CN102258779B - 四种及四种以上hiv载体基因疫苗序贯及重复应用 - Google Patents
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Abstract
本发明涉及一种提供可以长期维持高水平特异性免疫反应的四种及四种以上HIV载体基因疫苗序贯及重复应用的策略。一种用于预防和/或治疗艾滋病的组合艾滋病疫苗，可以活化广谱的、抗艾滋病毒的细胞免疫反应，它由四种或四种以上的艾滋病疫苗组成，不同的艾滋病疫苗所包含的HIV蛋白基因可以相同，每次用一种艾滋病疫苗接种，每种疫苗可连续接种两次。在四种或四种以上不同载体疫苗序贯应用后，可以用上述载体疫苗重复序贯应用。
Description
技术领域
本发明属于生物医药领域。本发明涉及四种或四种以上HIV载体基因疫苗序贯及重复应用的免疫策略。在具体应用中将不限于本前期研究中所使用的四种疫苗。
背景技术
人类免疫缺陷病毒(Human immunodeficiency virus，HIV)属于逆转录病毒科慢病毒属中的人类慢病毒组，是获得性免疫缺陷综合征(acquired immunodeficiency syndrome，AIDS)即艾滋病的病原，于1983年首次被鉴定。艾滋病被认为是世界上直接威胁人类健康的第一大传染病，已经成为世界范围内的重要公共卫生问题。联合国艾滋病规划署和世界卫生组织联合发布的2009年全球艾滋病流行趋势报告显示，目前全球有大约3340万艾滋病感染者，其中2008年新增感染者270万人，200万人死于与艾滋病相关的疾病。自1983年中国出现首例HIV感染者以来，截至2010年10月底，累计报告艾滋病病毒感染者和病人37万余例，其中病人13万余例，死亡6.8万余例。
艾滋病疫苗是控制艾滋病的重要手段。长期以来科学家们希望能研究出一种预防艾滋病感染的疫苗。在艾滋病流行初期曾经认为产生中和抗体足以预防HIV感染。在早期进行的试验表明由单体HIV膜蛋白Env产生的抗体并不能预防和控制HIV感染，究其原因主要是不能很好地产生广谱中和抗体。因此重心又从诱导中和抗体转向诱导细胞免疫反应。在此基础上进行的研究表明特异性的细胞毒性T细胞反应与病毒载量降低有关，但诱导HIV特异性细胞免疫的预防性疫苗在临床试验中均未观察到明显的保护效果。疫苗免疫可以产生较好的特异性细胞免疫反应，但持续时间较短，即使部分有效，也无法预防HIV感染，这些因素可能和疫苗失败有关。由于对艾滋病免疫保护的机理的认识进展缓慢，完全预防HIV-1感染的疫苗研制还有待突破，因此正确认识和评价疫苗的作用是十分重要的。越来越多的研究表明降低HIV感染者的病毒载量可以延缓其发展为艾滋病的时间，降低其传染给别人的可能性，从而限制HIV-1的流行。由于HIV感染者体内已经产生了针对病毒的免疫反应，通过疫苗免疫来增强对特异抗原的免疫反应，理论上在控制病毒复制，降低病毒载量，对延缓疾病进展方面能起到一定作用。目前已有50多种HIV治疗性疫苗已经完成或正在进行临床试验，这些疫苗大多采取一种疫苗单独应用或者两种疫苗以初免-加强的方案联合应用。在治疗性疫苗的设计中，很重要的一点是要考虑到疫苗的反复应用，因为这些病人可能需要接受终身治疗。载体疫苗能诱导很好的细胞免疫应答，但载体自身的免疫原性大大限制了疫苗的反复应用，因此有必要将多种能很好地诱导HIV特异性免疫应答的载体疫苗序贯应用，达到能够在病人体内多次治疗的效果。当一种疫苗免疫后产生的特异性免疫反应下降到一定水平时再给予另一种疫苗免疫，又可以再次加强已有的特异性免疫反应，依次类推免疫反应再下降时再给予第三种及第四种疫苗免疫。这样通过四种疫苗的序贯免疫即可使接种者体内长期维持较高水平的免疫反应。当一个周期的四联疫苗免疫结束后，可以再给予第二个周期的重复应用。这样可以更长时间的维持接种体内的特异性细胞免疫反应。必要时还可以增加新载体的疫苗。我们对这个设计思路进行了试验验证。
发明内容
本发明的目的是提供可以长期维持高水平特异性免疫反应的HIV多基因四种及四种以上多载体疫苗序贯及重复应用的策略。
本发明组合疫苗的技术方案是：一种抗艾滋病毒的组合疫苗，可以活化广谱的、抗艾滋病毒的细胞免疫反应，它由四种或四种以上的艾滋病疫苗组成，不同的艾滋病疫苗所包含的HIV基因可以相同，也可以不同，每一种艾滋病疫苗单独接种且至少接种一次，即接种总次数至少为四次。
所述艾滋病疫苗可为含有编码HIV膜蛋白或核心蛋白基因的DNA疫苗、重组病毒载体疫苗、重组细菌载体疫苗、重组酵母载体疫苗或重组病毒样颗粒疫苗等。
所述HIV膜蛋白编码基因至少可以为gp120、gp140、gp145、gp160或含有上述基因片断的编码基因；所述核心蛋白基因为gag全长或部分片段编码基因。
所述HIV膜蛋白或核心蛋白基因可来源于HIV-1或HIV-2型，或来源于不同的HIV-1亚型或毒株，也可以是来源于同一毒株的不同形式，例如(但不局限于)同一亚型来源的gp140与gp145被认为是不同的膜蛋白或膜蛋白编码基因。
所述不同的艾滋病疫苗进行先后序贯及重复免疫以活化针对病毒的细胞免疫反应。
本发明组合多载体基因疫苗序贯和重复应用的技术方案是：一种抗多种艾滋病毒的组合疫苗组合方法，采用四种或四种以上的艾滋病疫苗进行序贯免疫，以活化针对病毒的细胞免疫反应，将针对共同表位的细胞免疫反应连续依此提高，从而提高整体的特异性免疫反应；所述不同的艾滋病疫苗所包含的HIV膜蛋白或核心蛋白基因可以相同。每次用一种艾滋病疫苗接种，每种疫苗可连续接种两次。在四种或四种以上不同载体疫苗序贯应用后，可以用上述载体疫苗重复序贯应用。
所述艾滋病疫苗可为含有编码HIV膜蛋白或核心蛋白基因的DNA疫苗、各种重组病毒载体疫苗、重组细菌载体疫苗、重组酵母载体疫苗或重组病毒样颗粒疫苗等。
所述HIV膜蛋白编码基因至少可以为gp120、gp140、gp145、gp160或含有上述基因片断的编码基因；所述核心蛋白基因为gag全长或部分片段编码基因。
所述HIV膜蛋白或核心蛋白基因可来源于HIV-1或HIV-2型，或来源于不同的HIV-1亚型或毒株，也可以是来源于同一毒株的不同形式，例如(但不局限于)同一亚型来源的gp140与gp145被认为是不同的膜蛋白或膜蛋白编码基因。
在本发明的具体实施例中，我们使用如下策略：利用表达B’亚型gag基因的质粒DNA、重组腺病毒及重组仙台病毒疫苗和表达B’/C重组亚型HIV gag-pol和env基因的重组痘病疫苗进行序贯及重复应用。在恒河猴体内用DNA疫苗初免两次，重组腺病毒或重组仙台病毒疫苗作为第二或第三种疫苗加强免疫两次，最后用重组痘病毒疫苗免疫两次。用ELISPOT方法检测细胞免疫反应，结果发现接种四种疫苗的恒河猴体内可长期维持较高水平的特异性免疫反应。在一轮四种疫苗免疫接种结束后给予第二轮疫苗接种，结果第二轮第一种疫苗重组腺病毒免疫后即可较长时间维持体内特异性免疫反应水平。(参见附图1)
本发明的优势在于：
在我们前期三种疫苗序贯应用的基础上增加了第四种疫苗的序贯使用，而且第四种疫苗表达与前三种疫苗不同亚型的抗原，结果可以很好的诱导高水平亚型交叉性免疫反应，而且持续的时间延长。
四种疫苗序贯和重复使用在文献中未见有报道，在我们的研究中能更好的激发机体的特异性免疫反应，并且可长期维持高水平。
附图说明
图1为四种疫苗序贯及重复免疫恒河猴用ELISPOT方法检测不同时间点的特异性细胞免疫反应情况。
具体实施方式
HIV疫苗序贯及重复免疫恒河猴实验
1.疫苗免疫和分组
8只成年恒河猴随机分成3组，第1组2只为PBS对照组，在所有与实验组相同的免疫时间点，肌肉注射1ml无菌PBS；第2组5只为四种疫苗免疫组，先用DNA于0周，2周初免两次(所用DNA疫苗是表达密码子优化型gag基因的真核表达质粒，是本申请人于2006提交的申请号为200610098946.1的在先专利申请中所构建的pVR-mod.gag，由本元正阳基因技术有限公司生产)，再于6周和33周免疫相同剂量的腺病毒疫苗rAd5-gag(rAd5-gag为表达mod.gag基因的E1、E3缺失的复制缺陷型重组5型腺病毒，表达的启动子为mCMV，加尾信号为SV40polyA，是本申请人于2007年提交的申请号为200710000534.4的在先专利申请中所构建的rAd5-mod.gag，由本元正阳基因技术有限公司生产)(或仙台病毒疫苗rSeV-gag，rSeV-gag为表达wt.gag269基因的重组F基因缺陷的仙台病毒载体疫苗，由日本载体研究所提供)，于45周和87周加强免疫仙台病毒疫苗(腺病毒疫苗)(同前)，于206周及236周免疫重组痘病毒疫苗rMVA(rMVA为表达B’/C重组亚型HIV gag-pol和env基因的重组MVA疫苗，由长春百克药业有限责公司提供)，第282周开始第二轮免疫，先免疫腺病毒疫苗。第3组1只为两种疫苗免疫组，于0周、2周、6周、33周、45周和87周肌肉注射1ml无菌PBS，206周及236周免疫重组痘病毒疫苗rMVA(同前)，282周免疫腺病毒疫苗rAd5-gag(rAd5-gag为表达mod.gag基因的E1、E3缺失的复制缺陷型重组5型腺病毒，表达的启动子为mCMV，加尾信号为SV40polyA，是本申请人于2007年提交的申请号为200710000534.4的在先专利申请中所构建的rAd5-mod.gag，由深圳市源兴生物医药有限公司生产)。所用疫苗剂量分别为：DNA 5mg/只，rAd5-gag 109PFU/只，rSeV-gag 109CIU/只，rMVA 9×107PFU/只。具体分组及免疫情况见表1。
表1.恒河猴分组及免疫情况
2.ELISPOT方法检测免疫的恒河猴体内Gag特异性细胞免疫反应水平。
ELISPOT分析技术是当今公认的最灵敏的抗原特异性T细胞的体外检测技术。特异性细胞免疫是指T细胞受到抗原刺激后，增殖、分化并转化为致敏T细胞，当相同抗原再次进入机体，致敏T细胞可以对表达抗原的细胞进行直接杀伤或致敏T细胞释放淋巴因子协同杀伤，从而清除体内被感染的细胞。在ELISPOT实验中，在体外以特异性刺激物，如多肽或基因表达产物，提取的抗原等模拟体内的抗原刺激，检测受到这种特异性刺激后分泌IFN-γ等细胞因子的淋巴细胞数，以此来评价细胞免疫反应的水平。
在免疫后不同时间点采集EDTA抗凝外周静脉血10ml/只，在采血后8小时内用猴淋巴细胞分离液(EZ-SepTM Monkey 9X，深圳达科为公司，产品货号：DKW33-H0100)分离外周血淋巴细胞。采用酶联免疫斑点方法(ELISPOT)检测受Gag特异性肽库刺激后分泌IFN-γ的淋巴细胞数。Gag269肽库来源于日本东京大学(gag269基因由本科室管永军博士从二十世纪90年代HIV-1阳性感染者血液标本中分离得到，根据该基因的氨基酸序列由合作单位日本东京大学合成肽库(Yu S，Feng X，Shu T，Matano T，HasegawaM，Wang X，Ma H，Li H，Li Z，Zeng Y：Potent specific immune responses induced byprime-boost-boost strategies based on DNA，adenovirus，and Sendai virus vectorsexpressing gag gene of Chinese HIV-1 subtype B.Vaccine 2008，26：6124-6131.)，由124条长度为14～16aa的短肽组成，相邻两条肽重叠11aa，每条肽的纯度从52％到100％不等，贮存液溶于DMSO中。将Gag269肽库按照氨基酸序列N端到C端的顺序分成5个分肽库，分别命名为AB (aa1-105)，CD (aa95-203)，EF (aa193-305)，GH(aa295-408)和IJ (aa398-516)。将各组多肽调整浓度至2μM，依次加入包被有IFN-γ捕获抗体的96孔ELISPOT培养板中，每个多肽做2个复孔，每孔50μl，每个样品设1个阴性对照孔(加1％DMSO共同培养)和1个阳性对照孔(加入终浓度为50ng/ml的PMA(德国Sigma公司，产品货号：P1585)和1μg/ml的Ionomycin(德国Sigma公司，产品货号I9657)，然后每孔加入50μl浓度为4×106/ml的PBMCs。加完所有的样品之后，盖上板盖，放入二氧化碳培养箱，37℃ 5％CO2培养36h。培养结束后，按照试剂盒说明书分别与生物素标记的一抗和HRP标记的链酶亲和素反应，最后加入AEC显色液显色。
检测结果见图1，由于对照组所有检测点免疫反应为阴性，所以图中未显示对照组结果。四种疫苗组DNA疫苗免疫后产生的反应水平比较低，第一种病毒载体疫苗免疫一次后很快产生免疫反应的高峰，高峰值为2744SFCs/106PMBCs，反应水平维持在500SFCs/106PMBCs以上持续的时间约为18周，第二次免疫后又很快诱发免疫反应的高峰，高峰值为3424SFCs/106PMBCs，反应水平维持在500SFCs/106PMBCs以上持续的时间约为12周。第二种病毒载体疫苗免疫一次后，又很快产生免疫的高峰，尽管高峰值比第一种病毒载体疫苗稍弱，但反应水平维持在500SFCs/106PMBCs以上持续的时间明显延长，可达34周。第三种病毒载体疫苗rMVA免疫一次及两次后又分别激发了免疫反应的高峰，尤其第二次免疫后高峰值可达3339SFCs/106PMBCs，反应水平维持在500SFCs/106PMBCs以上持续的时间分别为30周及32周。在第三种疫苗开始免疫时，在一只以前接种过PBS的恒河猴中也进行了rMVA疫苗的免疫接种，第一次免疫后仅产生很弱的反应，第二次接种后免疫反应有所增强，但仍远远低于四联疫苗免疫组，并且很快降到较低水平。四种疫苗免疫组在266周(约5年)之前，第一种病毒载体疫苗免疫后，细胞免疫反应平均水平都在500SFCs/106PMBCs以上，仅用四种疫苗共免疫8次可使接种体内长期维持一定水平的细胞免疫反应。在第282周又开始了四种疫苗的重复免疫，第一种免疫的疫苗选用腺病毒疫苗，接种后四种疫苗组又很快产生免疫反应高峰，免疫后监测到14周仍在很高的水平。而接种过rMVA疫苗的恒河猴，再接种腺病毒疫苗后的反应要明显低于四种疫苗组。
以上多载体四种疫苗免疫恒河猴的实验表明四种疫苗序贯、重复使用能多次诱导特异性免疫反应，并且可持续较长时间。
应当指出，对于经充分说明的本发明来说，还可具有多种变换及改型的实施方案，并不局限于上述实施方式的具体实施例。上述实施例仅仅作为本发明的说明，而不是限制。总之，本发明的保护范围应包括那些对于本领域普通技术人员来说显而易见的变换或替代以及改型。
Claims (2)
1.一种用于预防和/或治疗艾滋病的组合艾滋病疫苗，其特征在于：由四种艾滋病疫苗组成，分别为：艾滋病疫苗pVR-mod.gag、腺病毒疫苗rAd5-gag、仙台病毒疫苗rSeV-gag、重组痘病毒疫苗rMVA。
2.一种权利要求1所述的组合艾滋病疫苗在制备药物中的应用，所述药物为预防和/或治疗艾滋病的药物。
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