The effects of polymerase-II-driven pSilencer4.1/HBV were compared with those of polymerase-III-driven pSilencer3.1/HBV at both the gene and protein level. RESULTS: pSilencer4.1/HBV inhibited the expression of viral protein, DNA and HBV subtype ayw mRNA in both HepG2.2.15 cells and transgenic mice. Toxicity, as well as off-target effects, did not occur after a short- to medium-term examination. Moreover, an HBV strain resistant to lamivudine, subtype adr, was suppressed by shRNA in both HepG2 cells and mice. In contrast to polymerase III, vectors that used polymerase II could drive efficient silencing without off-target effects. CONCLUSIONS: Silencing by shRNA dramatically inhibited HBV expression and replication regardless of strain type. ShRNA could therefore be a promising treatment for HBV infection.     

In vivo antiviral efficacy of prenylation inhibitors against hepatitis delta virus

Hepatitis delta virus (HDV) can dramatically worsen liver disease in patients coinfected with hepatitis B virus (HBV). No effective medical therapy exists for HDV. The HDV envelope requires HBV surface antigen proteins provided by HBV. Once inside a cell, however, HDV can replicate its genome in the absence of any HBV gene products. In vitro, HDV virion assembly is critically dependent on prenyl lipid modification, or prenylation, of its nucleocapsid-like protein large delta antigen. To overcome limitations of current animal models and to test the hypothesis that pharmacologic prenylation inhibition can prevent the production of HDV virions in vivo, we established a convenient mouse-based model of HDV infection capable of yielding viremia. Such mice were then treated with the prenylation inhibitors FTI-277 and FTI-2153. Both agents were highly effective at clearing HDV viremia. As expected, HDV inhibition exhibited duration-of-treatment dependence. These results provide the first preclinical data supporting the in vivo efficacy of prenylation inhibition as a novel antiviral therapy with potential application to HDV and a wide variety of other viruses.     

Inhibition of hepatitis B virus replication during schistosoma mansoni infection in transgenic mice

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.     

Effective inhibition of HBV replication in vivo by anti-HBx short hairpin RNAs.

Exploiting the RNA interference pathway has shown promise for developing novel and effective treatment of hepatitis B virus (HBV) infection. To advance this approach, we analyzed the antiviral efficacy of a panel of 10 Pol III U6 promoter-encoded short hairpin RNAs (shRNAs) that target conserved sequences of the oncogenic HBx open reading frame. To facilitate intracellular processing, the shRNAs included mismatches in the 25-bp stem region and a terminal loop of miRNA-23. Two shRNAs (shRNA 5 and shRNA 6) showed knockdown of HBV markers by 80-100% in transfected hepatocytes and also in a murine hydrodynamic injection model of HBV replication. Intracellular processing of hairpin RNA with the intended strand bias correlated with antiviral efficacy. Moreover, markers of HBV replication were inhibited without inducing genes associated with the nonspecific interferon response. To assess the antiviral efficacy of the shRNAs in a context that is similar to natural HBV infection, shRNA-encoding cassettes were tested against the virus in a HBV transgenic murine model. When delivered using recombinant adenovirus vectors, U6 shRNA 5 and U6 shRNA 6 mediated significant HBV knockdown. Collectively, these observations indicate that U6 shRNA 5 and U6 shRNA 6 are promising candidates for therapy of chronic HBV infection.     

The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection

Hepatitis B virus (HBV) is a noncytopathic virus, and the recognition of infected hepatocytes by HBV-specific CD8 cells has been assumed to be the central mechanism causing both liver damage and virus control. To understand the role of cytotoxic T cells in the pathogenesis of HBV infection, we used functional assays that require T cell expansion in vitro and human histocompatibility leukocyte antigen (HLA)-peptide tetramers that allow direct ex vivo quantification of circulating and liver-infiltrating HBV-specific CD8 cells. Two groups of patients with persistent HBV infection were studied: one without liver inflammation and HBV replication, the other with liver inflammation and a high level of HBV replication. Contrary to expectation, a high frequency of intrahepatic HBV-specific CD8 cells was found in the absence of hepatic immunopathology. In contrast, virus-specific T cells were more diluted among liver infiltrates in viremic patients, but their absolute number was similar because of the massive cellular infiltration. Furthermore, inhibition of HBV replication was associated with the presence of a circulating reservoir of CD8(+) cells able to expand after specific virus recognition that was not detectable in highly viremic patients with liver inflammation.These results show that in the presence of an effective HBV-specific CD8 response, inhibition of virus replication can be independent of liver damage. When the HBV-specific CD8 response is unable to control virus replication, it may contribute to liver pathology not only directly but by causing the recruitment of nonvirus-specific T cells.     

Small Interfering RNA Targeting M2 Gene Induces Effective and Long Term Inhibition of Influenza A Virus Replication

RNA interference (RNAi) provides a powerful new means to inhibit viral infection specifically. However, the selection of siRNA-resistant viruses is a major concern in the use of RNAi as antiviral therapeutics. In this study, we conducted a lentiviral vector with a H1-short hairpin RNA (shRNA) expression cassette to deliver small interfering RNAs (siRNAs) into mammalian cells. Using this vector that also expresses enhanced green fluorescence protein (EGFP) as surrogate marker, stable shRNA-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed siRNAs targeting to conserved regions of influenza A virus genome were assessed. The results showed that a siRNA targeting influenza M2 gene (siM2) potently inhibited viral replication. The siM2 was not only effective for H1N1 virus but also for highly pathogenic avian influenza virus H5N1. In addition to its M2 inhibition, the siM2 also inhibited NP mRNA accumulation and protein expression. A long term inhibition effect of the siM2 was demonstrated and the emergence of siRNA-resistant mutants in influenza quasispecies was not observed. Taken together, our study suggested that M2 gene might be an optimal RNAi target for antiviral therapy. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for human influenza virus infection.     

dsAAV8-mediated gene transfer and β-cell expression of IL-4 and β-cell growth factors are capable of reversing early-onset diabetes in NOD mice

Type-I diabetes is a chronic disease mediated by autoimmune destruction of insulin-producing β-cells. Although progress has been made towards improving diabetes-associated pathologies and the quality of life for those living with diabetes, no therapy has been effective at eliminating disease manifestations or reversing disease progression. Here, we examined whether double-stranded adeno-associated virus serotype 8 (dsAAV8)-mediated gene delivery to endogenous β-cells of interleukin (IL)-4 in combination with β-cell growth factors can reverse early-onset diabetes in NOD mice. Our results demonstrate that a single treatment with dsAAV8 vectors expressing IL-4 in combination with glucagon-like peptide-1 or hepatocyte growth factor/NK1 under the regulation of the insulin promoter enhanced β-cell proliferation and survival in vivo, significantly delaying diabetes progression in NOD mice, and reversing disease in ～10% of treated NOD mice. These results demonstrate the ability to reverse hyperglycemia in NOD mice with established diabetes by in vivo gene transfer to β-cells of immunomodulatory factors and β-cell growth factors.     

Antiviral effect of oral administration of tenofovir disoproxil fumarate in woodchucks with chronic woodchuck hepatitis virus infection

Tenofovir disoproxil fumarate (TDF) is a nucleotide analogue approved for treatment of human immunodeficiency virus (HIV) infection. TDF also has been shown in vitro to inhibit replication of wild-type hepatitis B virus (HBV) and lamivudine-resistant HBV mutants and to inhibit lamivudine-resistant HBV in patients and HBV in patients coinfected with the HIV. Data on the in vivo efficacy of TDF against wild-type virus in non-HIV-coinfected or lamivudine-na?ve chronic HBV-infected patients are lacking in the published literature. The antiviral effect of oral administration of TDF against chronic woodchuck hepatitis virus (WHV) infection, an established and predictive animal model for antiviral therapy, was evaluated in a placebo-controlled, dose-ranging study (doses, 0.5 to 15.0 mg/kg of body weight/day). Four weeks of once-daily treatment with TDF doses of 0.5, 1.5, or 5.0 mg/kg/day reduced serum WHV viremia significantly (0.2 to 1.5 log reduction from pretreatment level). No effects on the levels of anti-WHV core and anti-WHV surface antibodies in serum or on the concentrations of WHV RNA or WHV antigens in the liver of treated woodchucks were observed. Individual TDF-treated woodchucks demonstrated transient declines in WHV surface antigen serum antigenemia and, characteristically, these woodchucks also had transient declines in serum WHV viremia, intrahepatic WHV replication, and hepatic expression of WHV antigens. No evidence of toxicity was observed in any of the TDF-treated woodchucks. Following drug withdrawal there was prompt recrudescence of WHV viremia to pretreatment levels. It was concluded that oral administration of TDF for 4 weeks was safe and effective in the woodchuck model of chronic HBV infection.     

Host-virus interactions during malaria infection in hepatitis B virus transgenic mice

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.     

Hepatitis B virus genotypes in Korea: an endemic area of hepatitis B virus infection

OBJECTIVES: It has been reported that distribution of hepatitis B virus (HBV) genotypes shows geographic difference and are associated with clinical outcomes of HBV infection, including response to antiviral therapy and progression of chronic liver diseases. In this study, we analyzed the distribution of HBV genotypes according to the various clinical outcomes of chronic HBV infection in Korea, which is one of the most endemic areas of HBV infection. METHODS: A total of 200 patients with chronic HBV infection were enrolled. Clinical diagnoses of the 200 patients with chronic liver diseases were as follows: hepatitis B e antigen (HBeAg)-positive healthy carrier (defined as HBeAg(+), anti-HBe(-), HBV DNA(+) by hybridization, normal transaminase; n = 40); inactive HBsAg carrier (n = 40); chronic hepatitis B (n = 40); liver cirrhosis (n = 40); hepatocellular carcinoma (n = 40). HBV genotypes were determined by nested polymerase chain reaction using genotype-specific primers. RESULTS: All patients except 2 (inactive HBsAg carriers) were positive for nested PCR and they have genotype C regardless of clinical outcomes. CONCLUSIONS: HBV genotype was genotype C regardless of various clinical outcomes of chronic HBV infection in Korea. Considering that HBV genotypes have clinical relevance, distribution of HBV genotype in each area should be monitored when management for chronic HBV infection is planned.     

Rapid and highly efficient transduction by double-stranded adeno-associated virus vectors in vitro and in vivo

Adeno-associated virus (AAV) is a promising gene vector based on a single-stranded (ss) DNA virus. Its transgene expression requires the conversion of ssDNA to double-stranded (ds) genome, a slow process responsible for the delayed transduction and occasional inefficiency. By mutating the inverted terminal repeat, we have made novel AAV vectors that predominantly package the self-complementary dsDNA genome. The dsAAV consistently demonstrated superior and accelerated transduction in vitro and in vivo. Dramatic increases in transgene expression were observed in most of the cell lines examined, including B16 melanoma and 3LL lung cancer that are difficult to be transduced by the conventional ssAAV vectors. Similar increases were also observed in vivo in a variety of tissues including muscle and liver. The dsAAV transduced a vast majority of the hepatocytes for more than 6 months, while the ssAAV transduced only a small fraction. In addition to circumventing the requirement for DNA synthesis, the dsAAV exhibited higher in vivo DNA stability and more effective circularization than the ssAAV, suggesting potential molecular mechanisms for the faster, stronger and prolonged transgene expression.     

Cytokines induced during chronic hepatitis B virus infection promote a pathway for NK cell-mediated liver damage

Hepatitis B virus (HBV) causes chronic infection in more than 350 million people worldwide. It replicates in hepatocytes but is non-cytopathic; liver damage is thought to be immune mediated. Here, we investigated the role of innate immune responses in mediating liver damage in patients with chronic HBV infection. Longitudinal analysis revealed a temporal correlation between flares of liver inflammation and fluctuations in interleukin (IL)-8, interferon (IFN)-alpha, and natural killer (NK) cell expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) directly ex vivo. A cross-sectional study confirmed these findings in patients with HBV-related liver inflammation compared with healthy carriers. Activated, TRAIL-expressing NK cells were further enriched in the liver of patients with chronic HBV infection, while their hepatocytes expressed increased levels of a TRAIL death-inducing receptor. IFN-alpha concentrations found in patients were capable of activating NK cells to induce TRAIL-mediated hepatocyte apoptosis in vitro. The pathogenic potential of this pathway could be further enhanced by the ability of the IFN-alpha/IL-8 combination to dysregulate the balance of death-inducing and regulatory TRAIL receptors expressed on hepatocytes. We conclude that NK cells may contribute to liver inflammation by TRAIL-mediated death of hepatocytes and demonstrate that this non-antigen-specific mechanism can be switched on by cytokines produced during active HBV infection.     

Nitric oxide inhibits hepatitis B virus replication in the livers of transgenic mice

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.     

Blocking chemokine responsive to gamma-2/interferon (IFN)-gamma inducible protein and monokine induced by IFN-gamma activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus-specific cytotoxic T lymphocytes.

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.