不同佐剂对含S+PreS1融合抗原的乙型肝炎病毒颗粒疫苗免疫原性的影响

本研究在前期工作基础上,用CHO细胞表达的含PreS1+S融合抗原的新型基因工程HBV颗粒疫苗(HBSS1)与Al(OH)3、CpG及CpG+Al(OH)3等佐剂配伍,在Balb/C小鼠模型上研究不同佐剂对HBV颗粒疫苗肌肉注射后免疫应答的影响,主要包括抗体滴度、抗体亚型分类及特异性细胞免疫(γ-IFNELISpot检测)。结果表明:CpG佐剂结合HBSS1颗粒疫苗可快速诱导(单针免疫)高水平的抗PreS1及S抗体,IgG2a/IgG1比率1,同时可诱导较高抗原特异的细胞免疫应答;Al(OH)3+CpG双佐剂组一次免疫后可诱导产生最高的抗S抗体滴度(1:105),其产生的抗体亚类包括IgG1、IgG2a与IgG2b;在S抗原N端(13~49aa)存在优势CTL表位。结论:CpG佐剂结合HBSS1颗粒疫苗应是发展新型治疗性乙肝疫苗的较佳选项。     

钙调磷酸酶B亚单位(CnB)作为佐剂对新型乙型肝炎病毒疫苗免疫效果的影响

比较不同佐剂配伍的效果,探讨常规剂量(5μg)钙调磷酸酶B亚单位(Calcineurin subunit B,CnB)佐剂对含PreS1+S融合抗原的乙型肝炎病毒新型疫苗(HBSS1)免疫效果的影响。采用Al(OH)3、常规剂量(5μg)CnB及CnB+Al(OH)3等佐剂与HBV颗粒疫苗配伍初次免疫,重组腺病毒载体疫苗加强免疫的策略,在C57BL/6小鼠模型上研究不同佐剂对HBV颗粒疫苗肌肉注射后免疫应答的影响,主要包括抗体滴度、抗体亚型分类及特异性细胞免疫(γ-IFN ELISpot检测)。研究结果显示Al(OH)3佐剂存在明显免疫增强作用,而单独加入5μg CnB佐剂或CnB与Al(OH)3佐剂联合应用对Anti-PreS1抗体无明显的增强作用,但可显著降低anti-HBs抗体水平;各免疫组在重组腺病毒载体疫苗加强后,其抗体亚类包括IgG1、IgG2a和IgG2b;并可诱导高水平的细胞免疫应答反应。因而常规剂量(5μg)CnB单独或联合Al(OH)3佐剂对新型HBV疫苗无明显的免疫增强作用。     

钙调磷酸酶B亚单位(CnB)作为佐剂对新型乙型肝炎病毒疫苗免疫效果的影响北大核心CSCD

比较不同佐剂配伍的效果,探讨常规剂量(5μg)钙调磷酸酶B亚单位(Calcineurin subunit B,CnB)佐剂对含PreS1+S融合抗原的乙型肝炎病毒新型疫苗(HBSS1)免疫效果的影响。采用Al(OH)3、常规剂量(5μg)CnB及CnB+Al(OH)3等佐剂与HBV颗粒疫苗配伍初次免疫,重组腺病毒载体疫苗加强免疫的策略,在C57BL/6小鼠模型上研究不同佐剂对HBV颗粒疫苗肌肉注射后免疫应答的影响,主要包括抗体滴度、抗体亚型分类及特异性细胞免疫(γ-IFN ELISpot检测)。研究结果显示Al(OH)3佐剂存在明显免疫增强作用,而单独加入5μg CnB佐剂或CnB与Al(OH)3佐剂联合应用对Anti-PreS1抗体无明显的增强作用,但可显著降低anti-HBs抗体水平;各免疫组在重组腺病毒载体疫苗加强后,其抗体亚类包括IgG1、IgG2a和IgG2b;并可诱导高水平的细胞免疫应答反应。因而常规剂量(5μg)CnB单独或联合Al(OH)3佐剂对新型HBV疫苗无明显的免疫增强作用。     

HBV包膜-核心蛋白融合基因DNA疫苗诱导小鼠持久的细胞免疫应答

构建编码HBV包膜-核心蛋白融合基因的DNA疫苗pSC、pSS1S2C和编码HBV包膜蛋白或核心蛋白基因的DNA疫苗pHBs、pHBc,分别肌肉注射免疫BALB/c小鼠,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应,比较融合基因DNA疫苗与单基因DNA疫苗诱生免疫应答的强度,发现融合基因DNA疫苗诱生抗体的效率明显不及单基因DNA疫苗,但其能诱导更强、更持久的细胞免疫应答,表明HBV包膜-核心蛋白融合基因DNA疫苗对于治疗慢性乙型肝炎可能比单基因DNA疫苗更为有效.     

乙型脑炎病毒DNA初免-蛋白加强策略在小鼠模型上的免疫效应评价

为了评价基因Ⅰ型乙型脑炎病毒prM-E DNA疫苗与prM和EⅢ融合抗原亚单位疫苗采用DNA初免-蛋白加强免疫策略对小鼠的免疫效果,本研究将prM-E融合基因插入到pVAX1真核表达载体中,构建重组表达载体prM-E-pVAX1作为DNA疫苗进行初免,利用原核表达系统获得的prM和EⅢ融合抗原作为亚单位疫苗进行加强免疫。将32只4−6周龄雌性BALB/c小鼠随机分成4组,设置prM-E-pVAX1 DNA疫苗组、DNA初免-蛋白加强免疫组、prM和EⅢ融合抗原亚单位疫苗组及pVAX1载体对照组,通过ELISA检测血清中特异性抗体水平;通过噬斑减少中和试验滴定中和抗体滴度;通过细胞因子表达丰度和淋巴细胞增殖试验分析不同疫苗免疫组诱导产生的细胞免疫反应。结果表明,用DNA初免-蛋白加强策略免疫的小鼠诱导产生的中和抗体滴度略高于prM和EⅢ融合抗原亚单位疫苗免疫组,显著高于prM-E-pVAX1 DNA疫苗免疫组。DNA初免-蛋白加强策略在小鼠模型中诱导产生了有效的Th1/Th2型免疫反应,特别是显著诱导了Th1型细胞免疫反应。本研究为预防流行性乙型脑炎提供了新的免疫策略和理论参考依据。     

电转联合热休克蛋白佐剂疫苗引发抗HBV的免疫应答

针对HBV感染的治疗性DNA疫苗虽然具有很好的应用前景,但目前抗病毒效果并不高,表明在病毒长期感染过程中存在免疫抑制机制。以HBV的表面蛋白(HBsAg)和核心蛋白(HBcAg)为DNA疫苗抗原,采用gp96和HSP70作为佐剂联合电转以提高疫苗的活性。将gp96为佐剂的HBsAg/HBcAg DNA疫苗免疫HBV转基因鼠后引发抗原特异性的细胞免疫和体液免疫应答。使用gp96和HSP70佐剂引起Treg下调20%。与没有免疫的小鼠相比,以gp96和HSP70为佐剂的DNA疫苗显著降低血清中病毒S抗原水平和DNA拷贝数,大幅降低小鼠肝脏中HBc的表达。该研究为设计以gp96为佐剂的乙肝治疗性DNA疫苗提供了依据。     

含H5N1多个结构抗原的DNA疫苗与重组痘苗病毒疫苗联合免疫小鼠可明显增强免疫原性

本研究旨在研发经济、高效的人高致病性禽流感病毒H5N1实验疫苗并优化免疫方案。利用本实验室前期构建的含有H5N1(安徽株)结构基因的多个单顺反子(HAop和NAop)和双顺反子(HAop/M2和NAop/M1)DNA疫苗及重组痘苗病毒(天坛株)疫苗,采用不同方案(单独或联合)免疫BALB/c小鼠,初步分析了抗原特异性体液免疫(HA血凝抑制抗体,NA特异性抗体,中和抗体及M1与M2特异性抗体)和细胞免疫应答(IFN-γELIS-pot)的特点。结果表明:DNA疫苗与重组痘苗病毒(天坛株)疫苗联合免疫可以激发较强的多个抗原特异的免疫应答,尤其是体液免疫应答,明显优于DNA疫苗或重组痘苗病毒(天坛株)疫苗单独免疫;联合免疫方案中DNA疫苗初免所激发的HA与NA特异的体液免疫应答强于重组痘苗病毒(天坛株)疫苗初免,然而M1与M2特异的体液免疫应答则反之。本研究为新型H5N1疫苗的研发及免疫方案的优化奠定了基础。     

携带HBV包膜大蛋白DNA疫苗的减毒沙门菌在小鼠诱导免疫应答

探索一种简便、有效的乙型肝炎病毒DNA疫苗免疫方法。将编码绿色荧光蛋白的真核表达质粒pEGFPN1转化到减毒鼠伤寒沙门菌SL7207,灌胃饲服BALB/c小鼠,流式细胞术检测出小鼠脾细胞内表达的绿色荧光蛋白;构建编码HBV包膜大蛋白的DNA疫苗pCIS1S2S,分别以SL7207为载体的口服途径或直接肌肉注射途径免疫BALB/c小鼠,检测小鼠的血清抗体、T细胞增殖和细胞毒性T淋巴细胞反应,结果表明两种免疫途径均能在小鼠体内诱生细胞和体液免疫应答,但口服途径诱导免疫应答的强度明显强于肌肉注射途径。口服携带HBV DNA疫苗的减毒伤寒沙门菌可能代表一种简便、有效的治疗乙型肝炎的新方法。      

融合表达DEC205单链抗体和NS3蛋白的HCV DNA疫苗的免疫原性研究

为研发新型HCV DNA疫苗并探讨优化其免疫原性的策略,我们分析靶向树突状细胞(Dendritic cells,DC)的分子对HCV DNA疫苗免疫原性的影响。我们基于抗小鼠DC细胞表面分子DEC205/CD205的单克隆抗体DEC205的单链分子,构建可单独表达DEC205单链抗体或者与HCV非结构蛋白NS3融合表达的DNA表达质粒,并构建单独表达HCV非结构蛋白NS3的DNA表达质粒;经瞬时转染法鉴定HCV NS3及其与DEC205单链抗体融合蛋白的表达;随后采用注射结合电转的方式免疫Balb/C小鼠并研究各疫苗的体液(NS3特异性IgG抗体)与细胞免疫(IFN-γELISPOT)效果。结果表明:DEC205单链抗体基因与HCV NS3编码基因的融合可显著增强NS3特异的免疫应答;采用皮内注射加卡钳电极电转的方式可以产生最强的NS3特异性抗体和T细胞免疫反应。因此,通过DEC205单链抗体与HCV DNA疫苗靶抗原融合可明显增强免疫应答效果。该策略为HCV及其他类似病原的新型DNA疫苗研制提供重要依据。     

电脉冲和布吡卡因增强A型肉毒毒素DNA核酸疫苗的免疫效果

目的：考查DNA疫苗注射免疫后电脉冲和布吡卡因佐剂化DNA疫苗递送方式对A型肉毒毒素DNA核酸疫苗免疫效果的影响。方法：A型肉毒毒素DNA复制子疫苗和传统DNA疫苗肌肉注射免疫小鼠后电脉冲和布吡卡因佐剂化DNA后再肌肉注射免疫小鼠;检测免疫小鼠的抗体和细胞水平,并分析抗体亚类。结果：电脉冲和布吡卡因这二种递送方式均增强DNA复制子疫苗和传统DNA疫苗的体液免疫和细胞免疫效果;电脉冲提高DNA疫苗免疫效果更为明显,并且电脉冲和布吡卡因组合这种递送方式增强DNA疫苗体液免疫和细胞免疫水平最高;与传统DNA疫苗相比,A型肉毒毒素DNA复制子疫苗在这些递送方式下均诱导产生了更好的特异性体液免疫和细胞免疫应答,并且这些递送方式没有改变DNA疫苗的Th1/Th2免疫应答特性,即DNA复制子疫苗诱导产生Th1/Th2混合免疫应答但偏向于Th2途经,而传统DNA疫苗则完全偏向于Th2途经。结论：电脉冲和布吡卡因增强DNA复制子疫苗和传统DNA疫苗的免疫效果,是提高DNA疫苗免疫原性的良好策略。     

乙肝PreS_2抗原决定簇和霍乱毒素B亚基基因的融合与表达

通过全化学法按大肠杆菌密码偏性合成了乙肝炎病毒（ＨＢＶ）前Ｓ２抗原（ＰｒｅＳ２）抗原决定簇基因,与霍乱毒素Ｂ亚基基因的３’端融合。重组质粒转化大肠杆菌后融合基因得到高效表达,表达量达３０μｇ／ｍＬ,表达产物９５％以上分泌到胞外。表达的融合蛋白能与神经节苷脂ＧＭ１结合,说明融合蛋白保持了霍乱毒素Ｂ亚基（ＣＴＢ）的基本高级结构和生物学功能;酶联免疫吸附实验证明融合蛋白具有ＣＴＢ和ＨＢＶＰｒｅＳ２的抗原性;应用亲和层析纯化后得到了电泳纯融合蛋白制品,为研究融合蛋白免疫原性并进一步构建基因工程肽苗奠定了基础。     

Optimisation of prime-boost immunization in mice using novel protein-based and recombinant vaccinia (tiantan)-based HBV vaccine

Background

A therapeutic vaccine for chronic hepatitis B virus (HBV) infection that enhances virus-specific cellular immune responses is urgently needed. The “prime–boost” regimen is a widely used vaccine strategy against many persistence infections. However, few reports have addressed this strategy applying for HBV therapeutic vaccine development.

Methodology/Principal Findings

To develop an effective HBV therapeutic vaccine, we constructed a recombinant vaccinia virus (Tiantan) containing the S+PreS1 fusion antigen (RVJSS1) combined with the HBV particle-like subunit vaccine HBVSS1 to explore the most effective prime–boost regimen against HBV. The immune responses to different prime–boost regimens were assessed in C57BL/C mice by ELISA, ELISpot assay and Intracellular cytokine staining analysis. Among the combinations tested, an HBV protein particle vaccine priming and recombinant vaccinia virus boosting strategy accelerated specific seroconversion and produced high antibody (anti-PreS1, anti-S antibody) titres as well as the strongest multi-antigen (PreS1, and S)-specific cellular immune response. HBSS1 protein prime/RVJSS1 boost immunization was also generated more significant level of both CD4+ and CD8+ T cell responses for Th1 cytokines (TNF-α and IFN-γ).

Conclusions

The HBSS1 protein-vaccine prime plus RVJSS1 vector boost elicits specific antibody as well as CD4 and CD8 cells secreting Th1-like cytokines, and these immune responses may be important parameters for the future HBV therapeutic vaccines.     

超抗原SEA增强小鼠对HBV DNA 疫苗的免疫反应

观察超抗原SEA(D227A)的真核表达载体(pmSEA),对HBVDNA疫苗诱导Balbc小鼠(H2d)免疫应答的调节作用。肌内注射空载体pcDNA3、HBVDNA疫苗加pmSEA佐剂(pHBVS2S+pmSEA)或不加佐剂(pHBVS2S);ELISA法测定血清抗HBs;ELISPOT检测分泌IFNγ的脾淋巴细胞;4h51Cr释放法检测小鼠脾细胞CTL活性。HBVDNA佐剂组免疫小鼠抗HBsAg抗体滴度明显高于不加佐剂组,其IgG1IgG2a的比例不同于多肽免疫组,二者分别为0.282与10。HBVDNA佐剂组均能增强IgG1和IgG2a的产生,是不加佐剂组的1.36、1.73倍。佐剂组小鼠脾淋巴细胞IFNγ的分泌量是不加佐剂组2～3倍。CTL细胞杀伤活性(E:T=100)佐剂组与不加佐剂组分别为:69.77%±7.5%、42.81%±7.7%,差异显著(P<0.05)。HBVDNA疫苗具有较强的免疫原性,能够诱导机体产生特异性的抗体及CTL反应;pmSEA佐剂能够提高小鼠对DNA疫苗的免疫应答,有望成为DNA疫苗的免疫佐剂。     

Overexpression and purification of PreS region of hepatitis B virus antigenic surface protein adr subtype in Escherichia coli

PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli DH5alpha cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-41 degrees C) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at 37 degrees C for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at -80 degrees.     

乙肝PreS_2抗原决定簇和霍乱毒素B亚基基因的融合与表达

本研究通过全化学法按大肠杆菌密码偏性合成了HBV PreS_2抗原决定簇基因,与ctxB基因的3’端融合。重组质粒转化大肠杆菌后融合基因得到高效表达,表达量达30μg/ml,表达产物95％以上分泌到胞外。表达的融合蛋白能与神经节苷脂GM1结合,说明融合蛋白保持了CTB的基本高级结构和生物学功能;ELISA实验证明融合蛋白具有CTB和HBV PreS_2的抗原性;应用亲和层析纯化后得到了电泳纯融合蛋白制品,为研究融合蛋白的免疫原性并进一步构建基因工程肽苗奠定了基础。     

Enhancing the potency of HBV DNA vaccines using fusion genes of HBV-specific antigens and the N-terminal fragment of gp96

BACKGROUND: Many clinical trials show that DNA vaccine potency needs to be greatly enhanced. We have reported that the N-terminal fragment of glycoprotein 96 (gp96) is able to produce an adjuvant effect for production of cytotoxic T-lymphocytes (CTLs) with hepatitis B virus (HBV)-specific peptides. Here, we report a new strategy for HBV DNA vaccine design using a partial gp96 sequence. MATERIALS AND METHODS: We linked the N-terminal 1-355aa (N355) of gp96 to HBV genes encoding for structural proteins, the major S and middle S2S envelope proteins and the truncated core HBcAg (1-149aa). ELISPOT, tetramer staining and intracellular IFN-gamma assay were performed to analyze the induced cellular immune responses of our DNA constructs in BALB/c mice and HLA-A2 transgenic mice. The relative humoral immune responses were analyzed in different IgG isotypes. RESULTS: The fusion genes induced 2- to 6-fold higher HBV-specific CD8(+) T cells as compared to the antigens alone. There was an approximate 10-fold decrease in the humoral immune responses with fusion genes based on HBV envelope proteins. Interestingly, the decreased humoral immune responses were not observed when antigens and plasmid encoding N355 were co-delivered. However, an approximate 20-fold higher antibody level was induced when linking N355 to a truncated HBcAg. Immunization by intramuscular injection resulted in predominantly IgG2a antibodies, which indicated that these vaccines preferentially prime Th1 responses. CONCLUSIONS: We constructed highly immunogenic fusions by linking the N-terminal fragment of gp96 to HBV antigens. Our results imply that the N-terminal fragment of gp96 may be used as a molecular adjuvant to enhance the potency of DNA vaccines.     

Immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis B and human immunodeficiency viruses

A synthetic chimeric gene, TBI-HBS, encoding the immunogenic ENV and GAG epitopes of human immunodeficiency virus (HIV-1) and the surface protein antigen (HBsAg) of hepatitis B virus (HBV), was expressed in tomato plants. Tomato fruits containing the TBI-HBS antigen were fed to experimental mice and, on days 14 and 28 post-feeding, high levels of HIV- and HBV-specific antibodies were present in the serum and feces of the test animals. Intraperitoneal injection of a DNA vaccine directing synthesis of the same TBI-HBsAg antigen boosted the antibody response to HIV in the blood serum; however, it had no effect on the high level of antibodies produced to HBV.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Cationic Lipid-Formulated DNA Vaccine against Hepatitis B Virus: Immunogenicity of MIDGE-Th1 Vectors Encoding Small and Large Surface Antigen in Comparison to a Licensed Protein Vaccine

Currently marketed vaccines against hepatitis B virus (HBV) based on the small (S) hepatitis B surface antigen (HBsAg) fail to induce a protective immune response in about 10% of vaccinees. DNA vaccination and the inclusion of PreS1 and PreS2 domains of HBsAg have been reported to represent feasible strategies to improve the efficacy of HBV vaccines. Here, we evaluated the immunogenicity of SAINT-18-formulated MIDGE-Th1 vectors encoding the S or the large (L) protein of HBsAg in mice and pigs. In both animal models, vectors encoding the secretion-competent S protein induced stronger humoral responses than vectors encoding the L protein, which was shown to be retained mainly intracellularly despite the presence of a heterologous secretion signal. In pigs, SAINT-18-formulated MIDGE-Th1 vectors encoding the S protein elicited an immune response of the same magnitude as the licensed protein vaccine Engerix-B, with S protein-specific antibody levels significantly higher than those considered protective in humans, and lasting for at least six months after the third immunization. Thus, our results provide not only the proof of concept for the SAINT-18-formulated MIDGE-Th1 vector approach but also confirm that with a cationic-lipid formulation, a DNA vaccine at a relatively low dose can elicit an immune response similar to a human dose of an aluminum hydroxide-adjuvanted protein vaccine in large animals.