乙型肝炎病毒特异性细胞毒性T淋巴细胞在病毒清除过程中的作用

在乙型肝炎病毒(HBV)感染过程中,适应性免疫与病毒的致病和清除密切相关。一般认为,体液免疫产生的抗体可以清除外周循环的病毒颗粒,从而阻止病毒在宿主体内的传播,细胞免疫主要清除被感染细胞中的病毒。HBV特异性的细胞毒性T淋巴细胞(CTL)在抑制HBV复制过程中发挥着重要的作用。CTL在肝内主要通过分泌γ干扰素抑制病毒,同时,当CTL识别HBV抗原后,HBV特异性CTL募集抗原非特异性炎症细胞对肝组织浸润,造成肝细胞的损伤。对CTL抗病毒作用进行深入研究,将为乙型肝炎的治疗开辟新的途径。     

组织特异抗原异位表达与免疫耐受

诱导和维持T细胞耐受是免疫系统区分自我和非我的关键。自身免疫调节因子(autoimmune regulator,AIRE)作为转录因子,在胸腺髓质上皮细胞中可驱动一系列组织特异抗原基因的表达,从而在诱导中枢免疫耐受的过程中发挥重要作用。外周免疫耐受的机制复杂一些,清除耐受是其重要机制之一。外周淋巴结的基质细胞可表达部分组织特异抗原,递呈给T淋巴细胞,激活并最终清除它。中枢免疫耐受和外周免疫耐受机制可清除潜在的自身反应性T淋巴细胞,维持对自身组织耐受。一旦免疫耐受被打破,将发生自身免疫反应和自身免疫疾病。     

小鼠的自身反应性T淋巴细胞系及其功能

本文报道了用同基因脾细胞和抗原在体外不断再刺激天花粉蛋白免疫过的C57BL/6J小鼠的T淋巴细胞,能刺激自身反应性的T细胞在体外增殖并长期存活。实验结果表明它们的增殖是依赖于同基因脾细胞的再刺激,C57BL/6J(H一2~b),B 10 ScSn(H-2~b)和129(H-2~b)小鼠的脾细胞都能引起它们明显的增殖,但对C3H/He(H-2~K)和Balb/c(H-2~b)小鼠的脾细胞很弱,说明识别的可能是H-2~b抗原。应用未经免疫的C 57 BL/6 J小鼠的脾和淋巴结T淋巴细胞,采用同样的体外刺激方法,未能引起它们对同基因脾细胞的增殖。从而提示自身反应性T细胞是存在于正常机体内的一种能识别自身抗原的T淋巴细胞。在无外来抗原刺激时,它们可能是处于静止或不激活状态;在外来抗原诱发免疫过程中,它们也随了抗原特异的淋巴细胞一同被激活,并可能起调节作用。它们在免疫系统中的地位还有待进一步阐明。     

EBV转化的人乳头瘤病毒感染者B淋巴母细胞系的建立及应用

建立EB病毒(EBV)转化的B淋巴母细胞系(LCL),应用此细胞系作为抗原递呈细胞筛选抗原特异性的T细胞克隆。收集人乳头瘤病毒(HPV)感染者外周血20份,分离外周血单个核细胞(PBMC),利用Pan T细胞试剂盒去除PBMC中的T细胞,获得non-T淋巴细胞,EBV病毒转化non-T淋巴细胞。应用转化的LCL细胞作为抗原递呈细胞,ELISPOT筛选可能的HPV抗原特异性的T细胞克隆。成功建立18个LCLs,建株成功率为90%。转化成功的LCL细胞体积增大,聚集成团状或簇状。建株失败的两份标本,用于转化的non-T淋巴细胞数少于2×106。应用LCL细胞作为抗原递呈细胞筛选出64个HPV抗原特异性的T细胞克隆。成功建立LCLs,此细胞系细胞可作为体外研究HPV特异性免疫反应的刺激细胞。     

治疗性结核病疫苗研究进展

治疗性结核病疫苗主要用于接种已感染结核分枝杆菌的个体,包括化学药物治疗的患者和潜伏感染者。治疗性疫苗可逆转发生在疾病进展期的非保护性免疫反应,使其向Th1型反应发展;能打破机体的免疫耐受,有效激发宿主针对结核分枝杆菌的以抗原为基础的细胞免疫反应,诱发抗原特异性的细胞毒性T淋巴细胞免疫反应,来清除胞内寄生的结核分枝杆菌。治疗性疫苗将有助于防止潜伏结核病的复发;与药物联合使用以提高药物的治疗效果,尤其是针对耐药结核病的治疗。     

嵌合HCV串联多表位的乙肝S基因真核载体构建及表达

[目的]构建丙型肝炎病毒(hepatitis C virus,HCV)串联多中和抗原表位与乙型肝炎病毒(hepatitis B virus,HBV)S抗原嵌合基因真核表达质粒,并在293T细胞中进行表达。[方法]将HCV基因中高度保守且具有广谱交叉中和活性的三个表位和一个HVR1模拟表位串连。串联表位嵌合于HBV S基因的氨基端胞外区,形成嵌合基因MEpS;将基因克隆至真核表达载体pCI-neo中,构建重组质粒pCI-MEpS。将质粒转染至293T细胞中,间接免疫荧光及Western Blot检测嵌合基因的表达情况。[结果]重组质粒经双酶切证实构建正确;pCI-MEpS转染的293T细胞胞浆内可见较强的绿色荧光,Western Blot显示pCI-MEpS在相对分子量约38 kDa处可见蛋白条带。[结论]构建了HBV S抗原嵌合HCV串联多中和抗原表位的重组质粒,成功在293T细胞中表达,为制备嵌合HCV串联多中和抗原表位的HBV S抗原VLPs,研究嵌合VLPs免疫动物后产生的中和抗体的保护作用奠定了实验基础。     

湖北咸宁地区乙型肝炎病毒基因型的调查

乙型肝炎病毒(Hepatitis B virus,HBV)是引起急性和慢性肝炎的最主要的病原[1].目前根据HBsAg的共同抗原决定簇"α"和两对相互排斥的抗原决定簇将HBV分为ayw1,ayw2,ayw3,ayw4,ayr,adw2,adw2,adw4,adrq+和adrq- 9种不同的血清学亚型,1988年Okamoto[2]等根据HBV基因组核苷酸的差异又提出了HBV基因型的概念,并以全基因组核苷酸差异≥8%,定为基因型分型标准.     

表面抗原和表面抗体检测双阳性CD重组型乙型肝炎病毒全基因组序列分析

西藏地区藏族人群乙型肝炎病毒(Hepatitis B virus,HBV)感染率较高,而针对感染者血清中HBV表面抗原(Hepatitis B surface antigen,HBsAg)和HBV表面抗原抗体(Hepatitis B surface antibody,HBsAb)双阳性的研究一直进展缓慢,尚无明确的研究结论。为探讨西藏地区藏族人群慢性HBV感染者血清中HBsAg和HBsAb双阳性与基因组核苷酸/氨基酸突变的关系,本研究在西藏选取7个地区作为研究区域,进行多阶段抽样,选取样本进行HBV血清五项指标检测,筛选HBsAg和HBsAb均为阳性的患者血清共24份作为双阳性组,以年龄和乙型肝炎e抗原(HBeAg)等感染指标进行匹配,选取96份HBsAg阳性,HBsAb阴性患者血清作为对照组。HBV全基因组序列通过聚合酶链式反应(Polymerase chain reaction,PCR)产物直接测序获得,并进行重组分析和突变分析。852名西藏HBV感染者中,HBsAg/HBsAb双阳性率为2.82%(24/852)。双阳性组在S蛋白N端和主要亲水区(Major hydrophilic region,MHR)的突变率以及PreS缺失发生率均显著高于对照组。T1753C、C1990T和C2002T等核苷酸突变;S蛋白中V224A、PreS区D103E等氨基酸突变在两组内分布存在显著差异。HBV/CD重组型的HBsAg/HBsAb双阳性发生率与中国乙肝主要流行区域接近。HBV感染者血清HBsAg和HBsAb共存可能与S蛋白,特别是MHR内的高氨基酸突变造成的免疫逃逸有关。PreS缺失、S抗原蛋白C端V224A突变和PreS区D103E突变可能对HBsAg/HBsAb双阳性的产生具有协同作用。     

信号肽和辅助性T细胞表位增强HBV核心抗原DNA疫苗诱导的免疫应答

为增强HBVDNA疫苗的免疫效率 ,于HBV核心抗原 (HBcAg)基因 5′末端引入人IL 2信号肽和一个通用型辅助性T淋巴细胞表位基因 ,并构建成DNA疫苗 ,转染COS7细胞后经ELISA检测出分泌型HBcAg。通过肌肉注射途径分别将这种DNA疫苗和编码天然HBcAg的DNA疫苗免疫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 ,结果表明前者诱导细胞和体液免疫应答的强度均明显超过后者 ,且更趋向于T辅助细胞 1(Th1)型免疫应答 ,故其对慢性HBV感染的治疗可能有潜在的应用价值     

血源性Dane颗粒的多肽分析

采用PAGE-Western Blot技术对血源性Dane颗粒的多肽组份进行了分析。并结合HBV感染的其它血清学指标,初步探讨了HBV感染者对这些多肽的抗体应答情况及意义。结果显示:Dane颗粒在PAGE图谱上有12条多肽表型;Western Blot证实除S区的6条多肽和来自C区的具有cAg和eAg两种抗原位点的同一多肽P21外,还有与抗Pol结合的P24-25多肽以及与抗人免疫球蛋白结合的多肽(P45-IgC,P76-IgM),推测血源性Dane颗粒上可能存在有Pol基因表达的产物和结合在表面的人免疫球蛋白或相似的抗原决定簇。调查29例隐性HBV感染自限后人群和185例HBV现症感染者对这些多肽的应答情况发现:P24、P27、P39、P42是引起免疫应答的多肽,两组对它们时应答存在着差异(P<0.01)。表明对它们的有效应答可能有利于HBV感染的清除。     

Improvement of hepatitis B virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and interleukin-2.

DNA vaccines encoding a viral protein have been shown to induce antiviral immune responses and provide protection against subsequent viral challenge. In this study, we show that the efficacy of a DNA vaccine can be greatly improved by simultaneous expression of interleukin-2 (IL-2). Plasmid vectors encoding the major (S) or middle (pre-S2 plus S) envelope proteins of hepatitis B virus (HBV) were constructed and compared for their potential to induce hepatitis B surface antigen (HBsAg)-specific immune responses with a vector encoding the middle envelope and IL-2 fusion protein or with a bicistronic vector separately encoding the middle envelope protein and IL-2. Following transfection of cells in culture with these HBV plasmid vectors, we found that the encoded major protein was secreted while the middle protein and the fusion protein were retained on the cell membrane. Despite differences in localization of the encoded antigens, plasmids encoding the major or middle proteins gave similar antibody and T-cell proliferative responses in the vaccinated animals. The use of plasmids coexpressing IL-2 and the envelope protein in the fusion or nonfusion context resulted in enhanced humoral and cellular immune responses. In addition, the vaccine efficacy in terms of dosage used in immunization was increased at least 100-fold by coexpression of IL-2. We also found that DNA vaccines coexpressing IL-2 help overcome major histocompatibility complex-linked nonresponsiveness to HBsAg vaccination. The immune responses elicited by HBV DNA vaccines were also modulated by coexpression of IL-2. When restimulated with antigen in vitro, splenocytes from mice that received plasmids coexpressing IL-2 and the envelope protein produced much stronger T helper 1 (Th1)-like responses than did those from mice that had been given injections of plasmids encoding the envelope protein alone. Coexpression of IL-2 also increased the Th2-like responses, although the increment was much less significant.     

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.     

重组质粒与重组蛋白共免疫诱导HBsAg特异性T细胞免疫抑制

摘要：【目的】为了探索治疗急性乙型肝炎和爆发性乙型肝的新策略,本研究将HBV DNA疫苗和相应抗原的蛋白质分子联合免疫小鼠,旨在探讨联合免疫对小鼠抗原特异性T细胞增殖反应的影响。【方法】我们将HBV DNA疫苗（pcDS2）和相应抗原蛋白质分子(HBsAg)联合免疫BALB/c小鼠;分别在第0、2和4周进行免疫,在第6周用ELISA方法检测抗-HBs IgG效价,MTT和流式细胞仪检测T细胞增殖反应,及流式细胞仪检测细胞因子表达水平。【结果】pcDS2和HBsAg联合免疫组小鼠的抗-HBs水平显著提高;免疫小鼠的T细胞体外经HBsAg刺激后, 联合免疫组刺激指数(SI)明显降低;经流式细胞仪检测进一步证实联合免疫组T细胞增殖反应被显著抑制;联合免疫组T细胞表达IL-10和Foxp3水平显著升高。【结论】pcDS2和HBsAg联合免疫能诱导产生特异性体液免疫应答,但不能诱导产生抗原特异性T细胞增殖反应;T细胞增殖反应被显著抑制可能与T细胞表达IL-10和Foxp3上调有关;本研究为急性乙型肝炎和爆发性乙型肝炎治疗及HBV疫苗的研究奠定了基础。     

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。      

Flt3配体增强HCV核心-包膜E2融合基因DNA疫苗诱导的细胞免疫应答

建立一种可高效诱导细胞免疫应答 ,对丙型肝炎病毒 (HCV)感染可能起预防和治疗作用的DNA疫苗。将小鼠Flt3配体 (FL)信号肽和胞外段cDNA插入结构优化的HCV核心 包膜E2融合抗原DNA疫苗pST CE2t,构建成pST CE2t FL。将pST CE2t FL转染COS7细胞 ,Westernblot和ELISA检测表明该重组质粒能表达HCV核心 包膜E2融合抗原和可溶性小鼠FL。分别将pST CE2t、pST CE2t FL和空载体pCI neo肌肉注射接种BALB c小鼠 ,检测小鼠的体液和细胞免疫应答。结果表明两种DNA结构均能在小鼠体内诱生细胞和体液免疫应答 ,但pST CE2t诱导的体液免疫应答强于pST CE2t FL ,而后者诱导的细胞免疫应答明显强于前者。FL能明显增强HCV核心 包膜E2融合抗原DNA疫苗诱导的细胞免疫应答 ,对于发展HCV预防和治疗性疫苗有潜在的应用价值。     

乙肝病毒DNA疫苗的构建及其诱导小鼠的免疫应答

构建含adr亚型HBV表面抗原基因的核酸疫苗 ,考察人白细胞介素II基因及重组白细胞介素II的免疫佐剂作用。用含有人白细胞介素II基因的真核表达质粒及基因重组白细胞介素II蛋白作为佐剂 ,将编码乙型肝炎病毒表面抗原的重组真核表达质粒 pVAX/HBS免疫BALB/C小鼠 (试验组 ) ,同时设置注射质粒pVAX的阴性对照组 ,并分别于第 2 ,4周后加强免疫各 1次。试验组在第 4周时开始有HBsAb产生 ,阴性对照组未测到HbsAb ,试验组和对照组均未检测到HBsAg。乙肝病毒DNA疫苗能引起小鼠特异性体液免疫应答 ,白细胞介素II的真核表达质粒的佐剂作用不明显 ,基因重组白细胞介素II蛋白具有提高小鼠对乙肝病毒核酸疫苗免疫应答水平的佐剂活性。     

DNA Prime-Adenovirus Boost Immunization Induces a Vigorous and Multifunctional T-Cell Response against Hepadnaviral Proteins in the Mouse and Woodchuck Model

Induction of hepatitis B virus (HBV)-specific cytotoxic T cells by therapeutic immunization may be a strategy to treat chronic hepatitis B. In the HBV animal model, woodchucks, the application of DNA vaccine expressing woodchuck hepatitis virus (WHV) core antigen (WHcAg) in combination with antivirals led to the prolonged control of viral replication. However, it became clear that the use of more potent vaccines is required to overcome WHV persistence. Therefore, we asked whether stronger and more functional T-cell responses could be achieved using the modified vaccines and an optimized prime-boost vaccination regimen. We developed a new DNA plasmid (pCGWHc) and recombinant adenoviruses (AdVs) showing high expression levels of WHcAg. Mice vaccinated with the improved plasmid pCGWHc elicited a stronger WHcAg-specific CD8⁺ T-cell response than with the previously used vaccines. Using multicolor flow cytometry and an in vivo cytotoxicity assay, we showed that immunization in a DNA prime-AdV boost regimen resulted in an even more vigorous and functional T-cell response than immunization with the new plasmid alone. Immunization of naïve woodchucks with pCGWHc plasmid or AdVs induced a significant WHcAg-specific degranulation response prior to the challenge, this response had not been previously detected. Consistently, this response led to a rapid control of infection after the challenge. Our results demonstrate that high antigen expression levels and the DNA prime-AdV boost immunization improved the T-cell response in mice and induced significant T-cell responses in woodchucks. Therefore, this new vaccination strategy may be a candidate for a therapeutic vaccine against chronic HBV infection.     

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。     

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。     

The adjuvant effects of co-stimulatory molecules on cellular and memory responses to HBsAg DNA vaccination

Because DNA vaccines on their own tend to induce weak immune responses in humans, adjuvant methods are needed in order to improve their efficacy. The co-stimulatory molecules 4-1BBL, OX40L, and CD70 have been shown to induce strong T cell activities; therefore, in this study, we investigated whether they may be used as molecular adjuvants for a hepatitis B surface antigen (HBsAg) DNA vaccine (pcDS2) in eliciting strong cellular and memory responses. Compared to mice immunized with pcDS2 alone, addition of the co-stimulatory molecules increased T cell proliferation and an HBsAg-specific antibody response that was marked with a higher ratio of IgG2a/IgG1. Importantly, pcDS2 plus these co-stimulatory molecules elicited a higher level of IFN-gamma and IL-4 in CD4(+) T cells and a higher level of IFN-gamma in CD8(+) T cells. In addition, a significantly robust antigen-specific cytotoxic T lymphocyte (CTL) response and the production of long-term memory CD8(+) T cells were also observed in the groups immunized with pcDS2 plus 4-1BBL, OX40L, or CD70. Consistently, as late as 100 days after immunization, upregulated expressions of BCL-2, Spi2A, IL-7Ra, and IL-15Ra were still observed in mice immunized with pcDS2 plus these co-stimulatory molecules, suggesting the generation of memory T cells in these groups. Together, these results suggest that the co-stimulatory molecules 4-1BBL, OX40L, or CD70 can enhance the immunogenicity of HBsAg DNA vaccines, resulting in strong humoral, cellular, and memory responses. This approach may lead to an effective therapeutic vaccine for chronic hepatitis B virus (HBV) infection.