Recombinant heat shock protein 65 carrying PADRE and HBV epitopes activates dendritic cells and elicits HBV-specific CTL responses

BCG Hsp65 and PADRE have been shown to be potent to enhance antigen specific immunity. In order to explore the possibility to utilize them for the development of HBV therapeutic vaccine, a chimeric protein, Hsp65-HBV, was created by fusing PADRE and epitopes from HBV to the carboxyl-terminus of BCG Hsp65 and expressed in E. coli. We evaluated its effects on human dendritic cell maturation and specific CTL induction in vitro. Results showed that Hsp65-HBV could activate human dendritic cells by up-regulating the expressions of HLA-A2, HLA-DR and CD86, companioning with high level of IL-12 secretion. Furthermore, Hsp65-HBV matured DCs could significantly stimulate human autologous CD8⁺ T cell proliferation and induce HBV-specific CTLs. Hsp65-HBV was also shown to generate HBsAg-specific CTLs in vivo in mice. These results indicated that Hsp65-HBV might be a candidate for the treatment of chronic HBV infection.     

Recombinant hepatitis B core antigen carrying preS1 epitopes induce immune response against chronic HBV infection

Many studies have provided evidence that core antigen of hepatitis B virus (HBcAg) is extremely immunogenic, HBcAg can be function as both a T-cell-dependent antigen and a T-cell-independent antigen, and thus may be a promising candidate for therapeutic vaccine for control of chronic HBV infection. HBcAg is also an effective carrier for heterologous peptide epitopes. The preS1 is a surface protein of HBV and is immunogenic at the T and B cell level. The amino acid sequence 21-47 of preS1 is crucial for HBV binding to human hepatocytes as well as to PBMC and haematopoietic cell lines of the B cell lineage. Here we expressed a chimeric protein named HBVCS1, created by fusing the preS1 sequence 3-55 to the carboxyl terminus of the truncated HBcAg sequence 1-155 in E. coli. Analysis of its antigenicity and immunogenicity revealed that both HBc and preS1 epitopes are surface accessible, and that fusion of preS1 did not affect the HBc antigenicity and immunogenicity of the truncated HBc sequence. HBVCS1 induced strong anti-HBc and moderate anti-preS1 immune responses as well specific T-cell response in Balb/c mice. HBVCS1 vaccination reduced of the titer of HBsAg and HBV DNA in sera of HBV-Tg mice. These results indicate that HBVCS1 may have potential as a therapeutic vaccine for treatment of HBV chronic infection.     

IgM-antibody response to the hepatitis B core antigen in acute and chronic hepatitis B.

A solid phase M-antibody capture radioimmunoassay (MACRIA) and a serum fractionation method were used to quantitate the IgM response to the hepatitis B core antigen (IgM anti-HBc) in acute and chronic hepatitis B infections. Antibody to the core antigen was predominantly of the IgM class during the acute phase of hepatitis B. Resolving acute infections remained positive by MACRIA, but at decreasing levels, for as long as 6 months. IgM anti-HBc persisted in HBsAg carriers but at levels very much lower than seen in acute infections. There was no correlation of IgM anti-HBc with severity of chronic liver disease in carriers. Measurement of IgM anti-HBc by MACRIA enabled accurate identification of acute hepatitis B on single serum specimens.     

Chimeric hepatitis B virus core particles carrying an epitope of anthrax protective antigen induce protective immunity against Bacillus anthracis

The major aim of present study is to develop and evaluate chimeric virus-like particles (VLPs) displaying a neutralizing epitope of anthrax protective antigen (PA) as a potential vaccine against anthrax. The truncated hepatitis B virus core (HBc) protein (aa 1-144) was used as a carrier, and the 2beta2-2beta3 loop of the PA domain 2 (aa 302-325) which has been shown contains a dominant neutralizing epitope was inserted into the major immunodominant region (MIR) of the HBc. The recombinant protein HBc-N144-PA-loop2 was expressed in Escherichia coli, and was able to form HBc-like particles confirmed by electron microscopy. The immunogenicity of these chimeric particles was evaluated in mice and guinea pigs. In mice the HBc-N144-PA-loop2 was able to induce PA-epitope specific antibodies; in guinea pigs it was able to induce PA-epitope specific antibodies and anthrax toxin-neutralizing antibodies regardless of whether alum adjuvant was used or not, and was able to partially protect the immunized guinea pigs against virulent anthrax spores challenge. This study suggests chimeric HBc particles carrying a neutralizing epitope of PA can induce protective immunity against Bacillus anthracis.     

在校大学生乙肝疫苗普种效果对比观察

目的 分析北京市农村居民乙肝疫苗接种状况及影响因素,为提高农村居民乙肝疫苗接种率和乙肝防控政策的制定提供参考依据。 方法 本研究数据采集于2011年1月 — 2012年4月,应用随机抽样的方法和结构式访谈问卷调查的方式在北京大兴区3个自然村进行入户调查 ≥ 16岁成年人,并采用方差分析、二元logistic回归模型进行统计分析。 结果 本研究共调查2 006人,总接种率29.86 %。其中男性1 004人（50.05 %）,接种率30.38 %,女性1 002人（49.95 %）,接种率29.34 %;平均年龄39.8岁。多因素分析中,结合回归系数和OR值分析可得,随着年龄的下降、家庭人均年收入的增加、乙肝认知水平的增加,乙肝疫苗的接种概率逐渐上升（P < 0.01）。职业分组中,接种率由高到低依次是学生、固定工作者、个体工商业者、打工者、农民（P < 0.01）。到达最近的医疗机构花费的时间越短,乙肝疫苗接种概率越高（P < 0.01）。 结论 高年龄、低收入、认知水平低都是阻碍乙肝疫苗接种的重要因素,农民和打工者群体接种率最低,医疗服务的可及性也会影响乙肝疫苗接种率。     

乙型和丙型肝炎患者及正常人外周血中热休克蛋白70的检测

热休克蛋白(heat shock protein,HSP)是一类结构和功能高度保守的蛋白质,广泛存在于原核及真核细胞内,如胞浆、内质网、线粒体及细胞核内。除了作为构成性表达外(约占生理状态下总蛋白含量的5~10%),HSP还可在各种应激条件下诱导表达,如高温、氧自由基、紫外线辐射、感染和恶性肿瘤等[1]。HSP根据分子量不同可分为若干类,病毒感染后产生的蛋白质可诱导宿主细胞表达HSP70。已报道的如腺病毒[2]、vaccinia病毒[3]、巨细胞病毒[4]等均可诱导HSP70蛋白质及mRNA的增高。在一些病毒(如水疱性口炎病毒、流感病毒A等)的成熟病毒颗粒中也可检…     

热休克蛋白Hsp70在抗原呈递过程中的作用

目的研究热休克蛋白Hsp70家族在抗原呈递中的作用，探讨其与肿瘤免疫反应的关系。方法利用反义RNA技术，将反义Hsp70、反义Hsc70、B7表达质粒导入肿瘤细胞B16中，24h后检测细胞中热休克蛋白表达水平。利用混合淋巴细胞培养，检测T淋巴细胞增殖能力。结果在黑色素瘤细胞B16中转染pLXSN—antihsp70、pLXSN—antihsc70及2者共转染，转染反义质粒能够抑制B16细胞中Hsp70／Hsc70的表达，导致Hsp70表达下调。其刺激T淋巴细胞增殖能力均大幅下降。而将pLXSNmB7转染入B16细胞中，其刺激T细胞增殖的能力均有所上升。转染pLXSNmB7后，B16细胞中Hsp70／Hsc70的表达量有所提高。结论抑制各热休克蛋白Hsp70家族的表达能够导致T细胞增值数降低。在肿瘤细胞中导入B7分子能够提高Hsp70的表达水平，从而提高其抗原呈递的能力。     

Recombinant hepatitis B large surface antigen, successfully produced in Escherichia coli, stimulates T-cell response in mice

A fusion protein consisting of the PA binding domain of anthrax lethal factor (LFn) and a codon optimized Hepatitis B virus large surface antigen (LHBsAg) expresses well in Escherichia coli. The LFn-LHBsAg fusion protein effectively elicits a cell-mediated immune (CMI) response to the hepatitis B viral antigens in mice.     

Recombinant ovine atadenovirus induces a strong and sustained T cell response against the hepatitis C virus NS3 antigen in mice

Ovine atadenovirus (OAdV) is a novel gene transfer vector with excellent in vivo gene transfer characteristics. In the present study, we have investigated the ability of an OAdV vector to mediate a T cell response to an antigen of the hepatitis C virus (HCV) in mice. Specifically, an expression cassette coding for non-structural protein 3 (NS3) of hepatitis C virus was inserted into the OAdV genome and the resulting recombinant virus (OAdV-ns3) was shown to propagate stably and to express the ns3 gene at a high level in vitro. A single injection of this non-replicating vector into BALB/c mice resulted in a strong induction of NS3-specific, IFN-gamma secreting T-lymphocytes as measured by direct ex vivo ELISpot assay. The number of IFN-gamma secreting lymphocytes remained nearly unaltered for a period of at least 10 weeks. The immune response was shown to depend on virus dose but a single intramuscular injection of less than 10(8) infectious particles of OAdV-ns3 was sufficient to induce a significant NS3-specific T cell response. Moreover, this response was not affected by prior immunisation of animals with human adenovirus type 5 (HAdV-5). The results of our study provide proof for the concept that OAdV vectors may be valuable tools for vaccination and immunotherapy even in the face of natural immunity to human adenoviruses.     

Plant heat shock protein 90 as carrier-adjuvant for immunization against a reporter antigen

Here, we evaluated the modulation of the immune response induced by Hsp90 of Nicotiana benthamiana (NbHsp90.3) against the Maltose Binding Protein (MBP) as a reporter antigen. Equimolar quantities of recombinant proteins were administered in mice as follows: MBP alone (MBP group), a mixture of MBP and rNbHsp90.3 (MBP + rNbHsp90.3 group) and the fusion of MBP to rNbHsp90.3 (MBP-rNbHsp90.3 group). The covalent linkage between NbHsp90.3 and MBP to bring a fusion protein was essential to induce the strong specific antibody response with predominance of IgG2a. Eighty-four days after the first immunization, splenocyte proliferation from MBP-rNbHsp90.3-immunized mice was consistently higher than that from MBP and MBP + rNbHsp90.3 groups. In addition, splenocytes from MBP-rNbHsp90.3 immunized mice produced higher levels of IFN-γ than controls. Finally, both formulations with rNbHsp90.3 significantly enhanced the MHC class I expression levels, but only rNbHsp90.3 covalent bound to MBP induced a specific cellular immune response against MBP measured as increased percentage of CD8⁺ T cells. Taken together, these results suggest that plant HSP90s could be incorporated as adjuvants in vaccines that require the generation of a Th1 response along with a CD8 cytotoxic cell response to confer immunity.     

Development of meningococcal polysaccharide conjugate vaccine that can elicit long-lasting and strong cellular immune response with hepatitis B core antigen virus-like particles as a novel carrier protein

Neisseria meningitidis caused meningitis is life-threatening acute infection with high fatality and high frequency of severe sequelae. Meningococcal capsular polysaccharides can be used to prevent meningococcal disease; while conjugating the polysaccharides to carrier protein was found necessary to improve the immunogenicity and induce memory responses in infants and young children. Nevertheless, repeated administration of glycoconjugate vaccines might lead to carrier-induced epitope suppression due to limited number of carrier proteins. Here in this study, full-length hepatitis B core antigen virus-like particles (HBc VLPs) was used as a novel potential carrier protein for conjugation of meningococcal group C polysaccharides (CPS) with heterobifunctional polyethylene glycol (PEG) of different length (2, 5 and 10?kDa) as linkers. The physiochemical properties of the CPS-PEG-HBc conjugate vaccines were fully characterized. The TEM, DLS, native agarose gel electrophoresis, and HPLC analyses all confirmed the successful conjugation. As compared to plain CPS and the physical mixture of CPS and HBc, the immunization with the conjugate vaccines can generate about 10 times increase in CPS specific IgG titers with a significant boosting effect. HBc conjugation induced a shift to a Th1 cellular immune type response, as assessed by the increased IgG2a subclass production. In addition, vaccination of the conjugate vaccines elicited much enhanced avidity functional antibody and long-lasting immunological memory. IgG titers elicited by CPS-P2k-HBc, CPS-P5k-HBc and CPS-P10k-HBc at week 18 maintained 38.1%, 17.9% and 33.3% of their peak values. All these results demonstrated that HBc VLPs can be used as potential carrier protein to develop polysaccharide conjugate vaccines effective in eliciting long-lasting and strong cellular immune response.     

原发性肝癌患者HBV前C/BCP区变异与HBeAg的相关性研究

目的 分析原发性肝癌(HCC)患者乙型肝炎病毒(HBV)前C区和C基因基本核心启动子(BCP)变异情况及与HBeAg的关系,为HCC的发病机制及预后提供理论依据.方法 收集39例原发性肝癌患者血清,采用巢式PCR法扩增前C/BCP区基因片段,PCR产物直接测序以检测前C/BCP区基因变异情况.结果 BCP区T1762/A1764双变异28例,前C区A1896变异19例,T1762、A1764、A1896聚集变异16例,在HBeAg阴性者中分别出现17、10、10例,HBeAg阳性者则分别为11、9、6例,上述3种变异HBeAg阴性组与HBeAg阳性组比较,差异无统计学意义;其他变异位点有C1753、T1846、A1899和T1846变异,其中T1846变异7例HBeAg阴性者中出现6例,HBeAg阳性者中出现1例,比较差异有统计学意义(P＜0.05).结论 原发性肝癌HBV前C/BCP区A1896、T1762、A1764双变异及T1762、A1764、A1896聚集变异在HBeAg阴性者中较常见,但对HBeAg表达影响不明显.     

L-Threonine induces heat shock protein expression and decreases apoptosis in heat-stressed intestinal epithelial cells

ObjectivesOsmotically acting amino acids can be cytoprotective following injury. As threonine (THR) induces osmotic cell swelling, our aim was to investigate the potential for THR to induce cellular protection in intestinal epithelial cells and evaluate possible mechanisms of protection.MethodsCells treated with a range of THR doses were evaluated following heat stress (HS) injury. Alpha-aminoisobutyric acid (AIB), a non-metabolizable amino acid analog, was used as an osmotic control. MTS assays were used to assess cell survival. Heat shock protein (HSP) expression and cleaved caspase-3 (CC3) were evaluated via Western blot. Cell morphology and cell size were analyzed via microscopy.ResultFollowing HS, THR treatment increased cell viability in a dose dependent manner vs. non-THR treated cells (CT). The non-metabolized amino acid analogue, AIB, also increased cell survival in heat-stressed cells versus HS controls. HSP70 and HSP25 expression increased with THR and AIB treatment versus HS controls. THR also increased HSP25 in non-stressed cells. Microscopic evaluation revealed both THR and AIB preserved the structural integrity of the actin cytoskeleton in heat-stressed cells versus HS controls. THR, but not AIB, enhanced nuclear translocation of HSP25 during HS. This nuclear translocation was associated with a 60% decrease in apoptosis in heat-stressed cells with THR. No antiapoptotic effect was observed with AIB.ConclusionsThis is the first demonstration that THR increases HSP70 and HSP 25 and protects cells from HS. THR's mechanism of protection may involve cytoskeletal stabilization, HSP up-regulation and nuclear translocation, and decreased apoptosis. THR's protection appears to involve both cell-swelling–dependent and –independent processes.     

Influence of maternal antibody against hepatitis B surface antigen on active immune response to hepatitis B vaccine in infants

Transplacentally acquired maternal antibodies in infants may inhibit active immune responses to vaccines. In this study, we compared the immunogenicity of the recombinant hepatitis B vaccine, which was intramuscularly injected at 0, 1, and 6 months of age, in 71 infants born to mothers with positive or negative antibody against hepatitis B surface antigen (anti-HBs). Forty-one infants born to anti-HBs positive mothers were all positive at birth. At 2 months after the second injection, anti-HBs in 30 infants with negative maternal antibody was significantly higher than that in 41 infants with positive maternal anti-HBs (191.1mIU/ml vs. 96.2mIU/ml, P=0.018). At one month after the full immunization, the anti-HBs levels had no statistical difference between maternal anti-HBs negative and positive groups, but the antibody response in infants with high maternal anti-HBs (>1000mIU/ml) was significantly inhibited. Nevertheless, all infants had anti-HBs higher than the protective level. In conclusion, passively acquired maternal anti-HBs in infants may to some extent impair the antibody response to hepatitis B vaccine. The long-term efficacy of hepatitis B vaccine in infants with high titers of maternal anti-HBs remains to be further evaluated.     

Maternal antibody to hepatitis B core antigen detected in dried neonatal blood spot samples.

Despite Department of Health recommendations, universal antenatal testing for hepatitis B virus (HBV) is not performed throughout Scotland. We describe the evaluation of an assay to document past or present infection with HBV, by identifying maternal antibody in routine Guthrie dried neonatal blood spot samples taken when infants are 7 days old. A modified haemagglutination assay to detect antibody to hepatitis B core antigen (CORECELL, Green Cross) was validated and found to be 79% sensitive (44/56) and 100% (105/105) specific when used with dried blood spot samples made from panels of serum of known reactivity. Ninety-three percent (13/14) of HBV carriers were CORECELL positive. Sixty-six (0.5%) of 14044 routine Guthrie samples taken from babies born in Scotland from June August 1992 were CORECELL positive indicating past or present maternal infection with HBV. A cross-sectional survey would document the maternity hospitals where universal antenatal hepatitis B screening should be urgently established.     

Oral administration of Lactococcus lactis delivered heat shock protein 65 attenuates atherosclerosis in low-density lipoprotein receptor-deficient mice

Lactococcus is a genus of Gram positive food-grade bacteria that has been widely used as a vaccine platform for the safe delivery of heterologous antigens. Many reports support the involvement of inflammation and immunity in atherosclerosis as well as the role of autoimmunity to heat shock proteins (HSPs) in the progression of atherogenesis. In this study, experiments were specifically designed to investigate the effect of oral administration of mycobacterial heat shock protein 65 (HSP65) delivered by Lactococcus lactis (L. lactis) on atherogenesis. Two types of HSP65-encoding plasmids for intracellular expression or secretion were constructed, and then transformed into L. lactis NZ9000. Oral administration of two recombinant L. lactis strains both induced suppression of HSP65-specific proliferation, accompanied by elevation of Interleukin-10 (IL-10) production and reduction of interferon-gamma (IFN-γ) level. Inducible HSP65-specific tolerance exerted a protective effect on atherosclerotic lesion formation and endothelial damage in low-density lipoprotein receptor-deficient (LDL-RD) mice model, while no obvious pathological abnormalities were observed. In conclusion, delivery of HSP65 at the intestinal mucosa by recombinant L. lactis provides a novel approach for the prevention of atherosclerosis. The results further illustrate the potential of using genetically modified L. lactis as a safe and effective vaccine delivery to elicit antigen-specific tolerance for treatment of autoimmune diseases.     

Efficacy of antigen dosage on the hepatitis B vaccine response in infants born to hepatitis B-uninfected and hepatitis B-infected mothers

ObjectiveTo compare the safety and immunogenicity of two dosages of recombinant hepatitis B (HB) vaccine administered to infants born to HB-uninfected and HB-infected mothers.MethodsA phase III, controlled, single-blinded clinical trial was conducted with 506 healthy newborns. The newborns were assigned to three groups based on maternal levels of HB surface antigen (HBsAg) and HB e antigen (HBeAg): Group A, HBsAg negative; Group B, HBsAg positive and HBeAg negative; and Group C, HBsAg positive and HBeAg positive. Three doses of 10 or 5 μg recombinant HB vaccine were randomly administered by 1:1 within 24 h after birth, at 1 month and at 6 months. Safety data and pre- and postvaccination blood samples were collected.ResultsA total of 326, 93, and 87 subjects were included in Groups A, B, and C, respectively. Both dosages of HB vaccine were well tolerated by all subjects. The most common injection-site adverse reactions (ARs) and systemic ARs were pain and fever. After 1 month of the third dose, the Group A infants who received the 10 μg HB vaccine achieved a higher geometric mean concentration (GMC) of HB surface antibody (anti-HBs) than those who received the 5 μg dosage. Maternal anti-HBs serostatus did not influence HB vaccine immunogenicity at either dosage. In contrast, there was no significant difference in the anti-HBs seroconversion rate, GMCs, or estimated vaccine efficacy (EVE) against perinatal transmission between Groups B and C, regardless of dosage. However, the seroconversion rate and EVE of the 5 μg HB vaccine was lower in Group C than in Group B.ConclusionsBoth dosages of the HB vaccine were well tolerated and elicited a good immune response in infants of Group A, regardless of the maternal anti-HBs serostatus. EVE did not significantly differ between Groups B and C.Clinicaltrails.gov identifier: NCT02152709     

蛋白质芯片定量检测乙肝表面抗原系统的建立

目的：建立以蛋白质芯片检测乙肝表面抗原的方法,以期对乙肝患者血清中HBsAg进行定量检测,帮助动态分析病情和评估疗效。方法：用点样仪将一种抗-HBsAg单抗点到硝酸纤维素膜（NC膜）上制成芯片,HRP标记另一种抗-HBsAg单抗,以双抗夹心法捕获血清中HBsAg,建立HBsAg浓度与检测点灰度间关系的标准曲线并通过芯片阅读仪进行定量分析。结果：建立的蛋白质芯片检测系统能快速准确地对血清中HBsAg进行定性和定量检测,系统的定量检测范围是1-10000 ng/ml。结论：蛋白质芯片检测系统具有微型化和自动化的优点,与PCR HBV-DNA相比其操作更简便,所需时间更短,可以代替传统的ELISA方法,是更为科学的疗效评估方法。