Efficacy of cationic lipid-DNA complexes (CLDC) on hepatitis B virus in transgenic mice

Cationic lipid–DNA (non-coding) complexes (CLDC) are activators of the innate immune response that increase survival of rodents with some acute viral infections and cancers. CLDC were evaluated for their ability to impact viral DNA levels in transgenic mice carrying an infectious clone of hepatitis B virus (HBV). Mice used in the studies were diet-restricted as nursing pups from solid food, because the expression of HBV DNA in the liver was increased above background levels in some mice with this restriction. Survival surgery was performed on these mice to obtain liver biopsies from which to select animals with suitable levels of liver HBV DNA for entry into the experimental protocols. Intravenous administration of 5 μg/mouse of CLDC on days 1, 7 and 13 reduced liver HBV DNA to similar low levels achieved with the positive control, adefovir dipivoxil. In a subsequent experiment, the same treatment schedule was used to determine that the minimal effective CLDC dose was between 0.5 and 0.05 μg/mouse. Selective cytokines were increased in the livers of CLDC-treated compared to placebo-treated mice in a dose-responsive manner. CLDC were effective in reducing liver HBV DNA and could be considered for further evaluation in other hepatitis models.     

Neutralization of hepatitis B virus (HBV) by human monoclonal antibody against HBV surface antigen (HBsAg) in chimpanzees

The virus neutralizing efficacy of HB-C7A, a human monoclonal antibody raised against the surface antigen of hepatitis B virus (HBsAg), was proved using hepatitis B virus (HBV)-na?ve chimpanzees. One control chimpanzee which received 100CID(50) of HBV, subtype adw, without HB-C7A antibody became infected by HBV as evidenced by the appearance of HBV DNA on week 10 and subsequent appearance of HBsAg, anti-HBc and anti-HBs in the serum. Two experimental chimpanzees were inoculated intravenously with same dose of HBV as the control chimpanzee, which was previously incubated with 0.1mg and 10mg of HB-C7A antibody prior to inoculation. HBV infection was not observed in the antibody-treated chimpanzees during 12 months of follow-up, exhibiting neither detectable HBsAg nor anti-HBc antibody. This work demonstrates the neutralization of HBV by HB-C7A monoclonal antibody and shows the possibility of prevention of HBV infection using this antibody in liver transplantation and exposure to HBV.     

Characterization of antiviral activity of entecavir in transgenic mice expressing hepatitis B virus

Entecavir (ETV), a cyclopentyl guanosine nucleoside analog, was evaluated in transgenic mice expressing hepatitis B virus (HBV). ETV administered orally once daily for 10 days at a dosage of 3.2mg/kg significantly (P相似文献   

Humoral and cell-mediated immune-responses after administration of a single-shot recombinant hepatitis B surface antigen vaccine formulated with cationic poly(l-lactide) microspheres

The present investigations were aimed to compare the humoral and cell-mediated immune responses between recombinant hepatitis B surface antigens (HBsAg) adsorbed L-PLA microspheres (Ms) vaccine (single-shot) and marketed alum-HBsAg vaccine (two-doses). The blank cationic (cetyltrimethyammoniumbromide) microspheres were prepared by the double emulsion (w/o/w) solvent evaporation technique. The HBsAg was adsorbed onto the surface of blank cationic microspheres. These microspheres were characterized in vitro for their size, shape, adsorption-efficiency, in-process stability, and HBsAg release studies. Specific humoral immune responses (IgM and IgG) and cell-mediated immune responses (cellular-proliferation) assay including release of interferon-gamma (IFN-γ), interleukin-2 (IL-2), and nitric oxide (NO) from host’s cells stimulated with HBsAg or lipopolysaccharide (LPS)/ concanavalin A (con A) in-vitro were determined. Based on these findings, it was concluded that the single injection (using subcutaneous-route) of the polymeric microspheres produced better immune response (both humoral and cell-mediated) than two injections of a conventional alum-HBsAg vaccine. These data demonstrate high potential of polymeric microspheres for their use as a carrier adjuvant for hepatitis B vaccine.     

Inhibition of human hepatitis B virus (HBV) by a novel non-nucleosidic compound in a transgenic mouse model

BAY 41-4109 is a member of a class of heteroaryl-pyrimidines that was recently identified as potent inhibitors of human hepatitis B virus (HBV) replication. We have investigated the antiviral activity of BAY 41-4109 (methyl (R)-4-(2-chloro-4-fluorophenyl)-2-(3,5-difluoro-2-pyridinyl)-6-methyl-1,4-dihydro-pyrimidine-5-carboxylate) in HBV-transgenic mice (Tg [HBV1.3 fsX(-)3'5']). Bay 41-4109 was administered per os using different schedules (b.i.d. or t.i.d. for up to 28 days) and dosages ranging from 3 to 30 mg/kg. The compound reduced viral DNA in the liver and in the plasma dose-dependently with efficacy comparable to 3TC. In contrast to 3TC-treated mice, we found a reduction of cytoplasmic hepatitis B virus core antigen (HBcAg) in liver sections of BAY 41-4109-treated mice, which indicated a different mode of action. Pharmacokinetic studies in mice have shown rapid absorption, a bioavailability of 30% and dose-proportional plasma concentrations. We conclude that BAY 41-4109 is a new anti-HBV drug candidate.     

Role of silent hepatitis B virus in chronic hepatitis B surface antigen(-) liver disease.

Despite a number of studies documenting hepatitis B virus (HBV) infection in the absence of hepatitis B surface antigen (HBsAg) a causal relationship between silent HBV infection and liver disease remain difficult to establish. In particular, both the prevalence and clinical significance of this observation are poorly understood. Why is HBV replication apparently so low in these patients? A number of studies have tried to elucidate the mechanism of HBsAg negative infections, and considerable data documenting HBV infectivity or reinfection in the absence of detectable HBsAg support the hypothesis that in some of these cases, HBV is undergoing low-level replication in the liver and this, in several situations including: (1) chronic liver disease, alcoholic liver disease, hepatocellular carcinoma; (2) viral reactivation following cancer chemotherapy or immunosuppression and (3) transmission via transfusion or from human serum to chimpanzees. In a recent study including 50 patients with chronic liver disease of unknown etiology we could detect serum HBV DNA by nested polymerase chain reaction (PCR) in 15/50 patients (50% at the cirrhosis stage) in the absence of HBsAg; in the liver of the 15 patients both HBcAg and/or HBsAg can be detected at very low-level. Viral host factors allowing HBV persistence in the absence of HBsAg can depend on several mechanisms. Coinfections with HCV can explain only a proportion of HBsAg(-) HBV infections. Secondly, HBV mutations in the core promotor region leading to a minimal viral replication, or mutations in the HBsAg-encoding region might explain the absence of serological recognition. Finally, it is possible that in some cases host immune mechanisms can maintain HBV infection in a latent state until transmission to another individual who subsequently develops a more active infection especially when immunosuppressive therapy is employed. Existence of HBsAg(-) HBV infections should be taken into account by the use of sensitive PCR tests for prevention of viral transmission in the settings of blood donations and organ transplants.     

含PreS1、PreS2和S抗原的重组乙型肝炎疫苗辅以不同佐剂在HBV转基因小鼠中的免疫原性

目的 研究以免疫刺激复合物(immune stimulating complex,ISCOM)或Al(OH)3为佐剂的含PreS1、PreS2和S抗原的重组乙型肝炎疫苗在HBV转基因小鼠中诱导的免疫应答.方法 将纯化的含PreS1、PreS2和S抗原的重组HBsAg(SS1S2)分别辅以ISCOM或Al(OH)3佐剂制成疫苗.HBV转基因小鼠每月肌内注射1针疫苗,共注射7针.免疫前及每针后1个月称量小鼠体重,观察小鼠生长情况.免疫前及每针后1个月采血,用ELISA检测HBV S、PreS1、PreS2抗原及其相应抗体.7针后1个月分离小鼠脾淋巴细胞,用酶联免疫斑点试验分别测定分泌IL-4、IL-5、IFN-γ的效应T细胞频数.结果 小鼠生长状况良好,每针后体重均有增长.首针后1个月,10只ISCOM+ SS1S2免疫鼠分别有3和1只抗S和PreS1抗体阳转,无一小鼠抗PreS2抗体阳转;10只Al(OH)3+ SS1S2免疫鼠的抗体均为阴性.7针后1个月,10只ISCOM+ SS1 S2免疫鼠的抗S和PreS1抗体均阳转,8只抗PreS2抗体阳转;10只Al(OH)3+ SS1S2免疫鼠分别有9、7和1只抗S、PreS1和PreS2抗体阳转;ISCOM+SS1S2组和Al(OH)3+ SS1S2组的抗S抗体几何平均滴度分别为1∶15 647和1∶1 039,差异有统计学意义0=5.18,P=0.000).7针后1个月,ISCOM+ SS1S2组和Al(OH)3+ SS1S2组分泌IL-4的细胞频数分别为219和78斑点形成细胞(spot-forming cell,SFC)/106细胞(t=10.50,P=0.000),分泌IL-5的细胞频数分别为286和34 SFC/106细胞(t=19.53,P=0.000),差异有统计学意义.结论 含PreS1、PreS2和S抗原的重组乙型肝炎疫苗辅以ISCOM或Al(OH)3佐剂在HBV转基因小鼠中均具有免疫原性,且ISCOM+ SS1 S2的免疫原性强于Al(OH)3+ SS1S2.     

Modeling the T-helper cell response in acute and chronic hepatitis B virus infection using T-cell receptor transgenic mice.

Chronicity following hepatitis B virus (HBV) infection may be maintained by high levels of viral proteins circulating in the serum. To examine the characteristics of T cells capable of co-existing with the secreted hepatitis B e-antigen (HBeAg), T-cell receptor (TCR) transgenic (Tg) mice were produced. To insure that HBeAg-specific T cells would not be deleted in the presence of serum HBeAg, the TCR alpha and beta-chain genes used to produce the TCR-Tg mice were derived from T-cell hybridomas from HBeAg-Tg mice. A TCR-Tg lineage (11/4-12) was produced that possessed a high frequency (approximately 67%) of CD4(+) T cells that expressed a TCR-Tg specific for the HBeAg. As predicted, when 11/4-12 TCR-Tg mice were bred with HBeAg-Tg mice no deletion of the HBeAg-specific CD4(+) T cells occurred in the thymus or the spleen. Functional analysis of the TCR-Tg T cells revealed that the HBeAg-specific CD4(+) T cells escaped deletion in the thymus and periphery by virtue of low avidity. Regardless of their low avidity, HBeAg-specific TCR-Tg T cells could be activated by exogenous HBeAg as measured by cytokine production in vitro and T-helper cell function for anti-HBe antibody production in vitro and in vivo. Furthermore, activated TCR-Tg HBeAg-specific T cells polarized to the Th(1) subset were able to elicit liver injury when transferred into HBeAg or HBcAg-Tg recipients. Therefore, HBeAg-specific CD4(+) T cells that can survive deletion or anergy in the presence of circulating HBeAg nonetheless are capable of being activated and of mediating liver injury in vivo.     

Physicochemical properties of recombinant hepatitis B surface antigen expressed in mammalian cell (C127)

The physicochemical properties of recombinant hepatitis B surface antigen (r-HBsAg), which was expressed in C127 mammalian cell were studied. Using roller bottle culture in DMEM supplemented with fetal bovine serum, 10–15 mg/L of r-HBsAg was produced with about 31% of purification yield. The purity of r-HBsAG by HPLC was 99.8% and electron microscopic examination showed homogeneous spherical particle with 22 nm in diameter, a morphological characteristic of HBsAg. The density of r-HBsAg by CsCl density gradient method was 1.19 g/ml and the isoelectric point by Mono P^TM HR 5/20 column was 4.6. The analysis of subunit protein pattern using SDS-PAGE followed by scanning densitometry gave 81.3% of S protein and 18.7% of pre-S protein. Fluorophore-assisted-carbohydrate-electrophoresis analysis showed the relative amount of carbohydrate to protein was 1.7% and its major component was N-acetyl glucosamine, which was about 39% of total carbohydrate. The relative amount of lipid to protein determined by vanillin phosphoric acid method was 32.5% and its major component was phospholipid, which was about 70% of total lipid. The physicochemical properties of C127 mammalian cell-derived r-HBsAg are similar to those of p-HBsAg, suggesting that the r-HBsAg can be used in developing a new preventive vaccine against hepatitis B.     

Immunological properties of recombinant hepatitis B surface antigen expressed in mammalian cell (C127)

The physicochemical properties of recombinant hepatitis B surface antigen (r-HBsAg), which was expressed in C127 mammalian cell were studied. Using roller bottle culture in DMEM supplemented with fetal bovine serum, 10–15 mg/L of r-HBsAg was produced with about 31% of purification yield. The purity of r-HBsAG by HPLC was 99.8% and electron microscopic examination showed homogeneous spherical particle with 22 nm in diameter, a morphological characteristic of HBsAg. The density of r-HBsAg by CsCl density gradient method was 1.19 g/ml and the isoelectric point by Mono P^TM HR 5/20 column was 4.6. The analysis of subunit protein pattern using SDS-PAGE followed by scanning densitometry gave 81.3% of S protein and 18.7% of pre-S protein. Fluorophore-assisted-carbohydrate-electrophoresis analysis showed the relative amount of carbohydrate to protein was 1.7% and its major component was N-acetyl glucosamine, which was about 39% of total carbohydrate. The relative amount of lipid to protein determined by vanillin phosphoric acid method was 32.5% and its major component was phospholipid, which was about 70% of total lipid. The physicochemical properties of C127 mammalian cell-derived r-HBsAg are similar to those of p-HBsAg, suggesting that the r-HBsAg can be used in developing a new preventive vaccine against hepatitis B.     

乙肝疫苗无应答者外周血Treg细胞的PD-1表达研究

目的:检测乙肝疫苗无应答者外周血Treg细胞的PD-1表达情况及PD-1在Treg中发挥的作用.方法:用流式细胞术检测乙肝疫苗无应答者外周血Treg细胞的PD-1表达情况.结果:乙肝疫苗无应答者外周血Treg细胞的PD-1表达率为(48.7±2.9)%,显著高于乙肝疫苗应答者外周血Treg细胞的PD-1表达率(16.3+1.4)%.结论:乙肝疫苗无应答者外周血Treg细胞的PD-1表达率的显著上调,有可能参与了Treg的抑制机制.     

Strong and multi-antigen specific immunity by hepatitis B core antigen (HBcAg)-based vaccines in a murine model of chronic hepatitis B: HBcAg is a candidate for a therapeutic vaccine against hepatitis B virus

Experimental evidence suggests that hepatitis B core antigen (HBcAg)-specific cytotoxic T lymphocytes (CTL) are essential for the control of hepatitis B virus (HBV) replication and prevention of liver damage in patients with chronic hepatitis B (CHB). However, most immune therapeutic approaches in CHB patients have been accomplished with hepatitis B surface antigen (HBsAg)-based prophylactic vaccines with unsatisfactory clinical outcomes. In this study, we prepared HBsAg-pulsed dendritic cells (DC) and HBcAg-pulsed DC by culturing spleen DC from HBV transgenic mice (HBV TM) and evaluated the immunomodulatory capabilities of these antigens, which may serve as a better therapy for CHB. The kinetics of HBsAg, antibody levels against HBsAg (anti-HBs), proliferation of HBsAg- and HBcAg-specific lymphocytes, production of antigen-specific CTL, and activation of endogenous DC were compared between HBV TM vaccinated with either HBsAg- or HBcAg-pulsed DC. Vaccination with HBsAg-pulsed DC induced HBsAg-specific immunity, but failed to induce HBcAg-specific immunity in HBV TM. However, immunization of HBV TM with HBcAg-pulsed DC resulted in: (1) HBsAg negativity, (2) production of anti-HBs, and (3) development of HBsAg- and HBcAg-specific T cells and CTL in the spleen and the liver. Additionally, significantly higher levels of activated endogenous DC were detected in HBV TM immunized with HBcAg-pulsed DC compared to HBsAg-pulsed DC (p<0.05). The capacity of HBcAg to modulate both HBsAg- and HBcAg-specific immunity in HBV TM, and activation of endogenous DC in HBV TM without inducing liver damage suggests that HBcAg should be an integral component of the therapeutic vaccine against CHB.     

Suppression of humoral immune response to hepatitis B surface antigen vaccine in BALB/c mice by 1-methyl-tryptophan co-administration

Background and the purpose of the study

Indoleamine 2,3-dioxygenase (IDO) suppresses adaptive immune response. The purpose of this study was to determine the effect of the IDO inhibitor namely 1-methyl-DL-tryptophan (DL-1-MT) on antibody production after vaccination with hepatitis B surface (HBs) antigen.

Methods

Four groups of BALB/c mice were immunized with a HBs antigen vaccine. In the first group the vaccine had no DL-1-MT, whereas in the other three groups the vaccine contained 1 mg, 10 mg and 20 mg DL-1-MT. Blood samples were collected 5 weeks post-vaccination and anti-HBs antibodies in the serum were measured by ELISA.

Results

Compared to the three groups of mice that were immunized with the vaccines containing DL-1-MT, serum anti-HBs level was much higher in the mice that were immunized with the vaccine with out DL-1-MT.

Conclusions

Inhibition of IDO at the time of vaccination decreased humoral immune response to HBs antigen vaccine. The idea that IDO activity is simply immunosuppressive may need to be re-evaluated.     

Design, synthesis, and biological evaluation of triazolo-pyrimidine derivatives as novel inhibitors of hepatitis B virus surface antigen (HBsAg) secretion

The high levels of hepatitis B virus (HBV) surface antigen (HBsAg)-bearing subviral particles in the serum of chronically infected individuals play an important role in suppressing HBV-specific immune response and are only mildly affected by the current small molecule therapies. Thus, a therapy that specifically reduces HBsAg serum levels could be used in combination therapy with nucleos(t)ide drugs or permit therapeutic vaccination for the treatment of HBV infection. Herein, we report the design, synthesis, and evaluation of novel triazolo-pyrimidine inhibitors (1, 3, and 4) of HBsAg cellular secretion, with activity against drug-resistant HBV variants. Extensive SAR led to substantial improvements in the EC(50) of the parent compound, 5 (HBF-0259), with the best being 3c, with EC(50) = 1.4 ± 0.4 μM, SI ≥ 36. The lead candidates, both 1a (PBHBV-001) and 3c (PBHBV-2-15), were well-tolerated in both normal and HBV-transgenic mice and exhibited acceptable pharmacokinetics and bioavailability in Sprague-Dawley rats.     

慢性HBV感染者外周血T细胞亚群与病毒载量、HBeAg的相关性分析

目的：分析慢性HBV感染者外周血T细胞亚群与病毒载量、HBeAg的相关性。方法以40例慢性乙型肝炎（ CHB）、35例慢性重型乙型肝炎（ CSHB）、30例肝硬化（ LC）及32例正常对照者为研究对象,采用流式细胞仪和荧光定量PCR法分别检测各组外周血T细胞亚群、HBV DNA载量。结果 CHB组仅CD^4＋亚群明显降低,与对照组比较,差异有统计学意义（ P ＜0．05）;CSHB及LC组CD^3＋、CD^4＋亚群、CD^4＋/CD^8＋比值呈逐渐降低趋势,CD^8＋亚群呈逐渐增高趋势,与对照组比较差异有统计学意义（ P ＜0．05）。 HBV DNA载量与CD^4＋亚群、CD^4＋/CD^8＋比值呈负相关（ r ＝-0．638,-0．778, P ＜0．05）,与CD^8＋亚群呈正相关（ r ＝0．647, P ＜0．05）,与CD^3＋无关。与HBeAg阳性患者比较,HBeAg阴性患者外周血CD^＋3＋、CD^4＋和CD^8＋亚群显著降低（ P ＜0．05）。结论慢性HBV感染者随着疾病加重细胞免疫功能进行性降低,与病毒复制水平密切相关,这是导致疾病慢性化的原因之一。外周血T细胞亚群变化趋势可为判断疾病转归及预后提供可靠指标。     

Immunogenicity of a recombinant yeast-derived hepatitis B vaccine (Engerix B) in children

Eighty-one children seronegative for markers of hepatitis B virus infection aged 0-10 years received 10 micrograms of a yeast-based hepatitis B vaccine (Engerix B) at 0, 1, and 6 months. Eighty subjects responded with a geometric mean titre of 7640 IU/L one month after the third dose. All seroconverters produced high level of antibodies regardless of age, race, or sex. It is concluded that Engerix B used in the dose and schedule described is highly immunogenic.