Ag85A口服DNA疫苗的制备及其诱导T细胞亚群应答效应的研究
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摘要
Ag85A口服DNA疫苗的制备及其诱导T细胞亚群应
答效应的研究
实验目的
近年来随着人口流动增加、艾滋病蔓延以及新型结核菌株，多重耐药菌株的出现，结核病一改过去逐年递减的趋势，发生回潮，病例数明显增加，发病率以每年20％的速度递增，成为死亡率最高的感染性疾病。迄今为止，其中全球约三分之一人口受到结核杆菌感染，每年死亡人数约300万人，约500万人感染耐药结核杆菌。3％的结核新病例与人免疫缺陷病毒(HIV)感染有关。我国是22个结核病高发国家之一，现有活动性肺结核患者约450万，传染性强。因此预防接种对结核病的控制和消灭起着关键性的作用。目前卡介苗(BCG)作为唯一临床使用的疫苗，预防效果并不稳定。BCG是由一株患结核性乳腺炎的奶牛身上分离到的牛型结核分支杆菌，经过230次连续传代减毒而失去毒力，1928年起在全世界广泛使用，证明对儿童血行播散性结核病，结核性脑膜炎的保护力十分显著，但对肺结核的免疫保护力各家报告差异甚大，从2％-84％不等。80年代初在南印度的研究结果是无效的，几乎否定了BCG的免疫保护作用，而且有报告免疫缺陷病人(如AIDS)接种BCG可引起全身播散性感染而死亡。Mahairas等研究发现BCG在传代过程中为达到减毒目的，失去了一些编码具有免疫保护作用的抗原基因。同时BCG的接种会使结核菌素试验阳性，对结核病的诊断产生干扰，延误治疗时间，因此研制新疫苗成为当前结核病防治的首要任务。
1990年Wolff等首次将纯化的DNA肌肉注射小鼠发现可产生相应基因的表达产物。1992年Tang等进行了首次DNA疫苗实验，从此发展了DNA疫苗新技术。1998年英、法、美、丹麦四国专家合作完成了结核分支杆菌的染色体全DNA测序工作，结核分支杆菌共含4，411，529碱基对，约4000个基因，为结核病疫苗的改造及重组DNA疫苗提供了有利的条件。一些编码具有免疫保护作用的保护性抗原基因成为结核病DNA疫苗研究的热点。目前研究最多的是ESAT6、Ag85复合体、HSP70、PstS-1、PstS-2、PstS-3、HSP65、MPB／NIPT51 MPT64、CFP-21和38-kDa抗原。1996年美国的Huygen等首次报道用结核分支杆菌Ag85A编码基因的质粒DNA免疫小鼠，可诱导小鼠产生很强的细胞和体液免疫反应，抵御结核分支杆菌的攻击。
Ag85A抗原蛋白是结核杆菌的一种主要的分泌性蛋白，同时也是一种膜蛋白，多项研究表明Ag85A抗原是一种十分重要的免疫保护性抗原，其分子结构中包含多个T淋巴细胞和B淋巴细胞的特异性识别表位，因此对该抗原蛋白进行克隆与表达研究对发展新型结核疫苗有十分重要的现实意义。简单地说，DNA疫苗通常为携带有外源性基因的真核细胞表达质粒，将该质粒注射给动物，可直接转染体内细胞，并诱导免疫应答产生。近年来的一些应用DNA疫苗抗感染和抗肿瘤的临床研究也初步展露了可喜的苗头。概括已有的有关DNA疫苗的研究成果主要为：1、携带编码蛋白质或肽基因的质粒DNA转染真核细胞可进行表达，诱导机体产生免疫应答；2、DNA疫苗主要诱导T细胞介导的细胞免疫功能增强，如增强外周血及脾内的细胞毒性T细胞(CTL)的细胞毒活性，促进Th1型细胞因子(IFN—γ和TNF—β)的产生等；3、DNA疫苗也可刺激体液免疫应答，增强特异性抗体产生；4、DNA疫苗可通过多种途径接种机体。DNA疫苗不存在减毒疫苗所潜在恢复毒性的危险，也不需要病毒作为运载体运载至靶细胞，这样就避免了考虑病毒运载体的安全性及宿主对病毒运载体的免疫性，进而也使DNA疫苗比其他疫苗更具安全性。
自从1997年Jones和Pascual等人分别用PLG微粒和减毒伤寒杆菌为运载体制备口服DNA疫苗以来，已经有多篇报道对口服DNA疫苗在预防或治疗感染、肿瘤和过敏性疾病中的作用进行了研究，并取得了较好的效果。研究表明1、口服DNA疫苗后，其编码蛋白可在肠上皮细胞、脾和肠系膜淋巴结等处表达；2、口服DNA疫苗可明显刺激全身性的细胞免疫和体液免疫应答，如CTL活性和特异性IgG抗体产生；3、口服DNA疫苗还可以使肠粘膜局部产生特异性分泌型IgA(SIgA)，而经注射途径通常不能诱导此种SIgA的分泌。
脂质体可提高质粒表达主要由于：1、脂质体包裹DNA可有效降低机体细胞内核酸酶降解质粒DNA；2脂质体与受体细胞结合可促进质粒DNA被组织细胞吸收。脂质体作为一种可供选择的基因载体具有无毒、无免疫原性、可生物降解的特点，可保护质粒DNA被核酸酶降解，能将目的基因DNA特异传递到靶细胞中。阳性脂质体作为一种可供选择的基因传递载体具有下列优点：1、可防止核酸被体内物质降解，可将其特异性传递到靶细胞中；2、无毒、无免疫性，具有生物惰性，可生物降解；3、易于制备，使用方便，可将大的DAN片断转运到细胞中；4、基因转染率高，100％离体细胞可以瞬间表达外源基因。本研究选择以脂质体作为DNA疫苗运载体，能携带和表达外源抗原，激发出宿主的局部体液免疫，全身体液免疫和细胞免疫反应，以此包裹重组质粒，观察其介导的佐剂作用。
抗原85复合体(Ag85)是BCG合成的能够刺激机体产生细胞免疫和体液免疫的多种成分之一，国外已有研究者将其试用于免疫生物治疗。比利时布鲁塞尔巴斯德研究所Huygen研究组多年来在对Ag85进行的研究中发现，接种Ag85蛋白主要引起机体细胞免疫功能增强，他们还观察到携带Ag85基因的的质粒DNA疫苗具有：1、经肌肉注射给动物(小鼠)，主要引起Th1样的应答，产生IL-2、IFNγ和TNFα；经基因枪(gene gun)注射则主要产生Th2应答和抗体产生水平增加；2、肌肉注射治疗膀胱癌，近80％的病人外周血和脾脏中T淋巴细胞增殖，产生IL-2和IFNγ的量增加，未见明显的Th2细胞活化和抗体产生增多；3、可诱导长寿命的T细胞产生。4、可降低迟发型超敏反应的强度。但目前尚无关于该疫苗经口途径免疫所产生效应的相关报道。裸DNA疫苗经肌肉注射，其目的基因一般在肌细胞内表达，诱导肌体免疫应答的产生，但经口服DNA疫苗在消化道细胞内表达定位目前尚无报道。到目前为止，已有的一些研究表明DNA疫苗能诱导全身性细胞和体液免疫应答增强。同时，有关其是否产生负效应也有待进行研究。我们推论口服DNA疫苗所产生的全身性免疫效应实际是肠道粘膜局部免疫应答后的一系列反应。
本研究通过构建结核分枝杆菌Ag85A与真核表达载体pCDNA3.1~+的重组DNA疫苗，并检测其可在真核细胞内表达Ag85A蛋白，以阳离子脂质体为运载体，制成可供口服的DNA疫苗，经口途径投与小鼠，检测疫苗的体内体外免疫生物活性及其诱导T细胞亚群应答效应，为口服DNA疫苗的临床应用提供理论和实验依据。
实验材料
一、主要仪器
流式细胞仪，二氧化碳孵箱，倒置显微镜，酶标仪，离心机，低温冰箱，超速离心机，超滤器，超净工作台等。
二、主要试剂
减毒鼠伤寒沙门菌X4064，RPMI1640培养基，pUCm-T载体，真核表达载体pCDNA3.1~+，DH5α感受态细胞，鼠anti-TB Ag85A，柱式质粒小量抽提试剂盒，脂质体转染试剂盒，胶回收试剂盒，T4 DNA连接酶，小牛血清，FITC标记的羊抗鼠IgG，免疫组化ABC试剂盒，Western-blot相关试剂，结核分支杆菌H37Rv株，引物，PCR扩增试剂盒，IPTG、X-gal培养基，SDS-PAGE电泳相关试剂，XhoⅠ及BamHⅠ，细胞因子ELISA检测试剂盒等。
三、实验动物
C57BL／6小鼠30只，6-8周龄，SPF级，购自中科院上海实验动物中心。
实验方法
一、真核表达编码Ag85A基因重组体的构建
1、引物设计
根据GenBank中Ag85A基因序列，PCR所用引物：上游引物5’-CAGGATCCGCGCGCGCAGTCTGACCTAGTTGAGGATGC -3’，含BamHI酶切位点：下游引物5’-GTCTCGAGAGGGCCGCCGCCGTTAATCGCT-3’含XhoⅠ酶切位点，并在ORF终止密码子之后。Ag85A含信号肽序列。确保克隆基因开放读码框正确。
2、结核分支杆菌H37Rv株接种改良罗氏培养基，37℃培养8周，小量抽提DNA为模板，按照试剂盒操作说明书进行
3、PCR技术扩增Ag85A基因
94℃预变性15min，热启动后转入94℃变性1min，60℃退火1.5min，72℃延伸2min，进行30个循环，72℃延伸10min后终止反应。反应结束后取5ul产物于1％琼脂糖凝胶电泳，鉴定正确后采用胶回收试剂盒纯化PCR产物。
4、纯化PCR产物TA克隆入载体pUCm-T载体
按pUCm-T载体PCR扩增试剂盒建立反应体系：10xbuffer 1ul，pUCm-T载体1ul，PCR产物3ul，T4 DNA连接酶1ul，加ddH_2O至10ul，25℃连接30min。连接产物转化到受体菌DH 5α感受态细胞，取100ul转化后的菌液铺于含氨苄青霉素(60ug／ml)、IPTG 4ul(200mg／ml)、X-gal 40ul(20mg／ml)的LB琼脂平板，平放30min后，37℃倒置培养过夜(12-16小时)。
5、蓝白斑筛选
待蓝色充分显现，挑取白色菌落于含有氨苄青霉素(60ug／ml)的LB培养基，37℃200 RPM振摇培养过夜，并扩增Amp抗性克隆，质粒提取试剂盒小量抽提质粒。
6、亚克隆入真核表达载体pCDNA3.1~+鉴定正确后将重组质粒转化感受态大肠杆菌DH5α，构建重组真核表达质粒pCDNA3.1~+／Ag85A
从上述受体菌DH5α中提取纯化pUCm-Ag85A，经XhoⅠ及BamHⅠ双酶切，回收小片段作为目的基因，将其亚克隆入pCDNA3.1~+(也经XhoⅠ及BamHⅠ双酶切，回收大片段作为载体)。连接反应体系：载体1ul，目的基因4ul，10xbuffer 1ul，T4 DNA连接酶1ul，加ddH_2O至10ul，4℃过夜。第二天取连接产物转化到受体菌DH 5α感受态细胞，挑取阳性克隆，抽提质粒行双酶切及测序鉴定。
二、稳定高效表达Ag85A基因细胞系的建立
1、无内毒素重组质粒pCDNA3.1~+／Ag85A的提取
按照Promega和V-gene公司无内毒素质粒提取试剂盒说明书分别进行大量和中量重组质粒制备。
2、脂质体介导的K562细胞的转染验证重组质粒中目的基因的瞬时表达
2ul脂质体与2ug重组质粒在不含抗生素的RPMI1640培养基中共孵育15min后转染K562细胞，37℃、5％CO_2孵育24h后，用含10％小牛血清的RPMI1640培养基培养48h。
3、重组质粒pCDNA3.1~+／Ag85A在K562细胞中表达的Ag85A蛋白鉴定
在G418(400ug／ml)的细胞培养基中稳定生长的K562细胞生长密度达1x10~7／ml时，PBS洗涤、离心收集细胞，运用细菌蛋白提取试剂盒裂解细胞。以12％分离胶，5％浓缩胶，进行SDS-PAGE电泳，考马斯亮蓝染色，脱色液脱色，拍照分析。
4、Western-blot鉴定
硝酸纤维素膜于转移缓冲液中平衡15min，与15％分离装置于电泳转移器各层，电转移50V、120min。用去离子水漂洗硝酸纤维素膜5min后，置于20ml含5％脱脂奶粉的PBS-T缓冲液中封闭3h．用PBS-T缓冲液洗膜5minx3次，加1：500的一抗(chicken anti-TB Ag85A IgY)，37℃孵育1h，再PBS-T缓冲液洗膜5minx3次，加1：1000的二抗(HRP-goat-anti-chicken IgY)，37℃孵育1h，再PBS-T缓冲液洗膜5minx3次，DAB显色反应，拍照分析。
5、免疫细胞化学检测技术检测Ag85A蛋白的细胞内定位
用鼠抗Ag85A单克隆抗体加入上述单细胞悬液(1×10~6)孵育，PBS洗2次，再与FITC标记的羊抗鼠IgG的二抗孵育，PBS洗2次，滴加50ul在乙醇处理过的玻璃盖玻片上，经甘油封片固定后在荧光显微镜下观察Ag85A基因表达产物的定位及分布。
6、G418筛选48h后的转染K562细胞株
分别以0，50，100，200，400，800ug／ml浓度每隔3-4天换液一次，计数存活细胞，确定杀死未转染的正在分裂的细胞的最低浓度，以此浓度为选择压力，防止质粒丢失。
7、流式细胞术检测瞬时表达和稳定表达K562细胞株中Ag85A蛋白的细胞所占比例
制备活性高的细胞悬液，用10％FCS的RPMI1640调整细胞浓度为5×10~6～1×10~7／ml，用PBS洗2次，离心沉淀重溶于1g·L~(-1) Triton X溶液200μl中。37℃作用1h后，PBS洗2次，加入PBS 100μl稀释的chicken anti-Ag85A IgY抗体一抗(1：1000)，37℃孵育1h：再加入100μl PBS稀释的二抗FITC-goat-anti-chicken IgY荧光抗体(1：500)，最后用PBS稀释至总量为500μl，流式细胞法检测荧光强度和阳性百分率即可知Ag85A蛋白的密度。
三、口服Ag85A DNA疫苗的体内表达和对T细胞亚群的影响
1、大量抽提无内毒素重组质粒DCDNA3.1~+／Ag85A，包裹阳离子脂质体，制备成可供口服的DNA疫苗。
2、C57 BL／6小鼠30只，雄性，随机分为5组，每组6只，NS--生理盐水组：JO--空质粒组：L+JO--脂质体+空质粒组组：JA--重组DNA组：L+JA--脂质体包裹的重组质粒组，共免疫3次，每次间隔2周，末次免疫后7天收集各组免疫小鼠的血清及脾细胞。JA和JO组小鼠分别经口灌饲100μg重组质粒pCDNA3.1~+／Ag85A和100μg pCDNA3.1~+质粒载体。
3、流式细胞分析术检测观察口服DNA疫苗前后，小鼠脾细胞CD4~+CD8~-、CD4~-CD8~+T细胞亚群的数量变化
用完全培养基(10％胎牛血清的RPMI 1640培养液)调整上述小鼠脾脏淋巴细胞浓度至1×10~6／ml。每组细胞取4ml细胞悬液，均匀分成3管，4000 rpm，离心2min。以含有2％小牛血清的PBS洗液洗涤1次，去除大部分上清，留下30～40μl液体。轻弹将细胞悬起，加入FITC-抗CD4~+单抗、PE-抗CD8~+单抗，PE-CY5-抗CD3~+单抗各20μl，对照管共3管，分别加入单色荧光标记抗体，孵育20～30min，用含有2％小牛血清的PBS洗液洗涤3次，离心同上。细胞重悬于适当体积的洗液中。流式细胞分析仪分析测定T细胞亚群。
4、口服DNA疫苗后小鼠分泌IFN-γ和IL-4的脾脏淋巴细胞数量的检测
通过ELISPOT技术检测，将适量捕获性单抗或抗原预包被于96孔ELISPOT板上，每孔加入50μl Magi．Coating Buffer稀释好的包被抗体，4℃包被过夜。接种细胞，加入刺激物，培养，PBST洗涤10次，每孔加入100μl稀释好的生物素标记检测抗体，37℃孵育lh。PBST洗涤5次，每孔加入100μl稀释好的酶标亲和素，37℃lh。PBST洗涤5次，每孔加入100μlAEC显色液，室温避光静置25min。以去离子水洗涤2遍，终止显色过程，室温静置10-30min，将ELISPOT板置于CTL自动读板仪内，调节好合适的参数，斑点计数，并记录斑点的各种参数，统计分析。
5、小鼠眼球采血血清中Ag85A抗体产生及抗体滴度测定
通过ELISA法检测用K562细胞裂解液抗原包被96孔酶标板，待测血清作1：50稀释。正常鼠血清为阴性对照，Chicken anti-TB Ag85A单抗为阳性对照。HRP标记的羊抗鼠IgG作1：1000稀释，TMB底物显色10min，加终止液2 M H_2SO_4后，于酶标仪测定A_(450)值。
6、小鼠眼球采血血清中Th1型细胞因子IFNγ和Th2型细胞因子IL-4的分泌水平检测
通过ELISA法检测，按照ELISA试剂盒操作说明检测血清中IFN-γ和IIJ-4的含量。
实验结果
1、真核表达编码Ag85A基因重组体的构建
重组质粒经PCR及双酶切证实成功构建了携带Ag85A基因的重组真核表达质粒pCDNA3.1~+／Ag85A，后者成功转化感受态大肠杆菌DH5α。经测序分析所克隆1141bp Ag85A与GenBank中结核分枝杆菌Ag85A氨基酸同源性为100％。
2、脂质体最佳使用浓度验证
重组质粒pCDNA3.1~+／Ag85A DNA转染K562细胞时与脂质体Lipofectamine~(TM)2000的最适比例为2ug：2ul，此时脂质体能够成功介导转染且对细胞毒性较小。
3．Ag85A蛋白的表达
Western-blot及SDS—PAGE检测转染的K562细胞株存在分子量为32KD的Ag85A蛋白的表达，与成熟的Ag85A蛋白分子量一致，表明Ag85A基因在K562细胞中得到了表达，表达产物具有免疫反应性。以此作为构建口服脂质体疫苗的依据。
4、G418筛选稳定表达Ag85A蛋白的细胞株
G418筛选48h后的转染K562细胞株，800ug／ml浓度杀死未转染的正在分裂的细胞，400ug／ml为杀死未转染的正在分裂的细胞的最低浓度，以此浓度为选择压力，防止质粒丢失。经过12周筛选，获得稳定表达Ag85A蛋白的细胞株，以FITC标记的二抗进行间接免疫荧光试验，荧光显微镜下可观察到大量黄绿色荧光，而对照组未见特异性荧光，大部分荧光染色细胞表达在细胞膜：经流式细胞术分析表达Ag85A蛋白的细胞所占比例为41.73％。
5、口服免疫对小鼠脾细胞T细胞亚群的影响
脂质体对T细胞无毒性，CD4~+T细胞及CD8~+T细胞的百分率均出现下调，其中空质粒组无显著作用，无脂质体包裹的重组质粒组对CD4~+、CD8~+T细胞有降低作用，但脂质体+重组质粒组对CD4~+、CD8~+T细胞降低作用效果更明显。
6、口服DNA疫苗后小鼠脾淋巴细胞的Th1型细胞因子IFNγ和Th2型细胞因子IL-4分泌水平的检测结果
分泌IFN-γ的Th1型细胞比率和血清中的IFN-γ水平下降，而分泌IL-4的Th2型细胞比率和血清中的IL-4水平升高。
7、血清中Ag85A特异性抗体产生水平的检测结果
用间接ELISA方法检测血清中Ag85A特异性抗体的产生，血清中抗体滴度脂质体包裹的重组DNA组所产生的特异性抗体水平最高，抗体滴度为1：160：重组DNA组为1：80，而脂质体包裹的空质粒组和空质粒组无特异性抗体产生。说明该口服疫苗可诱导一定的体液免疫。
8、脂质体包裹的重组质粒组血清中Th1型细胞因子IFN-γ的产生水平受到抑制。
9、血清中Th2型细胞因子IL-4的产生水平脂质体包裹重组质粒组相对较高，而其它各组无显著差异。
讨论
本研究选择了pCDNA3.1~+作为真核表达载体，该载体含氨苄青霉素抗性基因和B-半乳糖苷酶启动子及该酶氨基末端126个氨基酸的编码序列，可用IPTG诱导表达，并与宿主菌所表达的β-半乳糖苷酶羧基末端片段形成A互补，在含IPTG和X-gal的LB平板上形成蓝色菌落，当外源基因插入其多克隆位点后可使β-半乳糖苷酶氨基末端片段失活，在含IPTG和X-gal的LB平板上形成蓝白相间的菌落，在生色底物X-gal存在下，含有载体质粒的细菌由于α互补现象而呈蓝色菌落：含有重组质粒的细菌则因外源DNA片段插入而无α互补能力，呈现白色菌落，挑出阳性克隆，从而为重组质粒的筛选和鉴定提供了抗性标记以外的另一种筛选标记。该载体同时具有CMV启动子和牛生长激素基因，非常适合重组外源基因在哺乳动物细胞中的高效表达，并且在CMV和LacZ的序列中含有7个未甲基化的CpG基序，对动物机体具有较强的免疫激活作用．该载体可在真核和原核细胞之间进行穿梭表达，这既为研究Ag85A在大肠杆菌中的表达产物的生化免疫特性奠定了基础，又为进一步研究Ag85A在真核细胞的表达及其在结核病核酸疫苗研制中的应用提供了方便。pCDNA3.1~+／Ag85A重组质粒的序列分析显示Ag85A基因编码的碱基序列与Genbank报道氨基酸序列完全一致，表明基因突变未改变三联体密码子的表型，免疫原性未受影响。
DNA纯度对转染效率影响非常大。脂质体转染技术基于电荷吸引原理~([13])，如果DNA不纯，如带少量的盐离子，蛋白，代谢物污染都会显著影响转染复合物的有效形成及转染的进行~([14])。我们用Vitagene的无内毒素质粒DNA中量提取试剂盒提取质粒，在质粒抽提过程中有效去除脂多糖分子，得到的质粒OD260／OD280比值都在1.8-2.0之间，有效的保证理想的转染效果，新霉素的类似物G418作用于80s核糖体，可抑制蛋白质合成，因此未转染的细胞可被G418杀死，通过阻断哺乳动物细胞蛋白质合成而杀死细胞。新霉素抗性基因编码的细菌磷酸转移酶可使筛选药物G418失活，所以当转染的细胞表达了这种neo抗性基因后，就会在含G418的选择性培养基中存活。
分泌IFN-γ的Th1型细胞比率和血清中的IFN—γ水平下降，而分泌IL-4的Th2型细胞比率和血清中的IL-4水平升高。这一结果提示，脾CD4~+T细胞数量下降主要为Th1型细胞数量的下降，而Th2型细胞数量下降不明显或没有下降。而且这一结果也进一步解释了体液免疫应答，即抗Ag85A蛋白的特异性抗体可以产生。本实验结果还发现L+JO组IFN—r的分泌水平显著高于L+JA组(P值＜0.0001)。出现这一结果可能是因为空质粒(pCDNA3.1~+)中所含的CpG基序具有明显刺激T细胞分泌IFN-r作用。而重组质粒(pCDNA3.1~+／Ag85A)由于有Ag85A基因片断的插入，CpG基序刺激IFN-r的分泌活性可能受到了抑制。本研究结果显示口服JO、L+JO二组之间对CD4~+T细胞和CD8~+T细胞数量影响无显著差异，JA和L+JA组也未见显著差异，进一步证明脂质体作为运载体对机体几乎无毒性。同时，我们在研究中还发现用脂质体包裹制备的口服DNA疫苗组(L+JA组)诱导Ag85A特异性抗体产生水平高于未用脂质体包裹重组质粒组(JA组)，表明脂质体可具有佐剂作用。ELISPOT技术可检出百万分之一的分泌细胞因子阳性细胞，灵敏度极高。实验中除应用抗原进行刺激之外，还使用了识别细胞因子和抗体的两个不同表位的单克隆抗体，因此ELISPOT技术有着高度的特异性。本次实验采用无血清ELISPOT技术。即细胞因子分泌细胞中，使用了不含血清的培养基，同时采用无内毒素质粒进行脂质体包裹，最大限度的降低内毒素的影响。ELISPOT技术检测中需用特异性刺激物应该是：成分尽可能简单，纯度尽可能高，内毒素的污染尽可能被避免。大肠杆菌中重组基因表达的蛋白质含有的大肠杆菌及内毒素成分，极易影响ELISPOT的检测结果。为此，本研究中选择在哺乳动物细胞中表达的蛋白质，即刺激物是稳定转染重组质粒并表达Ag85A蛋白的哺乳动物细胞K562的裂解物，其中的蛋白己通过SDS-PAGE及Western-blot验证，在保证纯度的前提下，为哺乳动物细胞中表达的蛋白质，内毒素含量在实验允许范围内，与ELISPOT的相容性较好。
结论
1、经DNA序列测定证实插入片段的序列与与GenBank中结核分枝杆菌Ag85A核苷酸编码的氨基酸同源性为100％，pCDNA3.1~+／Ag85A重组质粒构建成功。
2、重组质粒pCDNA3.1~+／Ag85A DNA经脂质体转染真核细胞表达的Ag85A蛋白具有免疫原性。
3、成功转染并获得稳定表达Ag85A蛋白的K562细胞株，此细胞可用于Ag85A蛋白的大量制备及其它方面的研究。
4、口服自制Ag85A DNA疫苗可在体内表达并刺激抗Ag85A特异性抗体的产生。
5、口服自制Ag85A DNA疫苗下调了脾CD4~+T细胞和CD8~+T细胞亚群的数量。
6、分泌IFN--r的Th1型细胞比率和血清中的IFN—r水平下降，而分泌IL-4的Th2型细胞比率和血清中的IL-4水平升高。提示脾CD4~+T细胞数量下降可能主要为Th1型细胞数量的下降，而Th2型细胞数量下降不明显或没有下降。
7、L+JA组IFN-r的分泌水平显著低于L+JO组，提示载体质粒pCDNA3.1~+中所含的CpG基序可能具有刺激T细胞分泌IFN-r作用，而重组质粒pCDNA3.1~+／Ag85A中，由于Ag85A基因片断的插入，CpG基序刺激IFN-r的分泌活性可能受到了抑制。
8、脂质体包裹制备的口服DNA疫苗组(L+JA组)诱导Ag85A特异性抗体产生水平高于未用脂质体包裹重组质粒组(JA组)，表明脂质体可具有免疫佐剂作用。
9、口服JO、L+JO二组之间对CD4~+T细胞和CD8~+T细胞数量影响无显著差异，JA和L+JA组间也未见显著差异，进一步证明脂质体作为运载体对机体几乎无毒性。
Objective
Tuberculosis (TB) is a chronic respiratory infectious disease that has afflicted humans for thousands of years. For 80 years, the bacillus Calmette-Gue'rin (BCG) vaccine has been the only licensed TB vaccine given to humans covering 86% of the world population in 2001. However, despite the use of BCG, TB remains a global epidemic with one-third of the world population being infected and an annual rate of 8 million new cases and 2-2.5 million deaths. Regardless of its protection from severe forms of childhood TB, BCG fails to confer protection from adult TB. Furthermore, BCG vaccine may cause severe complications in immunocompromised hosts. Thus, there is an urgent need for developing safe and effective TB vaccines that are able to confer potent protection at the mucosal site.
Presently Tuberculosis appears an increasing trend not only in developing countries but also in advanced countries. It is still a severe disease imperiling human health in the 21st century. Migration, HIV infection and multidrug resistants are the major dificulties for Tuberculosis control work.. The protective effect of BCG ranges from 0% to 85%, especially has a very low efficacy to adults and cannot be used for therpay. Protective ability of BCG varies in areas and populations, especially in developing countries such as in Africa areas, BCG is virtually usefuless. Since the global situation of Tuberculosis has become so severely and BCG is far from being an ideal vaccine against Tuberculosis, and there is no suitable vaccine available at moment, it is necessary to search for an efficient and safe and vaccine for us. As a new vaccine, DNA vaccine has been achieved inspiring progress in other infective diseases and tumor research. DNA vaccine can induce both of humoral and cellular immunity. Inoculation of DNA vaccines into muscle or skin by a syringe or a propulsion device such as a gene gun results in uptake of the DNA into cells, followed by transcription and translation of the pathogen's gene and, consequently, an immune response composed of antibodies, T helper cells and cytotoxic T lymphocytes (CTL). DNA vaccines have several advantages over traditional vaccines: they stimulate a full spectrum of immuneresponses including CTLs generally not induced by protein vaccines and they generate exceptionally long lasting immune responses. They provide their own adjuvant in the form of unmethylated bacterial CpG sequences that induce an innate immune response which in turn sponsors activation of an antigen-specific immune response. DNA vaccines, which were first described in 1992, have been shown to induce immune responses to a variety of viral, bacterial and parasitic antigens. In addition, they have shown efficacy in treatment of allergic diseases, autoimmunity and tumor models.
DNA vaccines, based on plasmid vectors ex-response to DNA vaccines can be enhanced by genetic engineering of the antigen to facilitate its presentation to pressing an antigen under the control of a strong promoter, which have been shown to induce protective immune B and T cells. Furthermore, the immune response can be modulated by genetic adjuvants in the form of vectors responses to a number of pathogens, including viruses, expressing biologically active determinants or by more bacteria and parasites. They have also displayed efficacy in treatment or prevention of cancer, allergic diseases and traditional adjuvants that facilitate uptake of DNA into autoimmunity. Immunologically, DNA vaccines induce cells. The ease of genetic manipulation of DNA vaccines a full spectrum of immune responses that invites their use not only as vaccines but also as research T cells, T helper cells and antibodies.
Vectors employed for the construction of DNA vaccines are bacterial plasmids which are otherwise commonly used for in vitro expression of proteins in Most DNA vaccine vectors contain an intron, which is an element that can increase expression of genes. This 'cassette' is followed by the gene encoding the antigen of interest flanked by the SV40 or bovine growth hormone 3%-untranslated region (BGH 3%-UTR) transcrip termination: polyadenylation sequences. This part of the vector is often referred to as the transcriptional unit responsible for antigen synthesis. The other part is the plasmid backbone that contains an origin of replication (ori) enabling high-yield production in Escherichia coli along with an antibiotic resistance gene, such as ampicillin (not approved by federal agencies for use in humans) or kanamycin (a resistance marker suitable for human vaccines), to confer antibiotic-selected growth in bacteria. This part of the plasmid backbone contains unmethylated CpG sequences that possess important immunomodulatory properties and provides an intrinsic adjuvant effect for DNA vaccines. It is thought that the magnitude of the immune response to DNA vaccines directly correlates with the level of antigen expression, measured in vitro upon transient transfection of cells.
Antigen 85A is one of the components of the Antigen 85 complex. This protein is expressed by a wide range of bacteria of genus Mycobacterium. The Antigen 85A protein is coded by the gene called fibronectin-binding protein-A (fbpA) gene. In Mycobacterium tuberculosis, this fibronectin-binding protein-A (fbpA) gene is 1014bp long and has a molecular weight of 32KD.
The fbpA gene codes for 338 amino acids which characterize the Antigen 85A protein. Antigen 85A has been applied differently in a wide range of research because of its role in the immunogenicity of the bacteria of genus Mycobacterium. The gene expressing the antigen 85A protein, fbpA, has been identified in different species of the mycobacteria such as M. leprae, M. gordonae, M. bovis, M. avium and they all show close homology to that of M. tuberculosis. Recent animal studies have shown that vaccination with Recombinant Antigen 85A DNA or the protein has powerful immunological properties, resulting in significant secretion of Th1 cells cytokines, particularly interleukins and interferon-γwhich are important in regulating the activities of a number of immune cells, especially the cytotoxic cells. These cytotoxic cells play significant roles in the elimination of viral infected and transformed cells.
Increasing evidence suggests that vaccination at the mucosal site is superior to vaccination at other sites in eliciting protection from mucosal infectious diseases. This is partially explained by the observation that memory T and B cells generated upon mucosal vaccination acquire mucosa-homing receptors and preferentially accumulate at the mucosal site of induction. Thus, it is believed that greater immune protection may be achieved if TB vaccine is given mucosally via oral route.
The cationic liposome acting as an adjuvant can greatly enhance the expression of recombinant plasmiddue to the function of preventing delivered DNA from digested by Dnase and can also promote the absorbance of cellular level. In addition, liposome has several advantages over traditional adjuvant., such as, easy preparated and ready to use, most important, with the high efficiency in gene transfection.
Materials and methods Materials
1. Mice
Male C57B1/6, 6-8 weeks old, were purchased from Animal Laboratories (Chinese Medical University). C57BL/6 mice were immunized and randomly separated 30 mice into 5 groups: (NS)saline, (JO) plasmid vector, (L+JO) liposomal plasmid vector, (JA)recombinant plasmid, (L+JA)liposomal recombinant plasmid.
2. Reagent
Lipofectamine~(TM) 200 were purchased from Invitrogen Co.(USA). Mammalian Cell Lysis Kit were purchased from Bio Basic Inc. (Canada) Anti-Chicken IgY, HRP Conjugate and Anti-Chicken IgY-FITC, PureYield Plasmid extraction kit were purchased from Promega Co.(USA). chichen anti-TB Ag85A IgY Monoclonal antibody were purchased from Prosci Co. Mouse CD4—FITC, CD8—PE, CD8—PE-CY5 antibodies were purchased from BD Co. (USA). IFN-r and IL-4 ELISA kit were purchased from R&D Co. (USA). Mouse IFN-r ELISPOT Kit, Mouse IL-4 ELISPOT Kit were purchased from U-CyTech Co. (Netherlands).
Method
1. The preparation of recombinant pCDNA3.1~+/Ag85A plasmid
The gene encoding Ag85A mature protein was amplified by polymerase chain reaction (PCR) using forward primer 5'-CAGGATCCGCGCGCGCAGTCTGACCTAGTTGAGGATGC-3', containing BamHI cloning site; reverse primer 5'-GTCTCGAGAGGGCCGCCGCCGTTAATCGCT-3' containing Xho I cloning site, while genome of mycobacterium tuberculosis H37Rv strain as template, and PCR product treated with DNA gel extraction was inserted into cloning vector pUCm-T After transformation into competent DH5α,. the pUCm-Ag85A plasmid was extracted and digested with restriction enzyme BamHI and XhoI, then was subcloned to the same sites of eukaryotic expressing vector pcDNA3.1. after transformation into competent DH5α, the clone growing in SOB agar with amp were selected then the plasmid was extracted. and determine the fragment was correctly inserted into the vector, which was confirmed by partial nucleotide sequencing and restriction endonuclease digestion with restriction enzyme BamHI and XhoI.
2.Screening of stably expressed K562 cells
(1) The preparation of endotoxin-free plasmid by Pure Yield Plasmid with extraction kit
(2) Lipofectamine~(TM) 2000 mediated transfection into K562 cells
Just prior to preparing complexes, plate 4×10~5 cells in 500ul of growth medium without antibiotics. Then dilute 0.2μg DNA in 50ul DMEM without serum and mix Lipofectamine~(TM) 2000 gently before use, dilute 0.5μl in 50μl DMEM without serum. Incubate for 5 minutes at room temperature. Combine the diluted DNA with diluted Lipofectamine~(TM) 2000. Mix gently and incubate foy 20 minutes at room temperature. Then add 100μl of complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth. Incubate cells at 37℃in a CO_2 incubator for 36 hours for testing transgene expression. For stable cell lines, passage cells at a 1:10 into fresh growth medium 24 hours after transfection. Add G418 into DMEM as selective medium the following day.
For K562 cells transfected afer 36 hours, centrifuge for minutes at 420×g, decant the supernatant. Wash the cells twice by resuspending the pellets with 1×PBS; centrifuge and discard the supernatant; Re-suspend the pellet in 1×cell lysis buffer, incubate for 5 minutes; Centrifuge the lysed cells for 45 minutes at 100000×g to prepare a protein solution. The extracted protein was used for SDS-PAGE and Western blotting analysis.; after the preliminary SDS-PAGE, the Recombinant Antigen 85A protein was transferred on to Nitrocellulose membrane and later incubated with chichen anti-TB Ag85A IgY Monoclonal antibody. For this technique, preliminary SDS-PAGE was run on two gels and one was stained using Coomasie stain while the other was used for the immunoblotting. After the transfer of the bands from the gel onto the nitrocellulose paper, the paper was properly blocked with BSA. This blocking step ensured that the binding potentials of non-specific antigens were eliminated. After the preliminary washing and the addition of the secondary antibody-horse radish peroxidase, a band corresponding to that on the Coomasie-stained gel was obtained.
(3) Screening of stably expressed K562 cells
passage cells at a 1:10 into fresh growth medium 24 hours after transfection. Add G418 into DMEM as selective medium the following day. The concentration of G418 in medium are 800μg/ml, 400μg/ml, 200μg/ml, 100μg/ml, 50μg/ml respectively. The medium were changed every 3-4 days. The screening pressure continues for 4 weeks, then we determine the lowest concentration of G418 to kill the non-transfection K562 cells as the appropriate pressure to culture another 6 weeks so that stably transfected K562 cells could be screened.
(4) Intracellular anti-chicken IgY-FITC staining were used to determine the expression efficacy in vitro.
3. The effect on the T cells subset of the oral recombinant plasmid pCDNA3.1~+/Ag85A mediated with LipofectamineTM 2000
(1) Recombinant plasmid containing Ag85A using liposome as a vector was constructed and administered to C57BL/6 mice via oral route.. C57BL/6 mice were immunized three times at 14 days intervals, 30 mice were randomly separated into 5 groups: (NS)saline, (JO)plasmid vector,(L+JO)liposomal plasmid vector, (JA)recombinant plasmid, (L+JA)liposomal recombinant plasmid.
(2) Determination of the contents of IFN-r, IL-4 in serum of C57BL/6 mice by double antibody sandwich ELISA, furthermore, the ability of splenocytes secreting IFN-γand IL-4 was tested by ELISPOT method..
(3) When the mice were vaccinated with recombinant eukaryotic expressing vector 5 weeks later, titers of serum antibody against Ag85A were detected by ELISA.
(4) Determination by flow cytometry of the percentage of the CD4~+ and CD8~+ T cell subsets in the splenocytes.
(5) Intracellular anti-chicken IgY-FITC staining were used to determine the expression efficacy in vitro.
Results
1. The nucleotide sequencing of the gene encoding Ag85A mature form of mycobacterium tuberculosis H37Rv strain was identical and there were no undesired mutations, the cloning gene was correctly inserted into the vector pcDNA3.1~+ as confirmed by restriction endonuclease digestion of Bam HI and XhoI and sequence analysis.
2. We obtain a stable cell line which can express the protein with molecular mass about 32KD by using SDS, and showed immunogenicity with monoclonal antibodies of Ag85A, as detected with western blot.
3. The effect on the T cells subset of the oral recombinant plasmid pCDNA3.1~+/Ag85A mediated with Lipofectamine~(TM) 2000
(1) Lymphocytes obtained from the spleen of pcDNA3.1~+/Ag85A vaccine mediated with lipofectamine~(TM) 2000 exhibited lower IFN-γproduction and higher IL-4 production than that for pcDNA3.1~+ vector immunized mice.
(2) The number of spleen MNC secreting IFN-γstimulated by Ag85A protein in vitro Ag85A protein were significantly lower than those of plasmid vector group.
(3) L+JA immunized mice elicited higher Ag85A-specific antibodies titres than that for JA immunized mice. The plasmid vector group and recombinant plasmid group both mediated with Lipofectamine~(TM) 2000, the titres are 1:80 and 1:160 respectively by EL ISA.
(4) Determination by flow cytometry of the percentage of the CD4~+ and CD8~+ T cell subsets in the splenocytes. The subset both shows the decrease of percentage compared with control group.
(5) Fluorescence-activated cell sorter (FACS) analysis after intracellular staining displayed that 41.3% pcDNA3.1~+/Ag85Atransfected stable K562 cells was FITC positive; whereas the controls only showed 1.5% positive cells.
Discussion
In this study, pCDNA3.1~+ was chosen as the expression vector of recombinant DNA vaccine due to the CMV promoter and BGH gene. CpG seguence can induce the immune activity, furthermore, this vector can shuttle between the DH5αand K562 cells, the Laz gene can supply another screening marker in addition to the Amp resistence. The sequence analysis of cloning Ag85A showed the complete same amino acid sequence coding.
The purity of DNA can greatly affect transfection efficiency. We chose the Vitagene kit to extract endotoxin-free plasmid to avoid the interference of the detection result. G418 can kill the growing cells, so we use it to screen the stably transfected K562 cells through dose selection as screening pressure.
CD4~+T and CD8~+T cell subsets can directly exhibit the celluar immune level.
In ELISPOT assay, we choose serum-free medium and endotoxin-free plasmid to assure the accuracy of the result, Ag85A protein which was isolated from K562 cells can lower the influence of endotoxin because the purity had been detected through SDS-PAGE and western-blot. ELISPOT assay has three typical advantanges. First, high sensitivity; second, single cell detection level; Last, easy operation.
The oral recombinant plasmid pCDNA3.1~+/ Ag85A. mediated with Lipofectamine~(TM) 2000 showed down-regulation effect of the subsets of CD4~+ T cells and CD8~+ T cells could induce Th2 type humoral response and suppressed the secretion of IFN-γin C57BL/6 mice by ELISPOT. In addition, the pCDNA3.1~+/Ag85A DNA vaccine can greatly enhance the titres of Ag85A-specific antibodies by ELISA. The down-regulation effect of the subsets of CD4~+ T cells and CD8~+ T cells reflect the oral immune tolerance. Because the immune system is a network, CD4~+CD25~+Treg and TGF-βtogether with apoptosis factors can lead to suppression of CD8~+T subsets, iDC and mDC can induce tolerance due to the secretion of chromosome. According to our result, liposome can act as adjunvant without toxicity to cells.
Conclusion
1. DNA vaccine encoding Ag85A mature protein ofmycobacterium tuberculosis was constructed and identified successfully.
2. The Ag85A protein was stably expressed after G418 screening transfected K562 cells.
3. The oral recombinant plasmid pCDNA3.1~+/Ag85A. mediated with Lipofectamine~(TM) 2000 shows down-regulation effect of the subsets of CD4~+ T cells and D8~+ T cells.
4. The oral recombinant plasmid pCDNA3.1~+/Ag85A has good immunogenecity and could induce Th2 type humoral response in C57BL/6 mice by ELISPOT.
5. The oral recombinant plasmid pCDNA3.1~+/Ag85A mediated with Lipofectamine~(TM) 2000 group suppressed the secretion of IFN-γ, while the plasmid pCDNA3.1~(+W) with Lipofectamine~(TM) 2000 group increased the IFN-γlevel due to the CpG sequences.
6. The oral recombinant plasmid pCDNA3.1~+/Ag85A mediated with Lipofectamine~(TM) 2000 can greatly enhance the titres of Ag85A-specific antibodies by ELISA.
7. Lipofectamine~(TM) 2000 can act as an adjuvant through comparation with negative control group.
答效应的研究
实验目的
近年来随着人口流动增加、艾滋病蔓延以及新型结核菌株，多重耐药菌株的出现，结核病一改过去逐年递减的趋势，发生回潮，病例数明显增加，发病率以每年20％的速度递增，成为死亡率最高的感染性疾病。迄今为止，其中全球约三分之一人口受到结核杆菌感染，每年死亡人数约300万人，约500万人感染耐药结核杆菌。3％的结核新病例与人免疫缺陷病毒(HIV)感染有关。我国是22个结核病高发国家之一，现有活动性肺结核患者约450万，传染性强。因此预防接种对结核病的控制和消灭起着关键性的作用。目前卡介苗(BCG)作为唯一临床使用的疫苗，预防效果并不稳定。BCG是由一株患结核性乳腺炎的奶牛身上分离到的牛型结核分支杆菌，经过230次连续传代减毒而失去毒力，1928年起在全世界广泛使用，证明对儿童血行播散性结核病，结核性脑膜炎的保护力十分显著，但对肺结核的免疫保护力各家报告差异甚大，从2％-84％不等。80年代初在南印度的研究结果是无效的，几乎否定了BCG的免疫保护作用，而且有报告免疫缺陷病人(如AIDS)接种BCG可引起全身播散性感染而死亡。Mahairas等研究发现BCG在传代过程中为达到减毒目的，失去了一些编码具有免疫保护作用的抗原基因。同时BCG的接种会使结核菌素试验阳性，对结核病的诊断产生干扰，延误治疗时间，因此研制新疫苗成为当前结核病防治的首要任务。
1990年Wolff等首次将纯化的DNA肌肉注射小鼠发现可产生相应基因的表达产物。1992年Tang等进行了首次DNA疫苗实验，从此发展了DNA疫苗新技术。1998年英、法、美、丹麦四国专家合作完成了结核分支杆菌的染色体全DNA测序工作，结核分支杆菌共含4，411，529碱基对，约4000个基因，为结核病疫苗的改造及重组DNA疫苗提供了有利的条件。一些编码具有免疫保护作用的保护性抗原基因成为结核病DNA疫苗研究的热点。目前研究最多的是ESAT6、Ag85复合体、HSP70、PstS-1、PstS-2、PstS-3、HSP65、MPB／NIPT51 MPT64、CFP-21和38-kDa抗原。1996年美国的Huygen等首次报道用结核分支杆菌Ag85A编码基因的质粒DNA免疫小鼠，可诱导小鼠产生很强的细胞和体液免疫反应，抵御结核分支杆菌的攻击。
Ag85A抗原蛋白是结核杆菌的一种主要的分泌性蛋白，同时也是一种膜蛋白，多项研究表明Ag85A抗原是一种十分重要的免疫保护性抗原，其分子结构中包含多个T淋巴细胞和B淋巴细胞的特异性识别表位，因此对该抗原蛋白进行克隆与表达研究对发展新型结核疫苗有十分重要的现实意义。简单地说，DNA疫苗通常为携带有外源性基因的真核细胞表达质粒，将该质粒注射给动物，可直接转染体内细胞，并诱导免疫应答产生。近年来的一些应用DNA疫苗抗感染和抗肿瘤的临床研究也初步展露了可喜的苗头。概括已有的有关DNA疫苗的研究成果主要为：1、携带编码蛋白质或肽基因的质粒DNA转染真核细胞可进行表达，诱导机体产生免疫应答；2、DNA疫苗主要诱导T细胞介导的细胞免疫功能增强，如增强外周血及脾内的细胞毒性T细胞(CTL)的细胞毒活性，促进Th1型细胞因子(IFN—γ和TNF—β)的产生等；3、DNA疫苗也可刺激体液免疫应答，增强特异性抗体产生；4、DNA疫苗可通过多种途径接种机体。DNA疫苗不存在减毒疫苗所潜在恢复毒性的危险，也不需要病毒作为运载体运载至靶细胞，这样就避免了考虑病毒运载体的安全性及宿主对病毒运载体的免疫性，进而也使DNA疫苗比其他疫苗更具安全性。
自从1997年Jones和Pascual等人分别用PLG微粒和减毒伤寒杆菌为运载体制备口服DNA疫苗以来，已经有多篇报道对口服DNA疫苗在预防或治疗感染、肿瘤和过敏性疾病中的作用进行了研究，并取得了较好的效果。研究表明1、口服DNA疫苗后，其编码蛋白可在肠上皮细胞、脾和肠系膜淋巴结等处表达；2、口服DNA疫苗可明显刺激全身性的细胞免疫和体液免疫应答，如CTL活性和特异性IgG抗体产生；3、口服DNA疫苗还可以使肠粘膜局部产生特异性分泌型IgA(SIgA)，而经注射途径通常不能诱导此种SIgA的分泌。
脂质体可提高质粒表达主要由于：1、脂质体包裹DNA可有效降低机体细胞内核酸酶降解质粒DNA；2脂质体与受体细胞结合可促进质粒DNA被组织细胞吸收。脂质体作为一种可供选择的基因载体具有无毒、无免疫原性、可生物降解的特点，可保护质粒DNA被核酸酶降解，能将目的基因DNA特异传递到靶细胞中。阳性脂质体作为一种可供选择的基因传递载体具有下列优点：1、可防止核酸被体内物质降解，可将其特异性传递到靶细胞中；2、无毒、无免疫性，具有生物惰性，可生物降解；3、易于制备，使用方便，可将大的DAN片断转运到细胞中；4、基因转染率高，100％离体细胞可以瞬间表达外源基因。本研究选择以脂质体作为DNA疫苗运载体，能携带和表达外源抗原，激发出宿主的局部体液免疫，全身体液免疫和细胞免疫反应，以此包裹重组质粒，观察其介导的佐剂作用。
抗原85复合体(Ag85)是BCG合成的能够刺激机体产生细胞免疫和体液免疫的多种成分之一，国外已有研究者将其试用于免疫生物治疗。比利时布鲁塞尔巴斯德研究所Huygen研究组多年来在对Ag85进行的研究中发现，接种Ag85蛋白主要引起机体细胞免疫功能增强，他们还观察到携带Ag85基因的的质粒DNA疫苗具有：1、经肌肉注射给动物(小鼠)，主要引起Th1样的应答，产生IL-2、IFNγ和TNFα；经基因枪(gene gun)注射则主要产生Th2应答和抗体产生水平增加；2、肌肉注射治疗膀胱癌，近80％的病人外周血和脾脏中T淋巴细胞增殖，产生IL-2和IFNγ的量增加，未见明显的Th2细胞活化和抗体产生增多；3、可诱导长寿命的T细胞产生。4、可降低迟发型超敏反应的强度。但目前尚无关于该疫苗经口途径免疫所产生效应的相关报道。裸DNA疫苗经肌肉注射，其目的基因一般在肌细胞内表达，诱导肌体免疫应答的产生，但经口服DNA疫苗在消化道细胞内表达定位目前尚无报道。到目前为止，已有的一些研究表明DNA疫苗能诱导全身性细胞和体液免疫应答增强。同时，有关其是否产生负效应也有待进行研究。我们推论口服DNA疫苗所产生的全身性免疫效应实际是肠道粘膜局部免疫应答后的一系列反应。
本研究通过构建结核分枝杆菌Ag85A与真核表达载体pCDNA3.1~+的重组DNA疫苗，并检测其可在真核细胞内表达Ag85A蛋白，以阳离子脂质体为运载体，制成可供口服的DNA疫苗，经口途径投与小鼠，检测疫苗的体内体外免疫生物活性及其诱导T细胞亚群应答效应，为口服DNA疫苗的临床应用提供理论和实验依据。
实验材料
一、主要仪器
流式细胞仪，二氧化碳孵箱，倒置显微镜，酶标仪，离心机，低温冰箱，超速离心机，超滤器，超净工作台等。
二、主要试剂
减毒鼠伤寒沙门菌X4064，RPMI1640培养基，pUCm-T载体，真核表达载体pCDNA3.1~+，DH5α感受态细胞，鼠anti-TB Ag85A，柱式质粒小量抽提试剂盒，脂质体转染试剂盒，胶回收试剂盒，T4 DNA连接酶，小牛血清，FITC标记的羊抗鼠IgG，免疫组化ABC试剂盒，Western-blot相关试剂，结核分支杆菌H37Rv株，引物，PCR扩增试剂盒，IPTG、X-gal培养基，SDS-PAGE电泳相关试剂，XhoⅠ及BamHⅠ，细胞因子ELISA检测试剂盒等。
三、实验动物
C57BL／6小鼠30只，6-8周龄，SPF级，购自中科院上海实验动物中心。
实验方法
一、真核表达编码Ag85A基因重组体的构建
1、引物设计
根据GenBank中Ag85A基因序列，PCR所用引物：上游引物5’-CAGGATCCGCGCGCGCAGTCTGACCTAGTTGAGGATGC -3’，含BamHI酶切位点：下游引物5’-GTCTCGAGAGGGCCGCCGCCGTTAATCGCT-3’含XhoⅠ酶切位点，并在ORF终止密码子之后。Ag85A含信号肽序列。确保克隆基因开放读码框正确。
2、结核分支杆菌H37Rv株接种改良罗氏培养基，37℃培养8周，小量抽提DNA为模板，按照试剂盒操作说明书进行
3、PCR技术扩增Ag85A基因
94℃预变性15min，热启动后转入94℃变性1min，60℃退火1.5min，72℃延伸2min，进行30个循环，72℃延伸10min后终止反应。反应结束后取5ul产物于1％琼脂糖凝胶电泳，鉴定正确后采用胶回收试剂盒纯化PCR产物。
4、纯化PCR产物TA克隆入载体pUCm-T载体
按pUCm-T载体PCR扩增试剂盒建立反应体系：10xbuffer 1ul，pUCm-T载体1ul，PCR产物3ul，T4 DNA连接酶1ul，加ddH_2O至10ul，25℃连接30min。连接产物转化到受体菌DH 5α感受态细胞，取100ul转化后的菌液铺于含氨苄青霉素(60ug／ml)、IPTG 4ul(200mg／ml)、X-gal 40ul(20mg／ml)的LB琼脂平板，平放30min后，37℃倒置培养过夜(12-16小时)。
5、蓝白斑筛选
待蓝色充分显现，挑取白色菌落于含有氨苄青霉素(60ug／ml)的LB培养基，37℃200 RPM振摇培养过夜，并扩增Amp抗性克隆，质粒提取试剂盒小量抽提质粒。
6、亚克隆入真核表达载体pCDNA3.1~+鉴定正确后将重组质粒转化感受态大肠杆菌DH5α，构建重组真核表达质粒pCDNA3.1~+／Ag85A
从上述受体菌DH5α中提取纯化pUCm-Ag85A，经XhoⅠ及BamHⅠ双酶切，回收小片段作为目的基因，将其亚克隆入pCDNA3.1~+(也经XhoⅠ及BamHⅠ双酶切，回收大片段作为载体)。连接反应体系：载体1ul，目的基因4ul，10xbuffer 1ul，T4 DNA连接酶1ul，加ddH_2O至10ul，4℃过夜。第二天取连接产物转化到受体菌DH 5α感受态细胞，挑取阳性克隆，抽提质粒行双酶切及测序鉴定。
二、稳定高效表达Ag85A基因细胞系的建立
1、无内毒素重组质粒pCDNA3.1~+／Ag85A的提取
按照Promega和V-gene公司无内毒素质粒提取试剂盒说明书分别进行大量和中量重组质粒制备。
2、脂质体介导的K562细胞的转染验证重组质粒中目的基因的瞬时表达
2ul脂质体与2ug重组质粒在不含抗生素的RPMI1640培养基中共孵育15min后转染K562细胞，37℃、5％CO_2孵育24h后，用含10％小牛血清的RPMI1640培养基培养48h。
3、重组质粒pCDNA3.1~+／Ag85A在K562细胞中表达的Ag85A蛋白鉴定
在G418(400ug／ml)的细胞培养基中稳定生长的K562细胞生长密度达1x10~7／ml时，PBS洗涤、离心收集细胞，运用细菌蛋白提取试剂盒裂解细胞。以12％分离胶，5％浓缩胶，进行SDS-PAGE电泳，考马斯亮蓝染色，脱色液脱色，拍照分析。
4、Western-blot鉴定
硝酸纤维素膜于转移缓冲液中平衡15min，与15％分离装置于电泳转移器各层，电转移50V、120min。用去离子水漂洗硝酸纤维素膜5min后，置于20ml含5％脱脂奶粉的PBS-T缓冲液中封闭3h．用PBS-T缓冲液洗膜5minx3次，加1：500的一抗(chicken anti-TB Ag85A IgY)，37℃孵育1h，再PBS-T缓冲液洗膜5minx3次，加1：1000的二抗(HRP-goat-anti-chicken IgY)，37℃孵育1h，再PBS-T缓冲液洗膜5minx3次，DAB显色反应，拍照分析。
5、免疫细胞化学检测技术检测Ag85A蛋白的细胞内定位
用鼠抗Ag85A单克隆抗体加入上述单细胞悬液(1×10~6)孵育，PBS洗2次，再与FITC标记的羊抗鼠IgG的二抗孵育，PBS洗2次，滴加50ul在乙醇处理过的玻璃盖玻片上，经甘油封片固定后在荧光显微镜下观察Ag85A基因表达产物的定位及分布。
6、G418筛选48h后的转染K562细胞株
分别以0，50，100，200，400，800ug／ml浓度每隔3-4天换液一次，计数存活细胞，确定杀死未转染的正在分裂的细胞的最低浓度，以此浓度为选择压力，防止质粒丢失。
7、流式细胞术检测瞬时表达和稳定表达K562细胞株中Ag85A蛋白的细胞所占比例
制备活性高的细胞悬液，用10％FCS的RPMI1640调整细胞浓度为5×10~6～1×10~7／ml，用PBS洗2次，离心沉淀重溶于1g·L~(-1) Triton X溶液200μl中。37℃作用1h后，PBS洗2次，加入PBS 100μl稀释的chicken anti-Ag85A IgY抗体一抗(1：1000)，37℃孵育1h：再加入100μl PBS稀释的二抗FITC-goat-anti-chicken IgY荧光抗体(1：500)，最后用PBS稀释至总量为500μl，流式细胞法检测荧光强度和阳性百分率即可知Ag85A蛋白的密度。
三、口服Ag85A DNA疫苗的体内表达和对T细胞亚群的影响
1、大量抽提无内毒素重组质粒DCDNA3.1~+／Ag85A，包裹阳离子脂质体，制备成可供口服的DNA疫苗。
2、C57 BL／6小鼠30只，雄性，随机分为5组，每组6只，NS--生理盐水组：JO--空质粒组：L+JO--脂质体+空质粒组组：JA--重组DNA组：L+JA--脂质体包裹的重组质粒组，共免疫3次，每次间隔2周，末次免疫后7天收集各组免疫小鼠的血清及脾细胞。JA和JO组小鼠分别经口灌饲100μg重组质粒pCDNA3.1~+／Ag85A和100μg pCDNA3.1~+质粒载体。
3、流式细胞分析术检测观察口服DNA疫苗前后，小鼠脾细胞CD4~+CD8~-、CD4~-CD8~+T细胞亚群的数量变化
用完全培养基(10％胎牛血清的RPMI 1640培养液)调整上述小鼠脾脏淋巴细胞浓度至1×10~6／ml。每组细胞取4ml细胞悬液，均匀分成3管，4000 rpm，离心2min。以含有2％小牛血清的PBS洗液洗涤1次，去除大部分上清，留下30～40μl液体。轻弹将细胞悬起，加入FITC-抗CD4~+单抗、PE-抗CD8~+单抗，PE-CY5-抗CD3~+单抗各20μl，对照管共3管，分别加入单色荧光标记抗体，孵育20～30min，用含有2％小牛血清的PBS洗液洗涤3次，离心同上。细胞重悬于适当体积的洗液中。流式细胞分析仪分析测定T细胞亚群。
4、口服DNA疫苗后小鼠分泌IFN-γ和IL-4的脾脏淋巴细胞数量的检测
通过ELISPOT技术检测，将适量捕获性单抗或抗原预包被于96孔ELISPOT板上，每孔加入50μl Magi．Coating Buffer稀释好的包被抗体，4℃包被过夜。接种细胞，加入刺激物，培养，PBST洗涤10次，每孔加入100μl稀释好的生物素标记检测抗体，37℃孵育lh。PBST洗涤5次，每孔加入100μl稀释好的酶标亲和素，37℃lh。PBST洗涤5次，每孔加入100μlAEC显色液，室温避光静置25min。以去离子水洗涤2遍，终止显色过程，室温静置10-30min，将ELISPOT板置于CTL自动读板仪内，调节好合适的参数，斑点计数，并记录斑点的各种参数，统计分析。
5、小鼠眼球采血血清中Ag85A抗体产生及抗体滴度测定
通过ELISA法检测用K562细胞裂解液抗原包被96孔酶标板，待测血清作1：50稀释。正常鼠血清为阴性对照，Chicken anti-TB Ag85A单抗为阳性对照。HRP标记的羊抗鼠IgG作1：1000稀释，TMB底物显色10min，加终止液2 M H_2SO_4后，于酶标仪测定A_(450)值。
6、小鼠眼球采血血清中Th1型细胞因子IFNγ和Th2型细胞因子IL-4的分泌水平检测
通过ELISA法检测，按照ELISA试剂盒操作说明检测血清中IFN-γ和IIJ-4的含量。
实验结果
1、真核表达编码Ag85A基因重组体的构建
重组质粒经PCR及双酶切证实成功构建了携带Ag85A基因的重组真核表达质粒pCDNA3.1~+／Ag85A，后者成功转化感受态大肠杆菌DH5α。经测序分析所克隆1141bp Ag85A与GenBank中结核分枝杆菌Ag85A氨基酸同源性为100％。
2、脂质体最佳使用浓度验证
重组质粒pCDNA3.1~+／Ag85A DNA转染K562细胞时与脂质体Lipofectamine~(TM)2000的最适比例为2ug：2ul，此时脂质体能够成功介导转染且对细胞毒性较小。
3．Ag85A蛋白的表达
Western-blot及SDS—PAGE检测转染的K562细胞株存在分子量为32KD的Ag85A蛋白的表达，与成熟的Ag85A蛋白分子量一致，表明Ag85A基因在K562细胞中得到了表达，表达产物具有免疫反应性。以此作为构建口服脂质体疫苗的依据。
4、G418筛选稳定表达Ag85A蛋白的细胞株
G418筛选48h后的转染K562细胞株，800ug／ml浓度杀死未转染的正在分裂的细胞，400ug／ml为杀死未转染的正在分裂的细胞的最低浓度，以此浓度为选择压力，防止质粒丢失。经过12周筛选，获得稳定表达Ag85A蛋白的细胞株，以FITC标记的二抗进行间接免疫荧光试验，荧光显微镜下可观察到大量黄绿色荧光，而对照组未见特异性荧光，大部分荧光染色细胞表达在细胞膜：经流式细胞术分析表达Ag85A蛋白的细胞所占比例为41.73％。
5、口服免疫对小鼠脾细胞T细胞亚群的影响
脂质体对T细胞无毒性，CD4~+T细胞及CD8~+T细胞的百分率均出现下调，其中空质粒组无显著作用，无脂质体包裹的重组质粒组对CD4~+、CD8~+T细胞有降低作用，但脂质体+重组质粒组对CD4~+、CD8~+T细胞降低作用效果更明显。
6、口服DNA疫苗后小鼠脾淋巴细胞的Th1型细胞因子IFNγ和Th2型细胞因子IL-4分泌水平的检测结果
分泌IFN-γ的Th1型细胞比率和血清中的IFN-γ水平下降，而分泌IL-4的Th2型细胞比率和血清中的IL-4水平升高。
7、血清中Ag85A特异性抗体产生水平的检测结果
用间接ELISA方法检测血清中Ag85A特异性抗体的产生，血清中抗体滴度脂质体包裹的重组DNA组所产生的特异性抗体水平最高，抗体滴度为1：160：重组DNA组为1：80，而脂质体包裹的空质粒组和空质粒组无特异性抗体产生。说明该口服疫苗可诱导一定的体液免疫。
8、脂质体包裹的重组质粒组血清中Th1型细胞因子IFN-γ的产生水平受到抑制。
9、血清中Th2型细胞因子IL-4的产生水平脂质体包裹重组质粒组相对较高，而其它各组无显著差异。
讨论
本研究选择了pCDNA3.1~+作为真核表达载体，该载体含氨苄青霉素抗性基因和B-半乳糖苷酶启动子及该酶氨基末端126个氨基酸的编码序列，可用IPTG诱导表达，并与宿主菌所表达的β-半乳糖苷酶羧基末端片段形成A互补，在含IPTG和X-gal的LB平板上形成蓝色菌落，当外源基因插入其多克隆位点后可使β-半乳糖苷酶氨基末端片段失活，在含IPTG和X-gal的LB平板上形成蓝白相间的菌落，在生色底物X-gal存在下，含有载体质粒的细菌由于α互补现象而呈蓝色菌落：含有重组质粒的细菌则因外源DNA片段插入而无α互补能力，呈现白色菌落，挑出阳性克隆，从而为重组质粒的筛选和鉴定提供了抗性标记以外的另一种筛选标记。该载体同时具有CMV启动子和牛生长激素基因，非常适合重组外源基因在哺乳动物细胞中的高效表达，并且在CMV和LacZ的序列中含有7个未甲基化的CpG基序，对动物机体具有较强的免疫激活作用．该载体可在真核和原核细胞之间进行穿梭表达，这既为研究Ag85A在大肠杆菌中的表达产物的生化免疫特性奠定了基础，又为进一步研究Ag85A在真核细胞的表达及其在结核病核酸疫苗研制中的应用提供了方便。pCDNA3.1~+／Ag85A重组质粒的序列分析显示Ag85A基因编码的碱基序列与Genbank报道氨基酸序列完全一致，表明基因突变未改变三联体密码子的表型，免疫原性未受影响。
DNA纯度对转染效率影响非常大。脂质体转染技术基于电荷吸引原理~([13])，如果DNA不纯，如带少量的盐离子，蛋白，代谢物污染都会显著影响转染复合物的有效形成及转染的进行~([14])。我们用Vitagene的无内毒素质粒DNA中量提取试剂盒提取质粒，在质粒抽提过程中有效去除脂多糖分子，得到的质粒OD260／OD280比值都在1.8-2.0之间，有效的保证理想的转染效果，新霉素的类似物G418作用于80s核糖体，可抑制蛋白质合成，因此未转染的细胞可被G418杀死，通过阻断哺乳动物细胞蛋白质合成而杀死细胞。新霉素抗性基因编码的细菌磷酸转移酶可使筛选药物G418失活，所以当转染的细胞表达了这种neo抗性基因后，就会在含G418的选择性培养基中存活。
分泌IFN-γ的Th1型细胞比率和血清中的IFN—γ水平下降，而分泌IL-4的Th2型细胞比率和血清中的IL-4水平升高。这一结果提示，脾CD4~+T细胞数量下降主要为Th1型细胞数量的下降，而Th2型细胞数量下降不明显或没有下降。而且这一结果也进一步解释了体液免疫应答，即抗Ag85A蛋白的特异性抗体可以产生。本实验结果还发现L+JO组IFN—r的分泌水平显著高于L+JA组(P值＜0.0001)。出现这一结果可能是因为空质粒(pCDNA3.1~+)中所含的CpG基序具有明显刺激T细胞分泌IFN-r作用。而重组质粒(pCDNA3.1~+／Ag85A)由于有Ag85A基因片断的插入，CpG基序刺激IFN-r的分泌活性可能受到了抑制。本研究结果显示口服JO、L+JO二组之间对CD4~+T细胞和CD8~+T细胞数量影响无显著差异，JA和L+JA组也未见显著差异，进一步证明脂质体作为运载体对机体几乎无毒性。同时，我们在研究中还发现用脂质体包裹制备的口服DNA疫苗组(L+JA组)诱导Ag85A特异性抗体产生水平高于未用脂质体包裹重组质粒组(JA组)，表明脂质体可具有佐剂作用。ELISPOT技术可检出百万分之一的分泌细胞因子阳性细胞，灵敏度极高。实验中除应用抗原进行刺激之外，还使用了识别细胞因子和抗体的两个不同表位的单克隆抗体，因此ELISPOT技术有着高度的特异性。本次实验采用无血清ELISPOT技术。即细胞因子分泌细胞中，使用了不含血清的培养基，同时采用无内毒素质粒进行脂质体包裹，最大限度的降低内毒素的影响。ELISPOT技术检测中需用特异性刺激物应该是：成分尽可能简单，纯度尽可能高，内毒素的污染尽可能被避免。大肠杆菌中重组基因表达的蛋白质含有的大肠杆菌及内毒素成分，极易影响ELISPOT的检测结果。为此，本研究中选择在哺乳动物细胞中表达的蛋白质，即刺激物是稳定转染重组质粒并表达Ag85A蛋白的哺乳动物细胞K562的裂解物，其中的蛋白己通过SDS-PAGE及Western-blot验证，在保证纯度的前提下，为哺乳动物细胞中表达的蛋白质，内毒素含量在实验允许范围内，与ELISPOT的相容性较好。
结论
1、经DNA序列测定证实插入片段的序列与与GenBank中结核分枝杆菌Ag85A核苷酸编码的氨基酸同源性为100％，pCDNA3.1~+／Ag85A重组质粒构建成功。
2、重组质粒pCDNA3.1~+／Ag85A DNA经脂质体转染真核细胞表达的Ag85A蛋白具有免疫原性。
3、成功转染并获得稳定表达Ag85A蛋白的K562细胞株，此细胞可用于Ag85A蛋白的大量制备及其它方面的研究。
4、口服自制Ag85A DNA疫苗可在体内表达并刺激抗Ag85A特异性抗体的产生。
5、口服自制Ag85A DNA疫苗下调了脾CD4~+T细胞和CD8~+T细胞亚群的数量。
6、分泌IFN--r的Th1型细胞比率和血清中的IFN—r水平下降，而分泌IL-4的Th2型细胞比率和血清中的IL-4水平升高。提示脾CD4~+T细胞数量下降可能主要为Th1型细胞数量的下降，而Th2型细胞数量下降不明显或没有下降。
7、L+JA组IFN-r的分泌水平显著低于L+JO组，提示载体质粒pCDNA3.1~+中所含的CpG基序可能具有刺激T细胞分泌IFN-r作用，而重组质粒pCDNA3.1~+／Ag85A中，由于Ag85A基因片断的插入，CpG基序刺激IFN-r的分泌活性可能受到了抑制。
8、脂质体包裹制备的口服DNA疫苗组(L+JA组)诱导Ag85A特异性抗体产生水平高于未用脂质体包裹重组质粒组(JA组)，表明脂质体可具有免疫佐剂作用。
9、口服JO、L+JO二组之间对CD4~+T细胞和CD8~+T细胞数量影响无显著差异，JA和L+JA组间也未见显著差异，进一步证明脂质体作为运载体对机体几乎无毒性。
Objective
Tuberculosis (TB) is a chronic respiratory infectious disease that has afflicted humans for thousands of years. For 80 years, the bacillus Calmette-Gue'rin (BCG) vaccine has been the only licensed TB vaccine given to humans covering 86% of the world population in 2001. However, despite the use of BCG, TB remains a global epidemic with one-third of the world population being infected and an annual rate of 8 million new cases and 2-2.5 million deaths. Regardless of its protection from severe forms of childhood TB, BCG fails to confer protection from adult TB. Furthermore, BCG vaccine may cause severe complications in immunocompromised hosts. Thus, there is an urgent need for developing safe and effective TB vaccines that are able to confer potent protection at the mucosal site.
Presently Tuberculosis appears an increasing trend not only in developing countries but also in advanced countries. It is still a severe disease imperiling human health in the 21st century. Migration, HIV infection and multidrug resistants are the major dificulties for Tuberculosis control work.. The protective effect of BCG ranges from 0% to 85%, especially has a very low efficacy to adults and cannot be used for therpay. Protective ability of BCG varies in areas and populations, especially in developing countries such as in Africa areas, BCG is virtually usefuless. Since the global situation of Tuberculosis has become so severely and BCG is far from being an ideal vaccine against Tuberculosis, and there is no suitable vaccine available at moment, it is necessary to search for an efficient and safe and vaccine for us. As a new vaccine, DNA vaccine has been achieved inspiring progress in other infective diseases and tumor research. DNA vaccine can induce both of humoral and cellular immunity. Inoculation of DNA vaccines into muscle or skin by a syringe or a propulsion device such as a gene gun results in uptake of the DNA into cells, followed by transcription and translation of the pathogen's gene and, consequently, an immune response composed of antibodies, T helper cells and cytotoxic T lymphocytes (CTL). DNA vaccines have several advantages over traditional vaccines: they stimulate a full spectrum of immuneresponses including CTLs generally not induced by protein vaccines and they generate exceptionally long lasting immune responses. They provide their own adjuvant in the form of unmethylated bacterial CpG sequences that induce an innate immune response which in turn sponsors activation of an antigen-specific immune response. DNA vaccines, which were first described in 1992, have been shown to induce immune responses to a variety of viral, bacterial and parasitic antigens. In addition, they have shown efficacy in treatment of allergic diseases, autoimmunity and tumor models.
DNA vaccines, based on plasmid vectors ex-response to DNA vaccines can be enhanced by genetic engineering of the antigen to facilitate its presentation to pressing an antigen under the control of a strong promoter, which have been shown to induce protective immune B and T cells. Furthermore, the immune response can be modulated by genetic adjuvants in the form of vectors responses to a number of pathogens, including viruses, expressing biologically active determinants or by more bacteria and parasites. They have also displayed efficacy in treatment or prevention of cancer, allergic diseases and traditional adjuvants that facilitate uptake of DNA into autoimmunity. Immunologically, DNA vaccines induce cells. The ease of genetic manipulation of DNA vaccines a full spectrum of immune responses that invites their use not only as vaccines but also as research T cells, T helper cells and antibodies.
Vectors employed for the construction of DNA vaccines are bacterial plasmids which are otherwise commonly used for in vitro expression of proteins in Most DNA vaccine vectors contain an intron, which is an element that can increase expression of genes. This 'cassette' is followed by the gene encoding the antigen of interest flanked by the SV40 or bovine growth hormone 3%-untranslated region (BGH 3%-UTR) transcrip termination: polyadenylation sequences. This part of the vector is often referred to as the transcriptional unit responsible for antigen synthesis. The other part is the plasmid backbone that contains an origin of replication (ori) enabling high-yield production in Escherichia coli along with an antibiotic resistance gene, such as ampicillin (not approved by federal agencies for use in humans) or kanamycin (a resistance marker suitable for human vaccines), to confer antibiotic-selected growth in bacteria. This part of the plasmid backbone contains unmethylated CpG sequences that possess important immunomodulatory properties and provides an intrinsic adjuvant effect for DNA vaccines. It is thought that the magnitude of the immune response to DNA vaccines directly correlates with the level of antigen expression, measured in vitro upon transient transfection of cells.
Antigen 85A is one of the components of the Antigen 85 complex. This protein is expressed by a wide range of bacteria of genus Mycobacterium. The Antigen 85A protein is coded by the gene called fibronectin-binding protein-A (fbpA) gene. In Mycobacterium tuberculosis, this fibronectin-binding protein-A (fbpA) gene is 1014bp long and has a molecular weight of 32KD.
The fbpA gene codes for 338 amino acids which characterize the Antigen 85A protein. Antigen 85A has been applied differently in a wide range of research because of its role in the immunogenicity of the bacteria of genus Mycobacterium. The gene expressing the antigen 85A protein, fbpA, has been identified in different species of the mycobacteria such as M. leprae, M. gordonae, M. bovis, M. avium and they all show close homology to that of M. tuberculosis. Recent animal studies have shown that vaccination with Recombinant Antigen 85A DNA or the protein has powerful immunological properties, resulting in significant secretion of Th1 cells cytokines, particularly interleukins and interferon-γwhich are important in regulating the activities of a number of immune cells, especially the cytotoxic cells. These cytotoxic cells play significant roles in the elimination of viral infected and transformed cells.
Increasing evidence suggests that vaccination at the mucosal site is superior to vaccination at other sites in eliciting protection from mucosal infectious diseases. This is partially explained by the observation that memory T and B cells generated upon mucosal vaccination acquire mucosa-homing receptors and preferentially accumulate at the mucosal site of induction. Thus, it is believed that greater immune protection may be achieved if TB vaccine is given mucosally via oral route.
The cationic liposome acting as an adjuvant can greatly enhance the expression of recombinant plasmiddue to the function of preventing delivered DNA from digested by Dnase and can also promote the absorbance of cellular level. In addition, liposome has several advantages over traditional adjuvant., such as, easy preparated and ready to use, most important, with the high efficiency in gene transfection.
Materials and methods Materials
1. Mice
Male C57B1/6, 6-8 weeks old, were purchased from Animal Laboratories (Chinese Medical University). C57BL/6 mice were immunized and randomly separated 30 mice into 5 groups: (NS)saline, (JO) plasmid vector, (L+JO) liposomal plasmid vector, (JA)recombinant plasmid, (L+JA)liposomal recombinant plasmid.
2. Reagent
Lipofectamine~(TM) 200 were purchased from Invitrogen Co.(USA). Mammalian Cell Lysis Kit were purchased from Bio Basic Inc. (Canada) Anti-Chicken IgY, HRP Conjugate and Anti-Chicken IgY-FITC, PureYield Plasmid extraction kit were purchased from Promega Co.(USA). chichen anti-TB Ag85A IgY Monoclonal antibody were purchased from Prosci Co. Mouse CD4—FITC, CD8—PE, CD8—PE-CY5 antibodies were purchased from BD Co. (USA). IFN-r and IL-4 ELISA kit were purchased from R&D Co. (USA). Mouse IFN-r ELISPOT Kit, Mouse IL-4 ELISPOT Kit were purchased from U-CyTech Co. (Netherlands).
Method
1. The preparation of recombinant pCDNA3.1~+/Ag85A plasmid
The gene encoding Ag85A mature protein was amplified by polymerase chain reaction (PCR) using forward primer 5'-CAGGATCCGCGCGCGCAGTCTGACCTAGTTGAGGATGC-3', containing BamHI cloning site; reverse primer 5'-GTCTCGAGAGGGCCGCCGCCGTTAATCGCT-3' containing Xho I cloning site, while genome of mycobacterium tuberculosis H37Rv strain as template, and PCR product treated with DNA gel extraction was inserted into cloning vector pUCm-T After transformation into competent DH5α,. the pUCm-Ag85A plasmid was extracted and digested with restriction enzyme BamHI and XhoI, then was subcloned to the same sites of eukaryotic expressing vector pcDNA3.1. after transformation into competent DH5α, the clone growing in SOB agar with amp were selected then the plasmid was extracted. and determine the fragment was correctly inserted into the vector, which was confirmed by partial nucleotide sequencing and restriction endonuclease digestion with restriction enzyme BamHI and XhoI.
2.Screening of stably expressed K562 cells
(1) The preparation of endotoxin-free plasmid by Pure Yield Plasmid with extraction kit
(2) Lipofectamine~(TM) 2000 mediated transfection into K562 cells
Just prior to preparing complexes, plate 4×10~5 cells in 500ul of growth medium without antibiotics. Then dilute 0.2μg DNA in 50ul DMEM without serum and mix Lipofectamine~(TM) 2000 gently before use, dilute 0.5μl in 50μl DMEM without serum. Incubate for 5 minutes at room temperature. Combine the diluted DNA with diluted Lipofectamine~(TM) 2000. Mix gently and incubate foy 20 minutes at room temperature. Then add 100μl of complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth. Incubate cells at 37℃in a CO_2 incubator for 36 hours for testing transgene expression. For stable cell lines, passage cells at a 1:10 into fresh growth medium 24 hours after transfection. Add G418 into DMEM as selective medium the following day.
For K562 cells transfected afer 36 hours, centrifuge for minutes at 420×g, decant the supernatant. Wash the cells twice by resuspending the pellets with 1×PBS; centrifuge and discard the supernatant; Re-suspend the pellet in 1×cell lysis buffer, incubate for 5 minutes; Centrifuge the lysed cells for 45 minutes at 100000×g to prepare a protein solution. The extracted protein was used for SDS-PAGE and Western blotting analysis.; after the preliminary SDS-PAGE, the Recombinant Antigen 85A protein was transferred on to Nitrocellulose membrane and later incubated with chichen anti-TB Ag85A IgY Monoclonal antibody. For this technique, preliminary SDS-PAGE was run on two gels and one was stained using Coomasie stain while the other was used for the immunoblotting. After the transfer of the bands from the gel onto the nitrocellulose paper, the paper was properly blocked with BSA. This blocking step ensured that the binding potentials of non-specific antigens were eliminated. After the preliminary washing and the addition of the secondary antibody-horse radish peroxidase, a band corresponding to that on the Coomasie-stained gel was obtained.
(3) Screening of stably expressed K562 cells
passage cells at a 1:10 into fresh growth medium 24 hours after transfection. Add G418 into DMEM as selective medium the following day. The concentration of G418 in medium are 800μg/ml, 400μg/ml, 200μg/ml, 100μg/ml, 50μg/ml respectively. The medium were changed every 3-4 days. The screening pressure continues for 4 weeks, then we determine the lowest concentration of G418 to kill the non-transfection K562 cells as the appropriate pressure to culture another 6 weeks so that stably transfected K562 cells could be screened.
(4) Intracellular anti-chicken IgY-FITC staining were used to determine the expression efficacy in vitro.
3. The effect on the T cells subset of the oral recombinant plasmid pCDNA3.1~+/Ag85A mediated with LipofectamineTM 2000
(1) Recombinant plasmid containing Ag85A using liposome as a vector was constructed and administered to C57BL/6 mice via oral route.. C57BL/6 mice were immunized three times at 14 days intervals, 30 mice were randomly separated into 5 groups: (NS)saline, (JO)plasmid vector,(L+JO)liposomal plasmid vector, (JA)recombinant plasmid, (L+JA)liposomal recombinant plasmid.
(2) Determination of the contents of IFN-r, IL-4 in serum of C57BL/6 mice by double antibody sandwich ELISA, furthermore, the ability of splenocytes secreting IFN-γand IL-4 was tested by ELISPOT method..
(3) When the mice were vaccinated with recombinant eukaryotic expressing vector 5 weeks later, titers of serum antibody against Ag85A were detected by ELISA.
(4) Determination by flow cytometry of the percentage of the CD4~+ and CD8~+ T cell subsets in the splenocytes.
(5) Intracellular anti-chicken IgY-FITC staining were used to determine the expression efficacy in vitro.
Results
1. The nucleotide sequencing of the gene encoding Ag85A mature form of mycobacterium tuberculosis H37Rv strain was identical and there were no undesired mutations, the cloning gene was correctly inserted into the vector pcDNA3.1~+ as confirmed by restriction endonuclease digestion of Bam HI and XhoI and sequence analysis.
2. We obtain a stable cell line which can express the protein with molecular mass about 32KD by using SDS, and showed immunogenicity with monoclonal antibodies of Ag85A, as detected with western blot.
3. The effect on the T cells subset of the oral recombinant plasmid pCDNA3.1~+/Ag85A mediated with Lipofectamine~(TM) 2000
(1) Lymphocytes obtained from the spleen of pcDNA3.1~+/Ag85A vaccine mediated with lipofectamine~(TM) 2000 exhibited lower IFN-γproduction and higher IL-4 production than that for pcDNA3.1~+ vector immunized mice.
(2) The number of spleen MNC secreting IFN-γstimulated by Ag85A protein in vitro Ag85A protein were significantly lower than those of plasmid vector group.
(3) L+JA immunized mice elicited higher Ag85A-specific antibodies titres than that for JA immunized mice. The plasmid vector group and recombinant plasmid group both mediated with Lipofectamine~(TM) 2000, the titres are 1:80 and 1:160 respectively by EL ISA.
(4) Determination by flow cytometry of the percentage of the CD4~+ and CD8~+ T cell subsets in the splenocytes. The subset both shows the decrease of percentage compared with control group.
(5) Fluorescence-activated cell sorter (FACS) analysis after intracellular staining displayed that 41.3% pcDNA3.1~+/Ag85Atransfected stable K562 cells was FITC positive; whereas the controls only showed 1.5% positive cells.
Discussion
In this study, pCDNA3.1~+ was chosen as the expression vector of recombinant DNA vaccine due to the CMV promoter and BGH gene. CpG seguence can induce the immune activity, furthermore, this vector can shuttle between the DH5αand K562 cells, the Laz gene can supply another screening marker in addition to the Amp resistence. The sequence analysis of cloning Ag85A showed the complete same amino acid sequence coding.
The purity of DNA can greatly affect transfection efficiency. We chose the Vitagene kit to extract endotoxin-free plasmid to avoid the interference of the detection result. G418 can kill the growing cells, so we use it to screen the stably transfected K562 cells through dose selection as screening pressure.
CD4~+T and CD8~+T cell subsets can directly exhibit the celluar immune level.
In ELISPOT assay, we choose serum-free medium and endotoxin-free plasmid to assure the accuracy of the result, Ag85A protein which was isolated from K562 cells can lower the influence of endotoxin because the purity had been detected through SDS-PAGE and western-blot. ELISPOT assay has three typical advantanges. First, high sensitivity; second, single cell detection level; Last, easy operation.
The oral recombinant plasmid pCDNA3.1~+/ Ag85A. mediated with Lipofectamine~(TM) 2000 showed down-regulation effect of the subsets of CD4~+ T cells and CD8~+ T cells could induce Th2 type humoral response and suppressed the secretion of IFN-γin C57BL/6 mice by ELISPOT. In addition, the pCDNA3.1~+/Ag85A DNA vaccine can greatly enhance the titres of Ag85A-specific antibodies by ELISA. The down-regulation effect of the subsets of CD4~+ T cells and CD8~+ T cells reflect the oral immune tolerance. Because the immune system is a network, CD4~+CD25~+Treg and TGF-βtogether with apoptosis factors can lead to suppression of CD8~+T subsets, iDC and mDC can induce tolerance due to the secretion of chromosome. According to our result, liposome can act as adjunvant without toxicity to cells.
Conclusion
1. DNA vaccine encoding Ag85A mature protein ofmycobacterium tuberculosis was constructed and identified successfully.
2. The Ag85A protein was stably expressed after G418 screening transfected K562 cells.
3. The oral recombinant plasmid pCDNA3.1~+/Ag85A. mediated with Lipofectamine~(TM) 2000 shows down-regulation effect of the subsets of CD4~+ T cells and D8~+ T cells.
4. The oral recombinant plasmid pCDNA3.1~+/Ag85A has good immunogenecity and could induce Th2 type humoral response in C57BL/6 mice by ELISPOT.
5. The oral recombinant plasmid pCDNA3.1~+/Ag85A mediated with Lipofectamine~(TM) 2000 group suppressed the secretion of IFN-γ, while the plasmid pCDNA3.1~(+W) with Lipofectamine~(TM) 2000 group increased the IFN-γlevel due to the CpG sequences.
6. The oral recombinant plasmid pCDNA3.1~+/Ag85A mediated with Lipofectamine~(TM) 2000 can greatly enhance the titres of Ag85A-specific antibodies by ELISA.
7. Lipofectamine~(TM) 2000 can act as an adjuvant through comparation with negative control group.
引文
1 Black GF, Weir RE, Chaguluka SD,et al.Gamma interferon responses induced by a panel of recombinant and purified mycobacterial antigens in healthy, non-mycobacterium bovis BCG-vaccinated Malawian young adults.Clin Diagn Lab Immunol. 2003;10(4):602-11
2 Lozes E, Huygen K, Content J,et al.Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex. Vaccine. 1997;15(8):830-3
3 .Rosseels V, Marche S, Roupie V,et al.Members of the 30- to 32-kilodalton mycolyl transferase family (Ag85) from culture filtrate of Mycobacterium avium subsp, paratuberculosis are immunodominant Th1-type antigens recognized early upon infection in mice and cattle.Infect Immun. 2006,74(1):202-12
4 D'Souza S, Rosseels V, Romano M,et al.Mapping of murine Th1 helper T-Cell epitopes of mycolyl transferases Ag85A,Ag85B, and Ag85C from Mycobacterium tuberculosis.Infect Immun. 2003;71(1):483-93
5 Godfrey HP, Feng Z, Mandy S,et al.Modulation of expression of delayed hypersensitivity by mycobacterial antigen 85 fibronectin-binding proteins. Infect Immun. 1992 Jun;60(6):2522-8.
6 D'Souza S, Romano M, Korf J,et al.Partial reconstitution of the CD4~+-T-cell compartment in CD4 gene knockout mice restores responses to tuberculosis DNA vaccines.Infect Immun. 2006 ;74(5):2751-9
7 Launois P, Vandenbussche P, M'Bayame NN,et al. IL-6 production in response to purified mycobacterial heat-shock proteins and to antigen 85 in leprosy. Cell Immunol. 1993 ;148(2):283-90
8 Rosseels V, Marche S, Roupie W,et al.Members of the 30- to 32-kilodalton mycolyl transferase family (Ag85) from culture filtrate of Mycobacterium avium subsp, paratuberculosis are immunodominant Th1-type antigens recognized early upon infection in mice and cattle.Infect Immun. 2006 ;74(1):202-12
9 Zlotta AR, Drowart A, Huygen K,et al.Humoral response against heat shock proteins and other mycobacterial antigens after intravesical treatment with bacille Calmette-Guerin (BCG) in patients with superficial bladder cancer.Clin Exp Immunol. 1997; 109( 1): 157-65
10 Tanghe A, Content J, Van Vooren JP,et al.Protective efficacy of a DNA vaccine encoding antigen 85A from Mycobacterium bovis BCG against Buruli ulcer. Infect Immun.2001;69(9):5403-11
11 Bentley-Hibbert SI, Quan X, Newman T,et al.Pathophysiology of antigen 85 in patients with active tuberculosis: antigen 85 circulates as complexes with fibronectin and immunoglobulin G.Infect Immun. 1999;67(2):581-8
12 Valle MT, Megiovanni AM, Merlo A,et al.Epitope focus, clonal composition and Th1 phenotype of the human CD4 response to the secretory mycobacterial antigen Ag85.Clin Exp Immunol. 2001;123(2):226-32
13 Kirman JR, Turon T, Su H,et al.Enhanced immunogenicity to Mycobacterium tuberculosis by vaccination with an alphavirus plasmid replicon expressing antigen 85A.Infect Immun. 2003;71(1):575-9
14 Romano M,Roupie V, Wang XM,et al. Immunogenicity and protective efficacy of tuberculosis DNA vaccines combining mycolyl-transferase Ag85A and phosphate transport receptor PstS-3.Immunology. 2006;118(3):321-32
15 Romano M, Roupie V, Hamard M,et al. Evaluation of the immunogenicity of pBudCE4.1 plasmids encoding mycolyl-transferase Ag85A and phosphate transport receptor PstS-3 from Mycobacterium tuberculosis.Vaccine. 2006 ;24(21):4640-3
16 Huygen K, Content J, Denis O,et al. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine.Nat Med. 1996;2 (8):893-8.
17 Sugawara I, Yamada H, Udagawa T,et al.Vaccination of guinea pigs with DNA encoding Ag85A by gene gun bombardment.Tuberculosis (Edinb). 2003;83(6):331-7
18 Tanghe A, D'Souza S, Rosseels V,et al.Improved immunogenicity and protective efficacy of a tuberculosis DNA vaccine encoding Ag85 by protein boosting. Infect Immun. 2001 ;69(5):3041-7
19 Romano M, D'Souza S, Adnet PY,et al. Priming but not boosting with plasmid DNA encoding mycolyl-transferase Ag85A from Mycobacterium tuberculosis increases the survival time of Mycobacterium bovis BCG vaccinated mice against low dose intravenous challenge with M.tuberculosis H37Rv.Vaccine. 2006;24(16):3353-64
20 Tarrant JP, Walsh MJ, Blanchard MC,et al .Reduced tumorigenicity of B16-F10 mouse melanoma cells transfected with mycobacterial antigen 85A.Int J Oncol. 2004;25(6): 1693-9
21 Dou J, Chen JS, Wang J,et al.Novel constructs of tuberculosis gene vaccine and its immune effect on mice.Cell Mol Immunol. 2005;2(1):57-62
22 Landowski CP,Godfrey HP,Bentley-Hibbert Sl,et al.Combinatorial use of antibodies to secreted mycobacterial proteins in a host immune system-independent test for tuberculosis.J Clin Microbiol. 2001;39(7):2418-24
23 Parida SK,Huygen K,Ryffel B,et al.Novel Bacterial Delivery System with Attenuated Salmonella typhimurium Carrying Plasmid Encoding Mtb Antigen 85A for Mucosal Immunization: Establishment of Proof of Principle in TB Mouse Model.Ann N Y Acad Sci. 2005; 1056:366-78
24 Ulmer JB,Liu MA,Montgomery DL,et al. Expression and immunogenicity of Mycobacterium tuberculosis antigen 85 by DNA vaccination.Vaccine. 1997; 15(8) :792-4
25 Ki K,Nagata T,Tanaka T,et al.Induction of protective cellular immunity against Mycobacterium tuberculosis by recombinant attenuated self-destructing Listeria monocytogenes strains harboring eukaryotic expression plasmids for antigen 85 complex and MPB/MPT51.Infect Immun. 2004;72(4):2014-21
26 Dermeier HM, Rhodes SG,et al.Cellular immune responses induced in cattle by heterologous prime-boost vaccination using recombinant viruses and bacille Calmette-Guerin.Immunology. 2004; 112(3):461-70
27 Gronevik E, Mathiesen I, Lomo T,et al.Early events of electroporation-mediated intramuscular DNA vaccination potentiate Th1-directed immune responses.J Gene Med. 2005;7(9): 1246-54
28 Dayball K, Millar J, Miller M, et al.Electroporation enables plasmid vaccines to elicit CD8+ T cell responses in the absence of CD4+ T cells.J Immunol. 2003;171(7):3379-84
29 Perrie Y, Obrenovic M, McCarthy D,et al.Liposome (Lipodine)-mediated DNA vaccination by the oral route.J Liposome Res. 2002;12(1-2):185-97
30 Ruozi B, Battini R, Montanari M,et al. DOTAP/UDCA vesicles: novel approach in oligonucleotide delivery.Nanomedicine. 2007;3(1):1-13
31 Zhang HW, Zhang L, Sun X,et al. Successful transfection of hepatoma cells after encapsulation of plasmid DNA into negatively charged liposomes.Biotechnol Bioeng. 2007;96(1):118-24
32 Fletcher S, Ahmad A, Perouzel E,et al. A dialkynoyl analogue of DOPE improves gene transfer of lower-charged, cationic lipoplexes.Org Biomol Chem. 2006;21;4(2): 196-9
33 Singh M, Ariatti M. A cationic cytofectin with long spacer mediates favourable transfection in transformed human epithelial cells.Int J Pharm. 2006; 17;309( 1-2): 189-98
34 Caracciolo G, Marchini C, Pozzi D,et al.Structural Stability against Disintegration by Anionic Lipids Rationalizes the Efficiency of Cationic Liposome/DNA Complexes.Langmuir.2007; 10;23(8):4498-508
35 Zhong ZR,ZhangZR, Liu J,et al.Characterization of transferrin-modified procationic-liposome protamine-DNA complexes.Yakugaku Zasshi. 2007;127(3):533-9
36 Yu JN, Ma SF, Miao DQ,et al. Effects of cell cycle status on the efficiency of liposome-mediated gene transfection in mouse fetal fibroblasts.J Reprod Dev. 2006;52(3):373-82
37 Zhang ZY,Ugwu S, Zhang A,et al.A novel cationic cardiolipin analogue for gene delivery. Pharmazie. 2006;61 (1): 10-4
38 Wang WR, Lin R, Yang YC,et al.Liposome-mediated human CD40 gene transfection and human umbilical vein endothelial ECV-304 cells,Di Yi Jun Yi Da Xue Xue Bao. 2005 ;25(12):1474-7
39 Hayes ME, Drummond DC, Kirpotin DB,et al.Genospheres: self-assembling nucleic acid-lipid nanoparticles suitable for targeted gene delivery.Gene Ther. 2006;13(7):646-51
40 Corsi K, Chellat F, Yahia L,et al.Mesenchymal stem cells, MG63 and HEK293 transfection using chitosan-DNA nanoparticles.Biomaterials.2003;24(7):1255-64
41 Dea-Ayuela MA, Rama-Iniguez S, Bolas-Fernandez F,et al.Vaccination of mice against intestinal Trichinella spiralis infections by oral administration of antigens microencapsulated in methacrilic acid copolymers.Vaccine. 2006;24(15):2772-80
42 Hatzifoti C, Roussel Y, Harris AG,et al.Mucosal immunization with a urease B DNA vaccine induces innate and cellular immune responses against Helicobacter pylori. Helicobacter. 2006; 11(2): 113-22
43 Wang R, Epstein J, Charoenvit Y,et al.Induction in humans of CD8+ and CD4+ T cell and antibody responses by sequential immunization with malaria DNA and recombinant protein.J Immunol. 2004; 172(9):5561-9
44 Wilson CC, McKinney D, Anders M,et al .Development of a DNA vaccine designed to induce cytotoxic T lymphocyte responses to multiple conserved epitopes in HIV-1.J Immunol. 2003;171(10):5611-23
45 Lladser A, Parraga M, Quevedo L,et al.Naked DNA immunization as an approach to target the generic tumor antigen survivin induces humoral and cellular immune responses in mice.Immunobiology. 2006;211(1-2):11-27
46 Gerdts V, Snider M, Brownlie R, et al .Oral DNA vaccination in utero induces mucosal immunity and immune memory in the neonate.J Immunol. 2002 ; 168(4): 1877-85
47 Somavarapu S, Bramwell VW, Alpar HO.Oral plasmid DNA delivery systems for genetic immunisation.J Drug Target. 2003;11(8-10):547-53
48 Faisant N, Akiki J, Siepmann F,et al. Effects of the type of release medium on drug release from PLGA-based microparticles: Experiment and theory. Int J Pharm. 2006;314(2): 189-97
49 Zheng BJ, Ng MH, Chan KW,et al .A single dose of oral DNA immunization delivered by attenuated Salmonella typhimurium down-regulates transgene expression in HBsAg transgenic mice.Eur J Immunol. 2002;32(11):3294-304
50 Naito T, Kaneko Y, Kozbor D, et al.Oral vaccination with modified vaccinia virus Ankara attached covalently to TMPEG-modified cationic liposomes overcomes pre-existing poxvirus immunity from recombinant vaccinia immunization.J Gen Virol. 2007;88:61-70
51 Sliedregt LA, Rensen PC, Rump ET,et al.Design and synthesis of novel amphiphilic dendritic galactosides for selective targeting of liposomes to the hepatic asialoglycoprotein receptor. J Med Chem 1999;42:609-618
52 Di Campli C, Wu J, Zern MA, et al.Targeting of therapeutics to the liver: liposomes and viral vectors. Alcohol Clin Exp Res 1999;23:950-954
53 Machluf M, Apte RN, Regev O,et al.Enhancing the immunogenicity of liposomal hepatitis B surface antigen (HBsAg) by controlling its delivery from polymeric microspheres. J Pharm Sci 2000;89:1550-1557
54 Perrie Y, Obrenovic M, McCarthy D,et al.Liposome (Lipodine)-mediated DNA vaccination by the oral route.J Liposome Res. 2002; 12(1-2): 185-97
55 Maier M, Seabrook TJ, Lemere CA.Modulation of the humoral and cellular immune response in Abeta immunotherapy by the adjuvants monophosphoryl lipid A (MPL), cholera toxin B subunit (CTB) and E.coli enterotoxin LT(R192G).Vaccine. 2005;23(44):5149-59
56 Helgeby A, Robson NC, Donachie AM,et al.The combined CTA1 -DD/ISCOM adjuvant vector promotes priming of mucosal and systemic immunity to incorporated antigens by specific targeting of B cells.J Immunol. 2006;176(6):3697-706
57 Kvarnstrom, M., M. C. Jenmalm, and C. Ekerfelt, et al .Effect of cryopreservation on expression of Th1 and Th2 cytokines in blood mononuclear cells from patients with different cytokine profiles, analysed with three common assays: an overall decrease of interleukin-4. Cryobiology. 2004;49:157-68
58 Alix-Panabieres, P. Brouillet, M. Fabbro,et al.Characterization and enumeration of cells secreting tumor markers in the peripheral blood of breast cancer patients. J Immunol Methods 2005;299:177-88
59 Anthony, P. V. Lehmann. T-cell epitope mapping using the ELISPOT approach. Methods 2003;29:260-9
60 Stanford M, Whittall T, Bergmeier LA,et al.Oral tolerization with peptide 336-351 linked to cholera toxin B subunit in preventing relapses of uveitis in Behcet's disease.Clin Exp Immunol. 2004;137(1):201-8
61 Roelofs-Haarhuis K, Wu X, Gleichmann E,et al.Oral tolerance to nickel requires CD4+ invariant NKT cells for the infectious spread of tolerance and the induction of specific regulatory T cells. J Immunol.2004; 173(2): 1043-50
62 Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells.Nat Rev Immunol. 2003;3(3):253-7
63 Steinman RM, Turley S, Mellman I, et al .The induction of tolerance by dendritic cells that have captured apoptotic cells.J Exp Med. 2000;191(3):411-6
64 Bonifaz L, Bonnyay D, Mahnke K,et al.Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8~+ T cell tolerance.J Exp Med. 2002; 196(12): 1627-38
65 Payette PJ, Ma X, Weeratna RD,et al.Testing of CpG-optimized protein and DNA vaccines against the hepatitis B virus in chimpanzees for immunogenicity and protection from hallenge.Intervirology. 2006;49(3): 144-51
1 Dea-Ayuela MA, Rama-Iniguez S, Bolas-Fernandez F,et al. Vaccination of mice against intestinal Trichinella spiralis infections by oral administration of antigens microencapsulated in methacrilic acid copolymers.Vaccine. 2006; 5;24(15):2772-80
2 Hatzifoti C, Roussel Y, Harris AG,et al.Mucosal immunization with a urease B DNA vaccine induces innate and cellular immune responses against Helicobacter pylori. Helicobacter. 2006; 11 (2): 113-22
3 Talavera A, Ano G, Pino Y,et al.Formulation in tablets of a cholera whole cells inactivated vaccine candidate.Vaccine.2006 12;24(16):3381-7
4 Karakasiliotis I, Paximadi E, Markoulatos P.Evolution of a rare vaccine-derived multirecombinant poliovirus.J Gen Virol. 2005;86:3137-42
5 Franco MA, Angel J, Greenberg HB,et al.Immunity and correlates of protection for rotavirus vaccines.Vaccine. 2006;24(15):2718-31
6 Robert Putnak J, Coller BA, Voss G,et al. An evaluation of dengue type-2 inactivated, recombinant subunit, and live-attenuated vaccine candidates in the rhesus macaque model.Vaccine.2005;23(35):4442-52
7 Sable SB, Verma I, Khuller GK,et al.Multicomponent antituberculous subunit vaccine based on immunodominant antigens of Mycobacterium tuberculosis.Vaccine. 2005;23(32):4175-84
8 Rouvio O, Dvorkin T, Amir-Kroll H,et al.Self HSP60 peptide serves as an immunogenic carrier for a CTL epitope against persistence of murine cytomegalovirus in the salivary gland.Vaccine.2005;23(27):3508-18
9 Ito N, Sugiyama M, YamadaK,et al.Characterization of M gene-deficient rabies virus with advantages of effective immunization and safety as a vaccine strain.MicrobiolImmunol. 2005;49(11):971-9
10 Lladser A, Parraga M, Quevedo L,et al.Naked DNA immunization as an approach to target the generic tumor antigen survivin induces humoral and cellular immune responses in mice.Immunobiology. 2006;211(1-2): 11-27
11 Parida SK, Huygen K, Ryffel B, et al.Novel Bacterial Delivery System with Attenuated Salmonella typhimurium Carrying Plasmid Encoding Mtb Antigen 85A for Mucosal Immunization: Establishment of Proof of Principle in TB Mouse Model.Ann N Y Acad Sci. 2005; 1056:366-78
12 Dus Santos MJ, Carrillo C, Ardila F,et al.Development of transgenic alfalfa plants containing the foot and mouth disease virus structural polyprotein gene P1 and its utilization as an experimental immunogen. Vaccine. 2005;23( 15): 1838-43
13 Johnson DC, McFarland EJ, Muresan P, et al. Safety and immunogenicity of an HIV-1 recombinant canarypox vaccine in newborns and infants of HIV-1-infected women. J infect Dis.2005;192(12):2129-33
14 Bellier B,Dalba C,Clerc B,et al.DNA vaccines encoding retrovirus-based virus-like particles induce efficient immune responses without adjuvant. Vaccine.2006;24(14):2643-55
15 Singh P, Destito G, Schneemann A,et al.Canine parvovirus-like particles, a novel nanomaterial for tumor targeting.J Nanobiotechnology. 2006;4:2
16 Faisant N, Akiki J, Siepmann F,et al. Effects of the type of release medium on drug release from PLGA-based microparticles: Experiment and theory.Int J Pharm. 2006;314(2): 189-197
17 Mollenkopf HJ, Dietrich G, Fensterle J,et al.Enhanced protective efficacy of a tuberculosis DNA vaccine by adsorption onto cationic PLG microparticles. Vaccine. 2004;22(21-22):2690-5
18 Stanford M, Whittall T, Bergmeier LA,et al.Oral tolerization with peptide 336-351 linked to cholera toxin B subunit in preventing relapses of uveitis in Behcet's disease.Clin Exp Immunol. 2004;137(1):201-8
19 Maier M, Seabrook TJ, Lemere CA,et al.Modulation of the humoral and cellular immune response in Abeta immunotherapy by the adjuvants monophosphoryl lipid A (MPL), cholera toxin B subunit (CTB) and E. coli enterotoxin LT(R192G). Vaccine. 2005;23(44):5149-59
20 Helgeby A, Robson NC, Donachie AM,et al.The combined CTA1-DD/ISCOM adjuvant vector promotes priming of mucosal and systemic immunity to incorporated antigens by specific targeting of B cells.J Immunol. 2006;176(6):3697-706
21 Payette PJ, Ma X, Weeratna RD,et al.Testing of CpG-optimized protein and DNA vaccines against the hepatitis B virus in chimpanzees for immunogenicity and protection from hallenge.Intervirology. 2006;49(3): 144-51
22 Roelofs-Haarhuis K, Wu X, Gleichmann E,et al.Oral tolerance to nickel requires CD4+ invariant NKT cells for the infectious spread of tolerance and the induction of specific regulatory T cells. J Immunol. 2004;173(2):1043-1048
2 Lozes E, Huygen K, Content J,et al.Immunogenicity and efficacy of a tuberculosis DNA vaccine encoding the components of the secreted antigen 85 complex. Vaccine. 1997;15(8):830-3
3 .Rosseels V, Marche S, Roupie V,et al.Members of the 30- to 32-kilodalton mycolyl transferase family (Ag85) from culture filtrate of Mycobacterium avium subsp, paratuberculosis are immunodominant Th1-type antigens recognized early upon infection in mice and cattle.Infect Immun. 2006,74(1):202-12
4 D'Souza S, Rosseels V, Romano M,et al.Mapping of murine Th1 helper T-Cell epitopes of mycolyl transferases Ag85A,Ag85B, and Ag85C from Mycobacterium tuberculosis.Infect Immun. 2003;71(1):483-93
5 Godfrey HP, Feng Z, Mandy S,et al.Modulation of expression of delayed hypersensitivity by mycobacterial antigen 85 fibronectin-binding proteins. Infect Immun. 1992 Jun;60(6):2522-8.
6 D'Souza S, Romano M, Korf J,et al.Partial reconstitution of the CD4~+-T-cell compartment in CD4 gene knockout mice restores responses to tuberculosis DNA vaccines.Infect Immun. 2006 ;74(5):2751-9
7 Launois P, Vandenbussche P, M'Bayame NN,et al. IL-6 production in response to purified mycobacterial heat-shock proteins and to antigen 85 in leprosy. Cell Immunol. 1993 ;148(2):283-90
8 Rosseels V, Marche S, Roupie W,et al.Members of the 30- to 32-kilodalton mycolyl transferase family (Ag85) from culture filtrate of Mycobacterium avium subsp, paratuberculosis are immunodominant Th1-type antigens recognized early upon infection in mice and cattle.Infect Immun. 2006 ;74(1):202-12
9 Zlotta AR, Drowart A, Huygen K,et al.Humoral response against heat shock proteins and other mycobacterial antigens after intravesical treatment with bacille Calmette-Guerin (BCG) in patients with superficial bladder cancer.Clin Exp Immunol. 1997; 109( 1): 157-65
10 Tanghe A, Content J, Van Vooren JP,et al.Protective efficacy of a DNA vaccine encoding antigen 85A from Mycobacterium bovis BCG against Buruli ulcer. Infect Immun.2001;69(9):5403-11
11 Bentley-Hibbert SI, Quan X, Newman T,et al.Pathophysiology of antigen 85 in patients with active tuberculosis: antigen 85 circulates as complexes with fibronectin and immunoglobulin G.Infect Immun. 1999;67(2):581-8
12 Valle MT, Megiovanni AM, Merlo A,et al.Epitope focus, clonal composition and Th1 phenotype of the human CD4 response to the secretory mycobacterial antigen Ag85.Clin Exp Immunol. 2001;123(2):226-32
13 Kirman JR, Turon T, Su H,et al.Enhanced immunogenicity to Mycobacterium tuberculosis by vaccination with an alphavirus plasmid replicon expressing antigen 85A.Infect Immun. 2003;71(1):575-9
14 Romano M,Roupie V, Wang XM,et al. Immunogenicity and protective efficacy of tuberculosis DNA vaccines combining mycolyl-transferase Ag85A and phosphate transport receptor PstS-3.Immunology. 2006;118(3):321-32
15 Romano M, Roupie V, Hamard M,et al. Evaluation of the immunogenicity of pBudCE4.1 plasmids encoding mycolyl-transferase Ag85A and phosphate transport receptor PstS-3 from Mycobacterium tuberculosis.Vaccine. 2006 ;24(21):4640-3
16 Huygen K, Content J, Denis O,et al. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine.Nat Med. 1996;2 (8):893-8.
17 Sugawara I, Yamada H, Udagawa T,et al.Vaccination of guinea pigs with DNA encoding Ag85A by gene gun bombardment.Tuberculosis (Edinb). 2003;83(6):331-7
18 Tanghe A, D'Souza S, Rosseels V,et al.Improved immunogenicity and protective efficacy of a tuberculosis DNA vaccine encoding Ag85 by protein boosting. Infect Immun. 2001 ;69(5):3041-7
19 Romano M, D'Souza S, Adnet PY,et al. Priming but not boosting with plasmid DNA encoding mycolyl-transferase Ag85A from Mycobacterium tuberculosis increases the survival time of Mycobacterium bovis BCG vaccinated mice against low dose intravenous challenge with M.tuberculosis H37Rv.Vaccine. 2006;24(16):3353-64
20 Tarrant JP, Walsh MJ, Blanchard MC,et al .Reduced tumorigenicity of B16-F10 mouse melanoma cells transfected with mycobacterial antigen 85A.Int J Oncol. 2004;25(6): 1693-9
21 Dou J, Chen JS, Wang J,et al.Novel constructs of tuberculosis gene vaccine and its immune effect on mice.Cell Mol Immunol. 2005;2(1):57-62
22 Landowski CP,Godfrey HP,Bentley-Hibbert Sl,et al.Combinatorial use of antibodies to secreted mycobacterial proteins in a host immune system-independent test for tuberculosis.J Clin Microbiol. 2001;39(7):2418-24
23 Parida SK,Huygen K,Ryffel B,et al.Novel Bacterial Delivery System with Attenuated Salmonella typhimurium Carrying Plasmid Encoding Mtb Antigen 85A for Mucosal Immunization: Establishment of Proof of Principle in TB Mouse Model.Ann N Y Acad Sci. 2005; 1056:366-78
24 Ulmer JB,Liu MA,Montgomery DL,et al. Expression and immunogenicity of Mycobacterium tuberculosis antigen 85 by DNA vaccination.Vaccine. 1997; 15(8) :792-4
25 Ki K,Nagata T,Tanaka T,et al.Induction of protective cellular immunity against Mycobacterium tuberculosis by recombinant attenuated self-destructing Listeria monocytogenes strains harboring eukaryotic expression plasmids for antigen 85 complex and MPB/MPT51.Infect Immun. 2004;72(4):2014-21
26 Dermeier HM, Rhodes SG,et al.Cellular immune responses induced in cattle by heterologous prime-boost vaccination using recombinant viruses and bacille Calmette-Guerin.Immunology. 2004; 112(3):461-70
27 Gronevik E, Mathiesen I, Lomo T,et al.Early events of electroporation-mediated intramuscular DNA vaccination potentiate Th1-directed immune responses.J Gene Med. 2005;7(9): 1246-54
28 Dayball K, Millar J, Miller M, et al.Electroporation enables plasmid vaccines to elicit CD8+ T cell responses in the absence of CD4+ T cells.J Immunol. 2003;171(7):3379-84
29 Perrie Y, Obrenovic M, McCarthy D,et al.Liposome (Lipodine)-mediated DNA vaccination by the oral route.J Liposome Res. 2002;12(1-2):185-97
30 Ruozi B, Battini R, Montanari M,et al. DOTAP/UDCA vesicles: novel approach in oligonucleotide delivery.Nanomedicine. 2007;3(1):1-13
31 Zhang HW, Zhang L, Sun X,et al. Successful transfection of hepatoma cells after encapsulation of plasmid DNA into negatively charged liposomes.Biotechnol Bioeng. 2007;96(1):118-24
32 Fletcher S, Ahmad A, Perouzel E,et al. A dialkynoyl analogue of DOPE improves gene transfer of lower-charged, cationic lipoplexes.Org Biomol Chem. 2006;21;4(2): 196-9
33 Singh M, Ariatti M. A cationic cytofectin with long spacer mediates favourable transfection in transformed human epithelial cells.Int J Pharm. 2006; 17;309( 1-2): 189-98
34 Caracciolo G, Marchini C, Pozzi D,et al.Structural Stability against Disintegration by Anionic Lipids Rationalizes the Efficiency of Cationic Liposome/DNA Complexes.Langmuir.2007; 10;23(8):4498-508
35 Zhong ZR,ZhangZR, Liu J,et al.Characterization of transferrin-modified procationic-liposome protamine-DNA complexes.Yakugaku Zasshi. 2007;127(3):533-9
36 Yu JN, Ma SF, Miao DQ,et al. Effects of cell cycle status on the efficiency of liposome-mediated gene transfection in mouse fetal fibroblasts.J Reprod Dev. 2006;52(3):373-82
37 Zhang ZY,Ugwu S, Zhang A,et al.A novel cationic cardiolipin analogue for gene delivery. Pharmazie. 2006;61 (1): 10-4
38 Wang WR, Lin R, Yang YC,et al.Liposome-mediated human CD40 gene transfection and human umbilical vein endothelial ECV-304 cells,Di Yi Jun Yi Da Xue Xue Bao. 2005 ;25(12):1474-7
39 Hayes ME, Drummond DC, Kirpotin DB,et al.Genospheres: self-assembling nucleic acid-lipid nanoparticles suitable for targeted gene delivery.Gene Ther. 2006;13(7):646-51
40 Corsi K, Chellat F, Yahia L,et al.Mesenchymal stem cells, MG63 and HEK293 transfection using chitosan-DNA nanoparticles.Biomaterials.2003;24(7):1255-64
41 Dea-Ayuela MA, Rama-Iniguez S, Bolas-Fernandez F,et al.Vaccination of mice against intestinal Trichinella spiralis infections by oral administration of antigens microencapsulated in methacrilic acid copolymers.Vaccine. 2006;24(15):2772-80
42 Hatzifoti C, Roussel Y, Harris AG,et al.Mucosal immunization with a urease B DNA vaccine induces innate and cellular immune responses against Helicobacter pylori. Helicobacter. 2006; 11(2): 113-22
43 Wang R, Epstein J, Charoenvit Y,et al.Induction in humans of CD8+ and CD4+ T cell and antibody responses by sequential immunization with malaria DNA and recombinant protein.J Immunol. 2004; 172(9):5561-9
44 Wilson CC, McKinney D, Anders M,et al .Development of a DNA vaccine designed to induce cytotoxic T lymphocyte responses to multiple conserved epitopes in HIV-1.J Immunol. 2003;171(10):5611-23
45 Lladser A, Parraga M, Quevedo L,et al.Naked DNA immunization as an approach to target the generic tumor antigen survivin induces humoral and cellular immune responses in mice.Immunobiology. 2006;211(1-2):11-27
46 Gerdts V, Snider M, Brownlie R, et al .Oral DNA vaccination in utero induces mucosal immunity and immune memory in the neonate.J Immunol. 2002 ; 168(4): 1877-85
47 Somavarapu S, Bramwell VW, Alpar HO.Oral plasmid DNA delivery systems for genetic immunisation.J Drug Target. 2003;11(8-10):547-53
48 Faisant N, Akiki J, Siepmann F,et al. Effects of the type of release medium on drug release from PLGA-based microparticles: Experiment and theory. Int J Pharm. 2006;314(2): 189-97
49 Zheng BJ, Ng MH, Chan KW,et al .A single dose of oral DNA immunization delivered by attenuated Salmonella typhimurium down-regulates transgene expression in HBsAg transgenic mice.Eur J Immunol. 2002;32(11):3294-304
50 Naito T, Kaneko Y, Kozbor D, et al.Oral vaccination with modified vaccinia virus Ankara attached covalently to TMPEG-modified cationic liposomes overcomes pre-existing poxvirus immunity from recombinant vaccinia immunization.J Gen Virol. 2007;88:61-70
51 Sliedregt LA, Rensen PC, Rump ET,et al.Design and synthesis of novel amphiphilic dendritic galactosides for selective targeting of liposomes to the hepatic asialoglycoprotein receptor. J Med Chem 1999;42:609-618
52 Di Campli C, Wu J, Zern MA, et al.Targeting of therapeutics to the liver: liposomes and viral vectors. Alcohol Clin Exp Res 1999;23:950-954
53 Machluf M, Apte RN, Regev O,et al.Enhancing the immunogenicity of liposomal hepatitis B surface antigen (HBsAg) by controlling its delivery from polymeric microspheres. J Pharm Sci 2000;89:1550-1557
54 Perrie Y, Obrenovic M, McCarthy D,et al.Liposome (Lipodine)-mediated DNA vaccination by the oral route.J Liposome Res. 2002; 12(1-2): 185-97
55 Maier M, Seabrook TJ, Lemere CA.Modulation of the humoral and cellular immune response in Abeta immunotherapy by the adjuvants monophosphoryl lipid A (MPL), cholera toxin B subunit (CTB) and E.coli enterotoxin LT(R192G).Vaccine. 2005;23(44):5149-59
56 Helgeby A, Robson NC, Donachie AM,et al.The combined CTA1 -DD/ISCOM adjuvant vector promotes priming of mucosal and systemic immunity to incorporated antigens by specific targeting of B cells.J Immunol. 2006;176(6):3697-706
57 Kvarnstrom, M., M. C. Jenmalm, and C. Ekerfelt, et al .Effect of cryopreservation on expression of Th1 and Th2 cytokines in blood mononuclear cells from patients with different cytokine profiles, analysed with three common assays: an overall decrease of interleukin-4. Cryobiology. 2004;49:157-68
58 Alix-Panabieres, P. Brouillet, M. Fabbro,et al.Characterization and enumeration of cells secreting tumor markers in the peripheral blood of breast cancer patients. J Immunol Methods 2005;299:177-88
59 Anthony, P. V. Lehmann. T-cell epitope mapping using the ELISPOT approach. Methods 2003;29:260-9
60 Stanford M, Whittall T, Bergmeier LA,et al.Oral tolerization with peptide 336-351 linked to cholera toxin B subunit in preventing relapses of uveitis in Behcet's disease.Clin Exp Immunol. 2004;137(1):201-8
61 Roelofs-Haarhuis K, Wu X, Gleichmann E,et al.Oral tolerance to nickel requires CD4+ invariant NKT cells for the infectious spread of tolerance and the induction of specific regulatory T cells. J Immunol.2004; 173(2): 1043-50
62 Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells.Nat Rev Immunol. 2003;3(3):253-7
63 Steinman RM, Turley S, Mellman I, et al .The induction of tolerance by dendritic cells that have captured apoptotic cells.J Exp Med. 2000;191(3):411-6
64 Bonifaz L, Bonnyay D, Mahnke K,et al.Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8~+ T cell tolerance.J Exp Med. 2002; 196(12): 1627-38
65 Payette PJ, Ma X, Weeratna RD,et al.Testing of CpG-optimized protein and DNA vaccines against the hepatitis B virus in chimpanzees for immunogenicity and protection from hallenge.Intervirology. 2006;49(3): 144-51
1 Dea-Ayuela MA, Rama-Iniguez S, Bolas-Fernandez F,et al. Vaccination of mice against intestinal Trichinella spiralis infections by oral administration of antigens microencapsulated in methacrilic acid copolymers.Vaccine. 2006; 5;24(15):2772-80
2 Hatzifoti C, Roussel Y, Harris AG,et al.Mucosal immunization with a urease B DNA vaccine induces innate and cellular immune responses against Helicobacter pylori. Helicobacter. 2006; 11 (2): 113-22
3 Talavera A, Ano G, Pino Y,et al.Formulation in tablets of a cholera whole cells inactivated vaccine candidate.Vaccine.2006 12;24(16):3381-7
4 Karakasiliotis I, Paximadi E, Markoulatos P.Evolution of a rare vaccine-derived multirecombinant poliovirus.J Gen Virol. 2005;86:3137-42
5 Franco MA, Angel J, Greenberg HB,et al.Immunity and correlates of protection for rotavirus vaccines.Vaccine. 2006;24(15):2718-31
6 Robert Putnak J, Coller BA, Voss G,et al. An evaluation of dengue type-2 inactivated, recombinant subunit, and live-attenuated vaccine candidates in the rhesus macaque model.Vaccine.2005;23(35):4442-52
7 Sable SB, Verma I, Khuller GK,et al.Multicomponent antituberculous subunit vaccine based on immunodominant antigens of Mycobacterium tuberculosis.Vaccine. 2005;23(32):4175-84
8 Rouvio O, Dvorkin T, Amir-Kroll H,et al.Self HSP60 peptide serves as an immunogenic carrier for a CTL epitope against persistence of murine cytomegalovirus in the salivary gland.Vaccine.2005;23(27):3508-18
9 Ito N, Sugiyama M, YamadaK,et al.Characterization of M gene-deficient rabies virus with advantages of effective immunization and safety as a vaccine strain.MicrobiolImmunol. 2005;49(11):971-9
10 Lladser A, Parraga M, Quevedo L,et al.Naked DNA immunization as an approach to target the generic tumor antigen survivin induces humoral and cellular immune responses in mice.Immunobiology. 2006;211(1-2): 11-27
11 Parida SK, Huygen K, Ryffel B, et al.Novel Bacterial Delivery System with Attenuated Salmonella typhimurium Carrying Plasmid Encoding Mtb Antigen 85A for Mucosal Immunization: Establishment of Proof of Principle in TB Mouse Model.Ann N Y Acad Sci. 2005; 1056:366-78
12 Dus Santos MJ, Carrillo C, Ardila F,et al.Development of transgenic alfalfa plants containing the foot and mouth disease virus structural polyprotein gene P1 and its utilization as an experimental immunogen. Vaccine. 2005;23( 15): 1838-43
13 Johnson DC, McFarland EJ, Muresan P, et al. Safety and immunogenicity of an HIV-1 recombinant canarypox vaccine in newborns and infants of HIV-1-infected women. J infect Dis.2005;192(12):2129-33
14 Bellier B,Dalba C,Clerc B,et al.DNA vaccines encoding retrovirus-based virus-like particles induce efficient immune responses without adjuvant. Vaccine.2006;24(14):2643-55
15 Singh P, Destito G, Schneemann A,et al.Canine parvovirus-like particles, a novel nanomaterial for tumor targeting.J Nanobiotechnology. 2006;4:2
16 Faisant N, Akiki J, Siepmann F,et al. Effects of the type of release medium on drug release from PLGA-based microparticles: Experiment and theory.Int J Pharm. 2006;314(2): 189-197
17 Mollenkopf HJ, Dietrich G, Fensterle J,et al.Enhanced protective efficacy of a tuberculosis DNA vaccine by adsorption onto cationic PLG microparticles. Vaccine. 2004;22(21-22):2690-5
18 Stanford M, Whittall T, Bergmeier LA,et al.Oral tolerization with peptide 336-351 linked to cholera toxin B subunit in preventing relapses of uveitis in Behcet's disease.Clin Exp Immunol. 2004;137(1):201-8
19 Maier M, Seabrook TJ, Lemere CA,et al.Modulation of the humoral and cellular immune response in Abeta immunotherapy by the adjuvants monophosphoryl lipid A (MPL), cholera toxin B subunit (CTB) and E. coli enterotoxin LT(R192G). Vaccine. 2005;23(44):5149-59
20 Helgeby A, Robson NC, Donachie AM,et al.The combined CTA1-DD/ISCOM adjuvant vector promotes priming of mucosal and systemic immunity to incorporated antigens by specific targeting of B cells.J Immunol. 2006;176(6):3697-706
21 Payette PJ, Ma X, Weeratna RD,et al.Testing of CpG-optimized protein and DNA vaccines against the hepatitis B virus in chimpanzees for immunogenicity and protection from hallenge.Intervirology. 2006;49(3): 144-51
22 Roelofs-Haarhuis K, Wu X, Gleichmann E,et al.Oral tolerance to nickel requires CD4+ invariant NKT cells for the infectious spread of tolerance and the induction of specific regulatory T cells. J Immunol. 2004;173(2):1043-1048