NLRX1 can counteract innate immune response induced by an external stimulus favoring HBV infection by competitive inhibition of MAVS-RLRs signaling in HepG2-NTCP cells.

INTRODUCTION: This study is aimed at the determination of the effect of the immune-regulatory factor NLRX1 on the antiviral activity of hepatocytes against an external stimuli favoring hepatitis B virus infection, and to explore its mechanism of action. METHODS: A HepG2-NTCP model was established using the LV003 lentivirus. Cells were transfected using an overexpression vector and NLRX1 siRNA to achieve overexpression and interference of NLRX1 expression (OV-NLRX1, si-NLRX1). Levels of HBsAg and HBcAg were determined using Western blotting analysis and immunohistochemical analysis. The levels of hepatitis B virus DNA and hepatitis B virus cccDNA were determined by real-time quantitative polymerase chain reaction. The expression and transcriptional activity of IFN-α, IFN-ß, and IL-6 were measured using real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and promoter-luciferase reporter plasmids. Co-immunoprecipitation was used to determine the effect of NLRX1 on the interaction between MAVS and RIG-1. Western blotting was used to obtain the phosphorylation of essential proteins in the MAVS-RLRs signaling pathways. RESULTS: NLRX1 promoted HepG2-NTCP cell hepatitis B virus infection. Compared to the control group, the levels of HBsAg, HBcAg, hepatitis B virus cccDNA, and hepatitis B virus DNA increased in the OV-NLRX1 group and decreased in the si-NLRX1. Co-immunoprecipitation results showed that NLRX1 competitively inhibited the interaction between MAVS and RIG-1, and inhibited the phosphorylation of p65, IRF3, and IRF7. Additionally, NLRX1 reduced the transcription activity and expression levels of the final products: IFN-α, IFN-ß, and IL-6. CONCLUSIONS: NLRX1 can counteract innate immune response induced by an external stimuli favoring hepatitis B virus infection by competitive inhibition of MAVS-RLRs signaling in HepG2-NTCP cells. Inhibition of the MAVS-RLR-mediated signaling pathways leads to a decline in the expression levels of I-IFN and IL-6.

Hepatitis B core antigen regulates dendritic cell proliferation and apoptosis through regulation of PKC/NF­κB signaling pathway.

Hepatitis B core antigen (HBcAg) possesses unusual immunologic features. However, the biological roles and mechanisms of HBcAg in dendritic cell proliferation and apoptosis remain to be elucidated. In the present study, DC2.4 cells were treated with different concentrations of HBcAg (10, 20 and 30 µg/ml). MTT assay and flow cytometry (Annexin V/propidium iodide analysis) were performed to investigate changes in cell proliferation and apoptosis. Western blot analysis was conducted to examine the changes in nuclear factor (NF)­κB and protein kinase C (PKC) signaling pathways. NF­κB inhibitor pyrrolidine dithiocarbamate (PDTC) and PKC inhibitor Chelerythrine were used to block these two signaling pathways. It was identified that HBcAg increased proliferation and decreased apoptosis in a dose­dependent manner. Western blotting results demonstrated that HBcAg upregulated p­PKC, p­IκB, p­P65, tumor necrosis factor­α and B­cell lymphoma 2 (Bcl­2) levels, and downregulated cleaved caspase 3, demonstrating that HBcAg activated the PKC and NF­κB signaling pathways. NF­κB inhibitor PDTC reduced the effects of HBcAg on DC2.4 proliferation (0.6 fold vs. 0.25 fold) and apoptosis (0.43 fold vs. 0.17 fold), and on Bcl­2 expression levels. PKC inhibitor Chelerythrine reduced the biological effects of HBcAg; it reduced proliferation (0.67 fold vs. 0.23 fold) and upregulated apoptosis (0.43 fold vs. 0.13 fold). Chelerythrine also blocked NF­κB activity and the HBcAg­induced Bcl­2 increase, suggesting the effect on Bcl­2 from HBcAg was dependent on the PKC/NF­κB signaling pathway. In conclusion, HBcAg promoted proliferation and inhibited apoptosis through the PKC/NF­κB/Bcl­2 signaling pathway in DC2.4 cells.

[Hepatitis B core antigen promotes invasion of hepatocellular carcinoma cell line HepG2.2.15 via Toll-like receptor 4].

Objective: To investigate the effect of hepatitis B core antigen (HBcAg) in promoting the invasion of hepatitis B virus (HBV)-related hepatocellular carcinoma cell line HepG2.2.15 and the role of Toll-like receptor 4 (TLR4) in the mechanism. Methods: TLR4 mRNA and protein expression in HepG2 cells and HepG2.2.15 cells was measured by reverse transcription real-time PCR and Western blot analysis, respectively. HepG2.2.15 cells were transfected with TLR4 specific small interfering RNA (siRNA) to silence TLR4 expression, and stimulated by recombinant HBcAg in culture. The invasion of cells was measured by Transwell invasion assay. The expression of TLR4 signaling pathway-related proteins in the cultured cells and proinflammatory cytokines in the supernatant was also determined. The student's t-test, one-way ANOVA, and SNK-q test were used for statistical analysis. Results: TLR4 mRNA and protein expression in HepG2.2.15 cells was significantly higher than that in HepG2 cells. TLR4 siRNA transfection remarkably down-regulated TLR4 expression in HepG2.2.15 cells. Inhibiting TLR4 expression and/or HBcAg stimulation did not affect the proliferation of HepG2.2.15 cells. However, HBcAg stimulation significantly increased the invasion ability of HepG2.2.15 cells and the secretion of proinflammatory cytokines [including interferon (IFN)-γ, interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α]. Inhibiting TLR4 expression significantly reduced HBcAg-induced cellular invasion. Meanwhile, HBcAg stimulation elevated the expression of MyD88 and TRIF in HepG2.2.15 cells. TLR4 silencing inhibited HBcAg-induced increase in the expression of MyD88, while it showed no significant impact on TRIF expression. Conclusion: HBcAg can promote the invasion of HepG2.2.15 cells. The TLR4/MyD88 signaling pathway may be involved in this process by inducing the expression of proinflammatory cytokines.

A virus-like particle-based connective tissue growth factor vaccine suppresses carbon tetrachloride-induced hepatic fibrosis in mice.

Connective tissue growth factor (CTGF) has been recognized as a central mediator and promising therapeutic target in hepatic fibrosis. In this study, we generated a novel virus-like particle (VLP) CTGF vaccine by inserting the 138-159 amino acid (aa) fragment of CTGF into the central c/e1 epitope of C-terminus truncated hepatitis B virus core antigen (HBc, aa 1-149) using a prokaryotic expression system. Immunization of BALB/c mice with the VLP vaccine efficiently elicited the production of anti-CTGF neutralizing antibodies. Vaccination with this CTGF vaccine significantly protected BALB/c mice from carbon tetrachloride (CCl4)-induced hepatic fibrosis, as indicated by decreased hepatic hydroxyproline content and lower fibrotic score. CCl4 intoxication-induced hepatic stellate cell activation was inhibited by the vaccination, as indicated by decreased α-smooth muscle actin expression and Smad2 phosphorylation. Vaccination against CTGF also attenuated the over-expression of some profibrogenic factors, such as CTGF, transforming growth factor-ß1, platelet-derived growth factor-B and tissue inhibitor of metalloproteinase-1 in the fibrotic mouse livers, decreased hepatocyte apoptosis and accelerated hepatocyte proliferation in the fibrotic mouse livers. Our results clearly indicate that vaccination against CTGF inhibits fibrogenesis, alleviates hepatocyte apoptosis and facilitate hepatic regeneration. We suggest that the vaccine should be developed into an effective therapeutic measure for hepatic fibrosis.

Hepatitis B virus peptide inhibitors: solution structures and interactions with the viral capsid.

Hepatitis B virus (HBV) infection remains a health problem globally despite the availability of effective vaccines. In the assembly of the infectious virion, both the preS and S regions of the HBV large surface antigen (L-HBsAg) interact synergistically with the viral core antigen (HBcAg). Peptides preS and S based on the L-HBsAg were demonstrated as potential inhibitors to block the viral assembly. Therefore, the objectives of this study were to determine the solution structures of these peptides and study their interactions with HBcAg. The solution structures of these peptides were solved using (1)H, (13)C, and (15)N NMR spectroscopy. Peptide preS has several structured regions of ß-turns at Ser7-Pro8-Pro9, Arg11-Thr12-Thr13 and Ser22-Thr23-Thr24 sequences whereas peptide S has only one structured region observed at Ser3-Asn4-His5. Both peptides contain bend-like structures surrounding the turn structures. Docking studies revealed that both peptides interacted with the immunodominant region of HBcAg located at the tip of the viral capsid spikes. Saturation Transfer Difference (STD) NMR experiments identified several aromatic residues in peptides preS and S that interact with HBcAg. This study provides insights into the contact regions of L-HBsAg and HBcAg at atomic resolution which can be used to design antiviral agents that inhibit HBV morphogenesis.

Silica nanoparticles as the adjuvant for the immunisation of mice using hepatitis B core virus-like particles.

Advances in nanotechnology and nanomaterials have facilitated the development of silicon dioxide, or Silica, particles as a promising immunological adjuvant for the generation of novel prophylactic and therapeutic vaccines. In the present study, we have compared the adjuvanting potential of commercially available Silica nanoparticles (initial particles size of 10-20 nm) with that of aluminium hydroxide, or Alum, as well as that of complete and incomplete Freund's adjuvants for the immunisation of BALB/c mice with virus-like particles (VLPs) formed by recombinant full-length Hepatitis B virus core (HBc) protein. The induction of B-cell and T-cell responses was studied after immunisation. Silica nanoparticles were able to adsorb maximally 40% of the added HBc, whereas the adsorption capacity of Alum exceeded 90% at the same VLPs/adjuvant ratio. Both Silica and Alum formed large complexes with HBc VLPs that sedimented rapidly after formulation, as detected by dynamic light scattering, spectrophotometry, and electron microscopy. Both Silica and Alum augmented the humoral response against HBc VLPs to the high anti-HBc level in the case of intraperitoneal immunisation, whereas in subcutaneous immunisation, the Silica-adjuvanted anti-HBc level even exceeded the level adjuvanted by Alum. The adjuvanting of HBc VLPs by Silica resulted in the same typical IgG2a/IgG1 ratios as in the case of the adjuvanting by Alum. The combination of Silica with monophosphoryl lipid A (MPL) led to the same enhancement of the HBc-specific T-cell induction as in the case of the Alum and MPL combination. These findings demonstrate that Silica is not a weaker putative adjuvant than Alum for induction of B-cell and T-cell responses against recombinant HBc VLPs. This finding may have an essential impact on the development of the set of Silica-adjuvanted vaccines based on a long list of HBc-derived virus-like particles as the biological component.

Inhibition of hepatitis B virus replication by the internal fragment of hepatitis B core protein.

The nucleocapsids formation is a pivotal step of hepatitis B virus (HBV) life cycle. The inhibition of HBV nucleocapsids assembly is a promising strategy for the anti-HBV treatment. HBc78-117 is an internal fragment of hepatitis B core protein (HBc). In this study, we used lentiviral vector to deliver HBc78-117 cDNA sequence into HepG2.2.15 cells and examined the effect of HBc78-117 on HBV replication. We confirmed by immunoprecipitation analysis that HBc78-117 interacted with full-length HBc in HepG2.2.15 cells. The nucleocapsids and HBV DNA replication intermediates were markedly reduced in the cells expressing HBc78-117, although HBV pregenome RNA was not affected. The level of HBV DNA was also significantly reduced in culture supernatant. These suggest that HBc78-117 can inhibit HBV DNA replication by interfering with nucleocapsids assembly.

[Human plasmacytoid dendritic cells from patients with chronic hepatitis B induce the generation of CD4+CD25+Treg in vitro].

OBJECTIVE: To compare the ability of plasmacytoid dendritic cells (pDCs) from chronic HBV patients and healthy controls to induce the the generation of CD4+CD25+Treg cells. METHODS: The pDCs were isolated from PBMCs of 46 chronic HBV patients, 10 resolved HBV patients and 25 healthy controls by magnetic cell sorting. Purified CD4+CD45RA+ naive T cells were incubated with allogeneic pDCs from chronic HBV infected patients, resolved HBV patients or healthy controls. The cells were stimulated with HBcAg or tetanus toxin. The proportion of CD4+CD25+Treg in CD4+ T cells primed by pDCs was determined by flow cytometry, the expression of Fox p3 mRNA was detected by RT-PCR, IL-10 and TGFb1 expression was quantified using ELISA kits. RESULTS: Compared with pDC isolated from healthy controls and the resolved HBV patients, pDC from chronic HBV patients was more effective in suppression of CD4+ T cells proliferation and interferon production when CD25-depleted PBMCs were stimulated with HBcAg, (7999.36+/-374.74 vs 11 282.56+/-1174.46; 7999.36+/-374.74 vs 12 304.58+/-1462.81, P less than 0.05 ). Depletion of CD4+CD25+ Treg from CD4+ T cells primed by pDC led to the lose of capability to suppress HBV-specific T-cell responses. When CD25- depleted PBMCs were stimulated with purified tetanus toxin, there was no significantly difference in proliferation between CD25-depleted PBMC co-cultured with pDC-primed CD4+ T cells and CD25-depleted PBMC cultured without pDC-primed CD4+ T cells. A higher percentage of CD4+CD25+ Treg was detected within the population of CD4+ T cells primed by pDC from chronic HBV patients compared with healthy controls and resolved HBV patients (5.99%+/-1.85% vs 3.04%+/-0.79%; 5.99%+/-1.85% vs 3.01%+/-1.53%, P less than 0.05). Accordingly, CD25+Treg from pDC-primed CD4+ T cells displayed a higher Fox P3 mRNA level. The IL-10 and TGFb1 could be also detectable in the supernatants of pDC-primed CD4+ T cells. CONCLUSION: pDCs from chronic hepatitis B induce the generation of a higher proportion of CD4+CD25+ Treg compared with pDCs from healthy controls.

Altered T cell costimulation during chronic hepatitis B infection.

T-cell response to hepatitis B virus (HBV) is vigorous, polyclonal and multi-specific in patients with acute hepatitis who ultimately clear the virus, whereas it is narrow and inefficient in patients with chronic disease, where inappropriate early activation events could account for viral persistence. We investigated the induction of activation receptors and cytokine production in response to HBcAg and crosslinking of CD28 molecules, in CD4+ cells from a group of chronically infected patients (CIP) and naturally immune subjects (NIS). We demonstrated that CD4+ cells from CIP did not increase levels of CD40L and CD69 following stimulation with HBcAg alone or associated to CD28 crosslinking, in contrast to subjects that resolved the infection (p<0.01). Furthermore, CD4+ cells from CIP produced elevated levels of IL-10 in response to HBcAg. These results suggest that a predominant inhibitory environment may be responsible for altered T cell costimulation, representing a pathogenic mechanism for viral persistence.

Coimmunization with IL-15 plasmid enhances the longevity of CD8 T cells induced by DNA encoding hepatitis B virus core antigen.

AIM: To test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for improving the immune responses induced by hepatitis B virus core gene DNA vaccine. METHODS: We used RT-PCR based strategies to develop IL-15 expression constructs. We first confirmed that the gene could be expressed in Escherichia coli due to the poor expression of IL-15. Then the bioactivity of IL-15 plasmid expression product was identified by CTLL-2 proliferation assay. One hundred micrograms of DNA from each of the IL-15 eukaryotic expressed plasmid and the recombinant plasmid harboring DNA encoding the 144 amino acids of the N-terminus of HBV core gene (abbreviated pHBc144) was used to co-immunize C57 BL/6 mice. The titer of anti-HBcIgG was detected by ELISA and the antigen-specific CD8(+) T cells (CD8(+)IFN-gamma(+) T cells) were detected by intracellular cytokine staining at different time points. RESULTS: After co-immunization by pIL-15 and pHBc144 DNA vaccine the antigen-specific CD8(+) cells of mice increased gradually, the first peak of immune response appeared 14 d later, then the number of antigen-specific CD8(+) Ts cells decreased gradually and maintained at a steady level in 3 mo. After boosting, the number of antigen-specific CD8(+) T cells reached the second peak 10 d later with a double of the 1st peak, then the number of antigen-specific CD8(+) T cells decreased slowly. IL-15 as a gene adjuvant had no significant effect on humoral immune responses induced by hepatitis B virus core gene DNA vaccine, but increased the memory antigen-specific CD8(+) T cells induced by hepatitis B virus core gene DNA vaccine. CONCLUSION: DNA vaccine constructed by HBc Ag 1-144 amino acid induces effective cell immunity, and cytokine plasmid-delivered IL-15 enhances the longevity of CD8(+) T cells.

Cytokine induction by the hepatitis B virus capsid in macrophages is facilitated by membrane heparan sulfate and involves TLR2.

The hepatitis B virus (HBV) core Ag (HBcAg) serves as the structural subunit of the highly immunogenic capsid shell. HBcAg harbors a unique arginine-rich C terminus that was implicated in immune responses induced by the capsid. In this study, we examined the capacity of the HBV capsid to induce proinflammatory and regulatory cytokines in human THP-1 macrophages and the possible underlying mechanism. Full-length HBc capsids, but not HBc-144 capsids lacking the arginine-rich domain of HBcAg, efficiently bound differentiated THP-1 macrophages and strongly induced TNF-alpha, IL-6, and IL-12p40. Capsid binding to macrophages and cytokine induction were independent of the RNA associated with the arginine-rich domain. Soluble heparin and heparan sulfate but not chondroitin sulfates greatly diminished cytokine induction through inhibition of capsid binding to THP-1 macrophages. Furthermore, serine phosphorylation in the arginine-rich domain modulates capsid binding to macrophages and the cytokine response. Induction of cytokines by the capsid involved activation of NF-kappaB, ERK-1/2, and p38 MAPK and did not require endosomal acidification. Finally, NF-kappaB activation by the capsid in HEK 293 cells specifically required expression of TLR2 and was compromised by soluble heparin. Thus, cytokine induction by the HBV capsid in macrophages is facilitated by interaction of its arginine-rich domain with membrane heparan sulfate and involves signaling through TLR2.

A study on interferon-gamma (IFN-gamma) response by T cells stimulated by hepatitis B virus core antigen.

Cell-mediated immune response by lymphocyte induced through recognition of HBV core antigen during acute HBV infection, chronic HBV infection and in subjects recovered from HBV infection was investigated in the present study by assessing the competence of IFN-gamma secretion by cultured PBMCs on stimulation by HBV nucleocapsid antigen (HBcAg). Fresh blood was collected in heparin from acute, chronic and recovered groups of HBV infected patients and uninfected vaccinated healthy controls aged between 18-50 years. PBMCs were separated by Ficoll-Hypaque density gradient centrifugation technique and were stimulated with hepatitis B virus core antigen (HBcAg) and mitogen (lectin). Stimulated PBMCs were cultured in CO2 and IFN-gamma levels were measured from the culture supernatant by an in-house ELISA technique. The mean+/-SE levels of IFN-gamma in HBcAg stimulated PBMCs in acute, chronic, recovered and controls groups were 875 pg/ml+/-297.56, 128.50 pg/ml+/-33.66, 905 pg/ml+/-172.51 and 235.33 pg/ml+/-111.28 respectively. IFN-gamma levels produced by HBcAg stimulated PBMCs of acute and recovered groups were significantly higher than that of chronic and control group (p<0.001). All groups responded strongly to lectin stimulation. Thus, it may be concluded that patients with acute HBV infection and those who had recovered from HBV infection show vigorous response to HBcAg stimulation in contrast to patients with chronic HBV infection.

Therapeutic polypeptides based on HBcAg(18-27) CTL epitope can induce antigen-specific CD(8)(+) CTL-mediated cytotoxicity in HLA-A2 transgenic mice.

AIM: To explore how to trigger an HLAI-restricted CD8(+) T cell response to exogenously synthesized polypeptides in vivo. METHODS: Three mimetic therapeutic polypeptides based on the immunodominant CTL epitope of HBcAg, the B- epitope of HBV PreS(2) region and a common T helper sequence of tetanus toxoid were designed and synthesized with Merrifield's solid-phase peptide synthesis method. Their immunological properties of inducing T( H1) polarization, CD8(+) HBV-specific CTL expansion and CD8(+) T cell mediated cytotoxicity were investigated in HLA-A2 transgenic mice. RESULTS: Results demonstrated that the mimetic polypeptides comprised of the immunodominant CTL, B-, and T helper epitopes could trigger specifically and effectively vigorous CD8(+) HBV-specific CTL-mediated cytotoxicity and T(H1) polarization of T cells in HLA-A2 transgenic mice. CONCLUSION: A designed universal T helper plus B-epitopes with short and flexible linkers could dramatically improve the immunogenicity of CTL epitopes in vivo. And that the mimetic therapeutic peptides based on the reasonable match of the above CTL, B- and T helper epitopes could be a promising therapeutic peptide vaccine candidate against HBV infection.

Interferon-gamma (IFN-gamma) response to different hepatitis B virus antigens in hepatitis B virus infection.

The IFN-gamma levels in serum and cultured supernatant of peripheral blood mononuclear cells (PBMCs) were compared after stimulation by HBsAg ad, HBsAg ay and HBcAg among 3 groups of subjects i.e. patients with acute HBV infection, patients with chronic HBV infection and subjects recovered from HBV infection. Uninfected vaccinated group was taken as control. Serum and PBMCs were obtained from 38 individuals between 18-50 years of age. PBMCs were separated from heparinised blood by Ficoll-Hypaque density gradient centrifugation technique and cultured in CO2 incubator after stimulation by HBV surface and core antigens. IFN-gamma concentration was measured in serum and culture supernatant of PBMCs by an in-house ELISA technique. The mean serum IFN-gamma levels in acute, chronic, recovered and control groups were 88 pg/ml, 96.6 pg/ml, 155 pg/ml and 205 pg/ml respectively. On stimulation by HBsAg ad, IFN-gamma levels in cultured PBMCs of the above mentioned groups were 282.50 pg/ml, 307.45 pg/ml, 915.62 pg/ml and 511.67 pg/ml respectively, while in the same group on HBsAg ay stimulation, IFN-gamma levels were 246.25 pg/ml, 374.70 pg/ml, 1040 pg/ml and 465.83 pg/ml respectively. On stimulation by HBcAg, the IFN-gamma levels were 875 pg/ml, 128.50 pg/ml, 905 pg/ml and 235.33 pg/ml respectively in the acute, chronic, recovered and control groups. When compared with serum, significantly higher levels of IFN-gamma in cultured supernatant of PBMCs were observed after stimulation by HBsAg ad and HBsAg ay subtype in cases of chronic (p<0.05) and recovered groups (p<0.01 and p<0.001 respectively). However, no statistically significant difference of IFN-gamma level was observed between serum and PBMCs amongst the acute and control groups when stimulated by either of the HBsAg subtypes or HBcAg. In the recovered group, IFN-gamma levels produced by PBMCs after stimulation by HBcAg were significantly higher than that of serum (p<0.01). The study concludes that on subsequent exposure, PBMCs of the recovered group produces higher levels of IFN-gamma in response to different hepatitis B antigens. This response perhaps is able to protect individuals who are unable to develop anti-HBs.

[Effect of IL-18 on peripheral blood monocytes from chronic hepatitis B patients].

OBJECTIVES: To explore the effect of IL-18 on peripheral blood monocytes (PBMCs) from chronic hepatitis B (CHB) patients and HBV DNA released by HepG2.2.15 cells, which were transfected with the gene of HBV. METHODS: PBMCs were isolated from 25 healthy persons and 25 CHB patients, which were co-cultured with HBcAg and IL-18 at different concentrations for 72 hours. The level of IFN-gamma in the culture supernatant of PBMCs was determined by ELISA. One patient' PBMCs were co-cultured for 96 hours with various concentrations of IL-18 and HepG2.2.15 cells which had been cultured for 24 hours, the supernatant was collected to detect HBV DNA level by PCR. RESULTS: When PBMCs were stimulated by HBcAg and IL-18 at various concentrations, the levels of supernatant IFN-gamma in the CHB group were much higher than those in the normal control group (at 0.2ng/ml: t=11.7, P<0.01; at 1.0ng/ml: t=16.19, P<0.01; at 5.0ng/ml: t=20.12, P<0.01), especially when the PBMCs were stimulated by HBcAg, IL-18 and IL-12 (1313.20pg/ml+-187.76pg/ml vs. 390.75pg/ml+-43.23pg/ml, t=23.94, P<0.01). The IFN-gamma level in the patients who were stimulated by HBcAg alone was much lower than the levels in the patients who were stimulated by HBcAg and IL-18 at various concentrations, and which were lower than those in the patients stimulated by HBcAg, IL-12 and IL-18 at the same concentrations (light: t=2.2, P<0.05; moderate: t=2.97, P<0.05). The HBV DNA content in the supernatant of co-cultivation with HepG2.2.15 cells and PBMCs was much higher than that of the two kinds of cells stimulated by HBcAg and IL-18 at various concentrations or HBcAg, IL-18 and IL-12/IFN-a1b. CONCLUSION: IL-18 can improve the PBMCs from CHB patients to produce a great deal of IFN-gamma, so it has a good application prospect in two aspects: immunoregulatory effects and increasing the ability to kill the cells infected with virus.

Inhibition of hepatitis B virus assembly with synthetic peptides derived from the viral surface and core antigens.

The long surface antigen (L-HBsAg) of hepatitis B virus (HBV) plays a central role in the production of infectious virions. During HBV morphogenesis, both the PreS and S domains of L-HBsAg form docking sites for the viral nucleocapsids. Thus, a compound that disrupts the interaction between the L-HBsAg and nucleocapsids could serve as a therapeutic agent against the virus based upon inhibition of morphogenesis. Synthetic peptides correspond to the binding sites in L-HBsAg inhibited the association of L-HBsAg with core antigen (HBcAg). A synthetic peptide carrying the epitope for a monoclonal antibody to the PreS1 domain competed weakly with L-HBsAg for HBcAg, but peptides corresponding to a linear sequence at the tip of the nucleocapsid spike did not, showing that the competing peptide does not resemble the tip of the spike.

Reduced virus specific T helper cell induction by autologous dendritic cells in patients with chronic hepatitis B - restoration by exogenous interleukin-12.

Insufficient stimulatory capacities of autologous dendritic cells (DC) may contribute in part to impaired T cell stimulation and therefore viral persistence in patients with chronic hepatitis B virus (HBV) infection. In order to characterize the antigen presenting functions of DC from chronic HBV carriers and controls antigen specific T cell responses were analysed. CD34+ peripheral blood progenitor cells were differentiated to immature DC in the presence of GM-CSF, IL-6/IL-6R fusion protein and stem cell factor. Proliferative CD4+ T cell responses and specific cytokine release were analysed in co-cultures of DC pulsed with HBV surface and core antigens or tetanus toxoid and autologous CD4+ T cells. Cultured under identical conditions DC from chronic HBV carriers, individuals with acute resolved hepatitis B and healthy controls expressed similar phenotypical markers but chronic HBV carriers showed less frequent and weaker HBV antigen specific proliferative T helper cell responses and secreted less interferon-gamma while responses to the tetanus toxoid control antigen was not affected. Preincubation with recombinant IL-12 enhanced the HBV specific immune reactivities in chronic HBV patients and controls. In conclusion, the weak antiviral immune responses observed in chronic hepatitis B may result in part from insufficient T cell stimulating capacities of DC. Immunostimulation by IL-12 restored the HBV antigen specific T cell responses and could have some therapeutical benefit to overcome viral persistence.

Cytokine release of peripheral blood mononuclear cells in children with chronic hepatitis B virus infection.

BACKGROUND: Immune response to hepatitis B virus (HBV) antigens or mitogens in Asian children with chronic HBV infection who are mainly perinatally infected has not been studied in connection with the production of various cytokines, although these patients are considered to be less responsive to antiviral therapy. METHODS: The production of the cytokines interferon (IFN)-gamma, lymphotoxin, interleukin (IL)-4, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta by peripheral blood mononuclear cells (PBMCs) was studied in 17 hepatitis B surface antigen (HBsAg) carrier children with raised alanine transferase levels (group 1), 17 HBsAg carrier children with normal alanine transferase levels (group 2), and 20 healthy noncarrier control subjects (group 3). RESULTS: Hepatitis B core antigen (HBcAg)-stimulated IFN-gamma production was significantly higher in group 1 than in groups 2 and 3, serum HBeAg cleared within 1 year in five of eight children in group 1 with stimulation indexes higher than 3, and HBcAg-induced IL-4 secretion was minimal in all groups. Interferon-gamma produced by PBMCs stimulated by purified HBsAg did not differ among the three groups. Higher lymphotoxin production by PBMCs stimulated by HBcAg was also noted in groups 1 and 2 than in group 3. Lipopolysaccharide (LPS)-stimulated TNF-alpha production by PBMCs was significantly higher in group 1 than in group 2. There was no association between HBeAg-anti-HBe status and production of various cytokines. No differences were seen in the profile of cytokines induced by HBV antigens or LPS in children of carrier mothers compared with children of HBsAg-negative mothers. CONCLUSION: Increased IFN-gamma production resulting from HBcAg-specific T-helper lymphocyte type 1 response, and increased TNF-alpha production may contribute to cell-mediated antiviral immune response in children with chronic hepatitis B. In HBV carrier children, the ability to produce the studied cytokines is related to whether an endogenous immune attempt to eliminate HBV infection emerges in the patients but is not related to the different modes of acquisition of HBV infection.

[Interleukin 10(IL-10) levels in serums and in culture supernatants of peripheral blood mononuclear cells from patients with chronic hepatitis B].

OBJECTIVE: To understand the impacts of interleukin 10(IL-10) in serums and in culture supernatants of peripheral blood mononuclear cells (PBMCs) from patients with chronic hepatitis B (CHB) on the persistent infection of hepatitis B virus (HBV). METHODS: The levels of IL-10 in serums and in culture supernatants of PBMCs from 15 patients with CHB and 6 normal controls were measured by ELISA method. PBMCs were cultured with or without Staphylococcus aureus enterotoxin B(SEB; 0.2 microgram/ml) or recombinant HBcAg(rHBcAg; 1.0 microgram/ml) for 48 hours in vitro. RESULTS: The levels of IL-10 in serums was greater in patients with CHB (123.11 +/- 13.89 ng/L) than in controls (95.97 +/- 11.68 ng/L, P < 0.01). Spontaneous, SEB-induced and rHBcAg-induced IL-10 production by PBMCs after culturing in vitro for 48 hours was also higher in patients with CHB (280.82 +/- 50.56 ng/L, 321.69 +/- 37.04 ng/L and 369.58 +/- 30.52 ng/L) than in controls (100.2 +/- 8.54 ng/L, 203.41 +/- 12.02 ng/L and 202.38 +/- 15.79 ng/L; P < 0.01). In addition, IL-10 production by rHBcAg-stimulated PBMCs was significantly greater than by SEB-stimulated PBMCs in patients with CHB. Patients with seropositive for HBV DNA had higher levels of IL-10 in serums and in culture supernatants of PBMCs than patients with seronegative for HBV DNA. But the levels of IL-10 were lower in patients with chronic heavy and severe hepatitis B than in patients with chronic mild and moderate hepatitis B. CONCLUSION: IL-10 may have an important role in persistent infection of HBV in patient with CHB.

HBV-specific lymphoproliferative and cytokine responses in patients with chronic hepatitis B.

BACKGROUND/AIMS: Hepatitis B virus specific T cell responses are crucial for viral elimination but their nature is not fully understood. METHODS: We studied the regulation of proliferation and cytokine production after antigenic stimulation in peripheral blood mononuclear cells from chronically HBV-infected patients and subjects with natural immunity after recovery from an acute infection. Proliferation and production of interferon-gamma, IL-10 and tumor necrosis factor-alpha were determined after stimulation with HBcAg, HBeAg or HBsAg in the absence or presence of IL-12 or neutralizing antibodies to IL-12, interferon-gamma, IL-4, IL-10 or tumor necrosis factor-alpha. RESULTS: Upon stimulation with HBcAg or HBeAg, peripheral blood mononuclear cells from chronic hepatitis B virus patients displayed a clear class-II restricted proliferative response (SI greater than 2.5). Both interferon-gamma (less than 50 IU/ml) and IL-10 levels up to 600 pg/ml were detected. Proliferative or cytokine responses to HBsAg were very weak or absent. Addition of IL-12 to HBeAg-stimulated cultures increased the production of interferon-gamma to more than 200 IU/ml in all patients and slightly increased the production of IL-10. Neutralization of IL-10 increased the HBeAg-induced interferon-gamma production but had no effect on tumor necrosis factor-alpha production. Addition of anti-IL-4 or anti-tumor necrosis factor-alpha had no significant influence on proliferation or cytokine release. Importantly, in both chronic hepatitis B virus patients and naturally immune subjects, IL-12 induced proliferative and interferon-gamma responses in peripheral blood mononuclear cells stimulated with HBsAg. CONCLUSIONS: Our data indicate that peripheral blood mononuclear cells from chronic hepatitis B virus patients proliferate and produce interferon-gamma and IL-10 upon HBeAg but not upon HBsAg stimulation. IL-12 augments the HBeAg-induced responses and, additionally, provokes proliferation and interferon-gamma production in HBsAg-stimulated cultures.