DNA免疫研究进展（下）

（续前）DNA免疫引起的免疫反应
    实验发现直接注射编码病毒蛋白的质粒DNA能够激起体液和细胞免疫反应。各种病毒疾病的动物模型实验已经证明这些反应具有保护性。在小鼠和非人类灵长类动物实验中，最早鉴定了流感病毒蛋白的抗体反应[15]。另外在小鼠、牛和非人类灵长类动物实验中也检测到其它病毒蛋白的抗体反应[26, 27]。编码流感病毒血凝素（HA）、基质蛋白和糖蛋白（NP）[15, 24]，HIV的gp120[72]和gp160[20, 78]，牛疱疹病毒的gpIV[27]，狂犬病毒表面糖蛋白[28]，乙肝表面抗原[29]，丙肝核心抗原[79]，淋巴细胞性脉络丛脑膜炎病毒的NP蛋白[80]，单纯疱疹病毒gB、gD[81]蛋白的DNA肌肉免疫显示可诱导产生抗体。一些实验直接注射溶于盐溶液中的质粒DNA64，而另一些实验在注射过毒素或局部麻醉剂后再注射DNA，以通过注射部位肌肉的坏死和再生来提高目的基因的表达[65, 66]。目前为止，已经在一些实验中成功地产生了针对流感病毒HA，HIVgp160，狂犬病毒表面糖蛋白等的病毒中和抗体。产生血凝素的抑制（HI）和中和抗体表明流感病毒HA以天然构象存在于被转染细胞的表面。非洲绿猴和小鼠肌肉注射流感病毒HA－DNA可以获得持续抗体反应[31]。一年后以该质粒再次免疫，非洲绿猴可激起HI抗体滴度的迅速剧增[82]。

    DNA免疫也可以用来产生针对非病毒病原体的抗原。通过肌肉注射质粒DNA，已经成功地产生了针对一种原生动物寄生虫Schistosoma japonicum[39]，原生生物Leishmania major[40]和Plasmodium joelii[83, 84]，细菌Mycobacterium tuberculosis[42, 85, 86]和Salmonella typhi，Mycoplasma pulmonis[43~45]的抗体反应。

DNA疫苗的保护性和治疗性作用
    DNA免疫产生保护性作用最早是在针对流感病毒的实验中被证实的。肌肉注射NP－DNA的小鼠可以获得保护，能够对抗致死剂量病毒的攻击[15]。实验中使用NP－DNA免疫的小鼠的血清被动免疫其它小鼠，不能使它们获得保护，尽管血清中含高滴度的NP抗体。而将免疫小鼠的脾细胞移植至其它小鼠体内，可以使它们获得前面提到的保护[87, 88]。说明这种保护作用的获得是针对NP蛋白的细胞免疫结果。T细胞剔除实验进一步证明CD4+及CD8+T细胞均对保护的获得都作出贡献。而以NP蛋白免疫，则既不能激起CTL反应也不会产生保护作用[89]。

    DNA疫苗不仅为预防许多感染性疾病开辟了一条极具前景的新路。DNA疫苗还可以用来治疗已经感染Mycoplasma pulmonis的动物[46]。实验中，被感染的BALB/C小鼠在5天内出现肺炎症状，DNA疫苗接种一周后，开始检测到病原生物的减少，4个月后完全清除，同时肺组织炎症症状逐渐减轻至消失[90]。

表达文库免疫（ELI）
    鉴定某个病原生物的保护性抗原通常是一种费力、困难的工作，特别是需要细胞免疫获得保护的情况，因为一些类型的疫苗（如亚单位蛋白或灭活病毒）不能产生CTL反应。此外，检测特异性抗原需要纯化形式的蛋白。而DNA疫苗不仅可以诱导CTL反应，而且只需编码抗原的基因即可。利用ELI也许可以使发现疫苗抗原的工作大大简化。这种方法发展起来，通过检测含病原体基因组片段质粒的混合物，以发现保护性抗原[44]。

    一组小鼠以来自M.pulmonis DNA文库的3000种不同质粒的混合物进行免疫，结果可以获得保护，能对抗野生型病原体的感染。说明其中至少含有一个质粒编码一个保护性抗原。接下来进行分部，进一步检测这些混合物，就有可能筛出具保护性的质粒。如前文所提，该方法有一定的缺陷，如同时表达大量抗原造成抗原竞争有可能掩盖重要表位。随着基因组研究飞快地进展，许多病原生物的序列信息可以迅速获得，这种途径将对鉴定疫苗抗原的工作有很大帮助。虽然该技术几年前已被提出，但到目前为止并没有利用此技术发现一个保护性抗原的基因，将来随着该方法的改进，可能会帮助更好地达到筛选目的[45]。有人已经预言抗原鉴定的ELI时代即将到来。

DNA作为免疫佐剂
    药物学家们对DNA疫苗感兴趣的方面还在于它的特性，因为它可能不仅是简单的传送编码目的抗原的多核苷酸序列的静态载体。DNA疫苗的质粒骨架较编码的目的蛋白的免疫原性小。目前实验结果表明DNA自身对于激起被编码抗原的免疫反应有其免疫效应。细菌DNA是一种淋巴细胞的有丝分裂原，且这种刺激作用可能与细菌DNA的序列和甲基化形式相关的观点早已被接受[91, 92, 93]。

    肌肉注射DNA疫苗导致诱导Th1类反应。各种DNA疫苗免疫动物的脾细胞经抗原特异性刺激后分泌IL-2和IFN-γ[94]，间接的证明是实验发现抗体主要以IgG2a亚型为主[95]。相反，以包被于金颗粒上的DNA重复皮内免疫激起了Th-2类帮助性T细胞反应。倾向何类Th细胞免疫反应，可能是由与抗原表达相关的各种因素，而不是质粒自身的直接因素。然而有趣的是，与重组蛋白共注射不编码任何蛋白的质粒DNA，可以改变激起抗体反应的亚型类型[95]。这可能显示与蛋白抗原共注射非编码质粒有影响免疫反应的作用，因此表明DNA自身有免疫原性作用[94]。此外，与编码流感病毒HA的DNA疫苗共注射非编码质粒导致免疫的非洲绿猴体内抗体滴度的明显增加[94]。特异性保护实验表明DNA疫苗激起特异性保护确实需要质粒编码相关蛋白抗原，而不是因为简单的非特异免疫反应而获得保护[96]。

    研究还表明DNA的免疫促进效应的机制可能在于质粒的特异序列和其甲基化形式。这一特异的序列模体（嘌呤－嘌呤－C－G－嘧啶－嘌呤）在体外实验中刺激NK细胞和淋巴细胞，激起它们分泌细胞因子[97]。CpG寡核苷与抗原共注射可以在体内产生显著作用。而是否该序列的双链形式也有类似效果，目前还并不清楚。一个特异的序列模体（AACGTT），插入编码β－半乳糖苷酶的DNA疫苗中，实验证明了促进针对编码蛋白的免疫反应[98]。然而编码其它抗原的类似载体并没发现有促进效应。因此，CpG模体的位置及结构，编码蛋白自身的抗原性，以及存在影响性或竞争性的核酸序列等因素，可能会影响到CpG模体是否对于DNA疫苗的功效有作用。

DNA疫苗在粘膜免疫方面的应用
    大多数病原体通过粘膜的表面进入宿主，因此如何诱导粘膜表面的免疫反应是免疫学家极为关注的问题。目前DNA疫苗可以通过肠胃外接种和粘膜给药的两种方式诱导粘膜免疫开始得到学者们的关注及讨论。

    在雪貂和鸡的免疫实验中，肠胃外DNA给药能够激起抵抗流感病毒攻击的保护性反应[99, 100, 101]。在其它实验中，肠胃外DNA免疫也能激起抵抗牛疱疹病毒攻击的保护反应，而使免疫后的动物不患感染性的牛鼻气管炎[102, 103]。以上这些实验都是粘膜系统被攻击的例子。特别值得一提的一个例子是：编码A/PR/8/34流感株HA的DNA免疫雪貂，在一些情况下可以完全防止同源流感病毒株的感染[104]。流感病毒DNA疫苗的一个特性在于可以在雪貂、鸡的动物实验中明显降低被免疫株相关的抗原突变株的感染（突变株与免疫株之间经血凝素抑制实验证明没有抗原交叉反应）的机率[99,105]。目前至少在流感病毒DNA疫苗研究中，肠胃外免疫可以激起保护反应，抵抗由粘膜系统进攻的病原体的侵入。这已得到广泛证实，并且事实上已经作为当前一些获批疫苗的基础。

    由于肠胃外免疫通常只能产生边际效应的粘膜免疫反应，所以粘膜免疫更为严格的检测是由单纯疱疹病毒和人免疫缺陷病毒以及肠道病原体（如Vibrio和Shigella spp.）的生殖系统感染实验提供。通过豚鼠阴道感染疱疹病毒建立了一个人生殖系统疱疹病毒感染的模型，提供了检测感染引起的粘膜损伤和测定其它参数（如后肢麻痹、泌尿停滞以及检测脊髓椎板神经节中病毒的存在）的方法。以编码HSV-2包膜糖蛋白的DNA进行免疫，在血清中产生了类似于HSV-2阴道感染导致的抗体水平[106]。而以编码病毒gD、gB糖蛋白的DNA共同或分别单独免疫豚鼠，可以明显地降低人HSV-2阴道感染后生殖系统粘膜的损伤程度[106, 107, 108]，此外在病毒攻击前的阴道分泌物含IgG亚型的gD－和gB－特异性抗体，这被认为可能是来自血清中抗体的渗出[108]。该模型中获得的保护作用被认为是抗体的作用，但细胞免疫反应可能也有一定的作用。编码HSV-2的gD或gB糖蛋白的DNA免疫小时或豚鼠后，免疫动物的淋巴细胞可以进行抗原特异地增殖，说明激起了一定的细胞免疫反应。此外，gB－特异的CTL反应也已经在编码gB的DNA免疫的C57BL/6小鼠实验中得到证实。Rouse和其同事证明了编码HSV-1 gB糖蛋白的DNA疫苗，在小鼠HSV感染的带状疱疹模型实验中诱导产生的CD4+细胞毒性T淋巴细胞具有保护性。在同样的感染模型中，他们还证实了以编码极早期反式激活因子ICP27的结构免疫能够激起CTL反应并获得保护[109]。

    目前，基于通过粘膜途径进行DNA免疫可能产生更有效的粘膜免疫的假设，已经有不少直接粘膜DNA免疫的工作报导。DNA疫苗直接鼻内或阴道内免疫，证明可以产生HIV抗原[110, 111, 112]和HSV抗原的局部抗体[113]。而鼻内接种编码流感病毒HA的DNA产生对抗病毒攻击的保护作用[101]。另一些研究中，粘膜免疫佐剂（如霍乱毒素[114,113]，使用阳离子脂整合DNA于脂质体中[115]或包裹DNA入多聚DL-lactidecoglycolide微粒中[116]）得到成功地应用，帮助粘膜系统呈递DNA疫苗。

总结
总之，应用DNA疫苗诱导保护性免疫反应已经在各种动物模型中获得成功，并且已经开始了早期一期临床试验。目前的临床前期研究主要包括，研究各种方法指导特异免疫反应（如粘膜免疫反应），调节或促进免疫反应（如利用CpG模体或共接种佐剂或细胞因子）。此外，DNA疫苗已经被证明是一个研究抗原加工和呈递的有力工具。因为构造的方便，使得易于在蛋白中整合进不同的定位信号（如进入分泌途径、MHCI类呈递的蛋白酶体、MHCII类呈递的溶酶体的信号序列），或者通过缺失一定区段（如穿膜区）改造蛋白。而接下来可以相当系统性地研究这些改变对于特定抗原的体液及细胞免疫原性的影响。
因此，该项技术不仅作为一种新型疫苗，而且作为进行免疫学基础研究的一种方法，都显示出诱人的前景。

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