乙型肝炎病毒DNA疫苗的研究进展

预防与控制乙型肝炎发病的乙型肝炎病毒(HBV)疫苗,是有重大的社会和经济意义。HBV的持续感染可引起慢性肝脏疾患,并逐步发展为肝硬化和肝细胞癌(HCC)。目前的乙肝重组亚单位疫苗可以使90％的接种产生保护性抗体;但是对慢性HBV携带,由于其机体对HBsAg蛋白产生耐受,不能产生体液和细胞免疫,因此它只能作为一种预防性的疫苗。DNA疫苗(基因疫苗)是一种新的疫苗技术,通过向体内递送编码抗原的细菌质粒,刺激产生特异的体液和细胞免疫反应。在小鼠和其他的肝炎病毒感染动物模型中,HBV DNA疫苗可以特异性地引起体液和细胞免疫,清除HBV转基因动物血循环中的HBsAg颗粒和HBV DNA。如果加入各种免疫调节细胞因子的基因,可以进一步提高HBV DNA疫苗的免疫效果,因此它不仅可作为预防性疫苗,也可作为治疗型疫苗。     

聚乙二醇作为乙型肝炎病毒DNA疫苗佐剂的体外试验

摘要：【目的】研究聚乙二醇作为佐剂是否能够增强HBV DNA疫苗体液和细胞免疫效果。【方法】我们将乙型肝炎病毒(HBV) DNA 疫苗（pVAX-S2）单独或PEG/pVAX-S2免疫C57BL/6小鼠,在最后一次免疫1 d检测抗-HBs IgG、T淋巴细胞增殖、细胞因子表达及体内细胞毒T淋巴细胞杀伤作用（CTL）等免疫学指标。【结果】结果表明,与仅免疫pVAX-S2比,PEG/ pVAX-S2能够增加免疫小鼠抗-HBs IgG水平; 混合免疫组的T淋巴细胞体外经乙肝表面抗原（HBsAg）刺激后,T淋巴细     

乙型肝炎病毒前S1抗原检测及其临床意义

目的:探讨乙型肝炎病毒前S1抗原与血清标志物之间的关系,了解其临床意义。方法:采用酶联免疫吸附实验检测乙型肝炎病毒血清标志物及前S1抗原,并采用速率法检测血清丙氨酸氨基转移酶。结果:乙肝表面抗原阳性率为9．2%,乙肝表面抗原阳性人群中前S1抗原阳性率为28．6%;乙肝e抗原阳性及阴性人群中前S1抗原阳性率分别为81．1%、13．9%(p＜0．01);前S1抗原阳性人群ALT(49．5U/L)高于前S1抗原阴性人群(43．2U/L,p＜0．01)。结论:前S1抗原与血清标志物e抗原有较高的一致性,是反映病毒复制的良好指标,能较早发现肝损伤。     

重组CHO细胞乙肝表面抗原诱导小鼠细胞免疫应答的研究

为了研究重组CHO细胞乙肝表面抗原(CHO-rHBsAg)在小鼠中诱导T细胞免疫应答的能力,全面评价疫苗的免疫原性,以CHO-rHBsAg免疫BALB/c小鼠,常规制备小鼠脾脏淋巴细胞并在体外以抗原或特异多肽刺激;采用ELISA法测定抗原特异性T淋巴细胞分泌的细胞因子,乳酸脱氢酶法(LDH)测定抗原特异性细胞毒T淋巴细胞(CTL)活性,酶联斑点法(ELISPOT)测定CTL频数(CTLp),应用流式细胞仪分析T淋巴细胞亚群。结果显示,rHBsAg可在小鼠中诱导Th1及Th2类细胞因子;加铝佐剂的rHBsAg较未加佐剂的抗原可诱导较高水平的IFN-γ、CTL克隆及较高百分比的CD8+T淋巴细胞亚群。重组CHO细胞来源的HBsAg可在BALB/c小鼠中诱导一定程度的细胞免疫应答。     

含体内激活启动子的重组减毒鼠伤寒沙门菌诱导转基因鼠细胞免疫应答

以庚型肝炎病毒 (hepatitisGvirus ,HGV)转基因小鼠为模型 ,探讨逆转免疫耐受的方法及与HGV致病性的关系。首先采用鼠伤寒沙门菌pagC基因的启动子 (PpagC)为转录调控元件 ,构建宿主体内表达HGVNS3抗原的重组减毒鼠伤寒沙门菌 ,并口服接种免疫HGV转基因小鼠。结果证明对诱导转基因小鼠血清HGVNS3抗体无明显影响 ,但对小鼠血清HGV抗原含量、肝组织HGV抗原及HGVmRNA表达量有明显抑制作用。体外培养脾细胞表现出针对HGVNS3抗原的细胞免疫反应 ,并检测到Th1 型细胞因子IFN γ。过继转移实验证明T细胞可能是通过IFN γ介导的机制抑制转基因鼠体内HGV的表达及复制。组织病理学检查显示 ,转基因小鼠肝组织见淋巴细胞浸润等轻度炎性变。T细胞免疫耐受消除的结果提示 ,这种宿主体内激活目的抗原表达的口服疫苗有望成为治疗病毒性肝炎的新方法。     

乙型肝炎病毒表面抗原基因在异源细胞中的表达研究

本研究构建了一系列乙型肝炎病毒表面抗原(HBsAg)基因的表达载体,其中HBsAg基因的启动区被小鼠金属硫蛋白启动子取代;而对HBsAg基因下游非编码区的几种基因元件则进行了不同的删除,并且添加异源基因调控元件。利用Hela细胞瞬时表达系统对各种构建物的表达水平进行分析。实验发现：除了必须取代HBsAg基因原有的启动子外,乙型肝炎病毒(HBv)增强子Ⅰ及其多聚腺苷酸化(poly A)信号对HBsAg在非肝脏细胞中表达也至关重要。但HBsAg的poIy A信号可用异源的poly A信号及猴空泡病毒(sV 40)T抗原基因的剪接信号取代。在HeLa细胞表达系统中,这种取代可进一步提高HBsAg的表达水平。反之,HBV增强子Ⅱ在HeLa细胞中对HBsAg基因的表达无显著作用。实验结果提示：除了已报道的HBV增强子Ⅰ能激活基因本身的启动子外,很可能还存在另外的功能,即对HBsAg转录体起稳定作用。     

大剂量HBsAg 对HBV 转基因小鼠T 细胞免疫调节的研究

目的:研究大剂量HBsAg对HBV转基因小鼠其T细胞免疫效果的影响。方法:用大剂量血源性HBsAg免疫HBV转基因小鼠,采用ELISA方法观察转基因小鼠所诱生的HBsAg特异性Th1类细胞因子的水平,ELISPOT方法检测不同免疫方案对小鼠HBsAg特异性分泌IFN-γT细胞数量的影响,同时检测对小鼠淋巴细胞增殖的影响。结果:HBsAg组免疫后脾细胞产生的Th1类细胞因子(IFN-γ、IL-2)、HBsAg特异性分泌IFN-γT细胞及T细胞增殖水平较对照组显著增加(P<0.05)。结论:大剂量的HBs-Ag可以诱导乙肝转基因小鼠产生高水平Th1类细胞因子并打破免疫耐受。     

表达丙型肝炎病毒(HCV)截短型NS3与core融合抗原的重组腺病毒疫苗在小鼠中的免疫原性与保护效果分析

为研发安全广谱有效的丙型肝炎病毒(Hepatitis C virus,HCV)T细胞疫苗,本研究构建了表达HCV截短型非结构蛋白3(Nonstructural protein 3,NS3)与核心蛋白(core)融合抗原的重组腺病毒疫苗。体外免疫荧光及蛋白印迹实验表明该融合抗原可有效表达;小鼠免疫结果表明该重组腺病毒疫苗除了激发抗原特异的抗体反应外,还可激发较强的针对NS3抗原特异的T细胞免疫应答。该T细胞免疫应答主要表现为IFN-γ+与TNF-α+CD4+T细胞亚群。采用异型(JFH1,2a型)HCV重组痘病毒接种小鼠进行保护效果分析,与对照组相比,表达截短型NS3与core融合抗原的重组腺病毒疫苗2针免疫后可产生明显的交叉免疫保护。本研究为进一步研究HCV免疫保护机制及新型疫苗研制提供了参考。     

IL-12增强HBV融合抗原DNA疫苗在小鼠诱导的细胞免疫应答

乙型肝炎病毒(hepatitis B virus,HBV)极易形成慢性感染,主要机制在于感染者不能产生强有力的细胞免疫应答以清除病毒[1].慢性HBV感染者体内虽然存在HBV抗原特异性T淋巴细胞,但对HBV抗原的反应性较低.研究发现,增强这类T淋巴细胞的反应性,可以促进HBV的清除[2].     

“a”决定簇145位点变异的HBsAg单克隆抗体制备及HBV变异株快速检测方法

HBsAg抗原145位突变,可能导致临床上广泛使用的HBsAg诊断试剂出现漏检.通过杂交瘤技术制备出了一种抗该突变体的单克隆抗体,属IgG1亚类(K型,效价为1:9×104)与其他类犁肝炎病毒抗原无交叉反应.采用该单抗与多克隆抗体所建立的AC-ELISA法,对临床上乙型肝炎病毒(HBV)的突变样品进行检测的特异性和敏感性较高.     

Platycodin D2 Improves Specific Cellular and Humoral Responses to Hepatitis B Surface Antigen in Mice

Platycodin D2 ( 1 ), a less hemolytic saponin from the root of Platycodon grandiflorum than platycodin D ( 2 ), was evaluated for the potential to enhance specific cellular and humoral immune responses to hepatitis B surface antigen (HBsAg) in mice. It significantly increased the concanavalin A (Con A)‐, lipopolysaccharide (LPS)‐, and HBsAg‐induced splenocyte proliferation in HBsAg‐immunized mice (P<0.05, P<0.01, and P<0.001, resp.). HBsAg‐specific IgG, IgG1, IgG2a, and IgG2b antibody titers in the serum were also markedly enhanced by 1 compared to the HBsAg control group (P<0.01 or P<0.001). Moreover, 1 significantly promoted the production of Th1 (IL‐2 and IFN‐γ) and Th2 (IL‐4 and IL‐10) cytokines from splenocytes in the HBsAg‐immunized mice (P<0.001). The adjuvant potential of 1 on splenocyte proliferation, serum HBsAg‐specific IgG2a and IgG2b antibody response, as well as Th1‐cytokine secretion from splenocytes in the HBsAg‐immunized mice was higher than that of Alum. The results suggest that 1 could improve both cellular and humoral immune responses to HBsAg in mice. Hence, 1 might be a promising adjuvant for hepatitis B vaccine with dual Th1‐ and Th2‐potentiating activity.     

Effect of hepatitis A virus infection on cell metabolism in vitro

Hepatitis A virus (HAV), when inoculated into cultures of the PLC/PRF/5 cell line which produces the surface antigen of hepatitis B virus (HBsAg), showed growth characteristics different from those of other picornaviruses. Antigen of HAV (HAAg) is expressed only about 10 days after infection. No major impact on the overall macromolecular biosynthesis of the host cells is observed. The growth rate of HAV-infected and uninfected cells was comparable, although the plating efficiency of infected cells was lower. Different hormonal factors were tested for their ability to stimulate viral antigen expression. Dexamethasone or prostaglandin E1 added to the culture medium increased HAAg expression; insulin reduced expression. Persistent infection of hepatoma cells by HAV never led to a cytolytic infection. In temperature-shift experiments, an adverse effect on the expression of HAAg and HBsAg was observed. In all experiments, the amounts of HBsAg in HAV-infected cells were reduced. On the whole, no major influence on host-cell metabolism is observed in cells persistently infected with HAV. Cell-mediated immunological response as a mechanism of pathological changes in HAV-infected liver is, therefore, more likely than a cytopathological effect.     

Hepatitis B virus-reactive human T lymphocyte clones: antigen specificity and helper function for antibody synthesis

To study immunity to hepatitis B surface antigen (HBsAg) at the cellular level, lymphocytes were obtained from the peripheral blood of hepatitis B vaccine recipients and were examined for various immune responses to HBsAg in vitro. The peripheral blood mononuclear cells (PBM) from most of the vaccinees did not proliferate to a great extent to HBsAg in vitro. However, HBsAg-reactive lymphocyte lines and clones were obtained from some of these individuals if the PBM were stimulated in vitro with HBsAg and were maintained in the presence of T cell growth supplement. Most of the HBsAg-reactive T cell clones obtained were found to be antigen-specific and some of them provided help in the production of anti-HBsAg antibodies by a cell population enriched for HBsAg-binding cells. These results indicate that HBsAg-specific T and B cells exist in the circulation of hepatitis B vaccine recipients, although they are at limiting concentrations for the in vitro cell proliferation and antibody production assays.     

Immunization of Woodchucks with Plasmids Expressing Woodchuck Hepatitis Virus (WHV) Core Antigen and Surface Antigen Suppresses WHV Infection

DNA vaccination can induce humoral and cellular immune response to viral antigens and confer protection to virus infection. In woodchucks, we tested the protective efficacy of immune response to woodchuck hepatitis core antigen (WHcAg) and surface antigen (WHsAg) of woodchuck hepatitis virus (WHV) elicited by DNA-based vaccination. Plasmids pWHcIm and pWHsIm containing WHV c- or pre-s2/s genes expressed WHcAg and WHsAg in transient transfection assays. Pilot experiments in mice revealed that a single intramuscular injection of 100 μg of plasmid pWHcIm DNA induced an anti-WHcAg titer over 1:300 that was enhanced by boost injections. However, two injections of 100 μg of pWHcIm did not induce detectable anti-WHcAg in woodchucks. With an increase in the dose to 1 mg of pWHcIm per injection, transient anti-WHcAg response and WHcAg-specific proliferation of peripheral mononuclear blood cells (PMBCs) appeared in woodchucks after repeated immunizations. Four woodchucks vaccinated with pWHcIm were challenged with 10⁴ or 10⁵ of the WHV 50% infective dose. They remained negative for markers of WHV replication (WHV DNA and WHsAg) in peripheral blood and developed anti-WHs in week 5 after challenge. In contrast, woodchucks not immunized or immunized with the control vector pcDNA3 developed acute WHV infection. Two woodchucks immunized with 1 mg of pWHsIm developed WHsAg-specific proliferative response of PBMCs but no measurable anti-WHsAg response. A rapid anti-WHsAg response developed during week 2 after virus challenge. Neither woodchuck developed any signs of WHV infection. These data indicate that DNA-based vaccination with WHcAg and WHsAg can elicit immunity to WHV infection.     

Delayed-Type Hypersensitivity Skin Reaction to HBsAg and Immunohistopathologic Study of Liver in Patients with Acute Type B Viral Hepatitis

In an attempt to clarify the role of cellular immunity in the pathogenesis of acute type B viral hepatitis (AHB), we studied delayed-type hypersensitivity (DTH) skin reaction to hepatitis B surface antigen (HBsAg) and immunohistopathology of livers in patients with AHB. DTH skin reaction to HBsAg developed early in the convalescent phase in all 14 patients with AHB. In contrast, the production of anti-HBs was significantly delayed in these patients, compared with healthy controls immunized with HB vaccine intradermally (P< 0.001). These observations suggest that DTH to HBsAg but not anti-HBs may be associated with recovery from AHB. In the biopsied livers obtained from patients with AHB, the proliferation of Kupffer cells was extensive and immunohistopathologic studies revealed an accumulation of CD-4 positive lymphocytes in the portal area, a finding which suggested a DTH reaction in the liver with AHB. CD-8 positive cells had infiltrated the lobule and made contact with affected hepatocytes, thereby indicating that a cytotoxic T cell response is involved in damaging of the infected cells. All these observations taken together, we propose that not only a cytotoxic T cell response but also a DTH reaction may be involved in the pathogenesis of AHB.     

乙肝病毒表面抗原基因在人参细胞中的表达

为获得表达乙肝病毒表面抗原(HBsAg)的人参细胞系,构建携带HBsAg基因的植物细胞表达载体pBIBSa,采用以农杆菌LBA4404感染的方法,经G418筛选后得到13株具有抗性的人参细胞.提取基因组DNA进行PCR反应,其中8株得到约700bp的HBsAg基因片段;提取mRNA进行RT-PCR反应,其中6株得到约700bp的HBsAg基因片段;用ELISA方法检测HBsAg,6株均为阳性.每克人参细胞中最高含HBsAg184 ng,占细胞可溶性蛋白的0.009%.免疫组化切片染色显示HBsAg主要分布在细胞膜上,少量位于细胞核中.这些结果表明人参细胞整合了HBsAg基因,并能稳定表达HBsAg.     

Hepatitis C virus subverts pattern recognition receptors-mediated control of adaptative immunity orchestrated by dendritic cells

Chronic hepatitis C virus (HCV) is a liver-borne infectious disease that remains a major global health threat. The mechanisms whereby HCV evades the host's immune defences and establishes persistent infection remain elusive; but they likely require a complex and coordinated interruption of the interplay between innate and adaptive immune actors. This review discusses the concept that HCV evades the host's immune response to its components partly because of its ability to inactivate the major orchestrator of the adaptive immune response - the DCs. It argues that DCs constitute an immunologically relevant cellular viral host actively targeted by HCV. This targeting disrupts TRIF- and IPS-1-dependent but not MyD88-coupled pathogen recognition receptors (PRR) sensing pathways in these infected cells to foil the networks by which innate immunity to HCV is translated into virus-specific adaptive immune-mediated host resistance. Thus, as a culprit, this cell-specific and numerically restrained DC defect offers a promising field of investigation in which to study and understand the HCV-restricted nature of the deficit in cellular immunity in persistently infected -individuals who have otherwise normal immune functions to unrelated pathogens. In this model, protective immunity is contingent on proper processing and delivery of danger signals by DCs presenting HCV antigens.     

Cell-mediated Immunity in Patients Positive for Hepatitis-associated Antigen

Cell-mediated immunity to antigens prepared from both serum and liver of patients positive for hepatitis-associated antigen (H.A.A.) was measured by using the leucocyte migration test. Altogether, 43 patients with H.A.A.-positive acute and chronic liver disease, eight with serum antibody to H.A.A., and 13 controls were studied. The cell-mediated immunity detected was specific for H.A.A. or other antigenic determinants of the associated infective agent and could be found only in patients with evidence of previous contact with H.A.A.Cell-mediated immunity to the H.A.A.-positive test antigens was found in all but one of the patients with acute hepatitis, in about half of the patients with chronic aggressive hepatitis or cirrhosis, rarely in those with chronic persistent hepatitis, and in none of the apparently healthy carriers.Our results support the hypothesis that the cellular immune response plays an important part in the clearance of the infective agent from H.A.A.-positive patients and in the pathogenesis of the associated liver cell injury.