中国人群HLA特异的HCMV串联表位腺病毒核酸疫苗的制备及免疫效果评价
|
摘要
研究背景
人巨细胞病毒(Human cytomegalovirus,HCMV)是人类先天性病毒感染的最常见病原体,人是其唯一的宿主。根据社会经济状况和地理位置的不同,人群中抗HCMV血清阳性率可达50%～90%。一般情况下,HCMV原发感染为隐性感染,无明显的临床症状,终生携带,病毒周期性地从黏膜部位释放。在器官和造血干细胞移植、肿瘤、艾滋病等免疫低下人群中,体内潜伏的病毒重新激活,引起HCMV活动性感染,其感染率约为50%～75%,死亡率约是25%,一般于术后1～4个月内发生,是器官移植失败和移植受体死亡的重要原因。对于孕妇来说,HCMV原发或复发感染均可引起胎儿宫内感染或围产期感染,而导致畸形、智力低下和发育迟缓,感染率达0.3-2.4%,其中发病率达15%-30%。在美国每年有8000名HCMV原发感染的婴儿出生,其中有10%～17%的婴儿出现耳聋或神经系统后遗症。在我国因先天HCMV感染造成的先天畸形患儿约4万人左右,占每年我国出生缺陷儿的6.2%。
巨细胞病毒的生物学特征
HCMV属于β-疱疹病毒亚科,为双链线状DNA病毒,直径约为120～300nm,核酸为235kb的双股线性DNA,有213个开放读码框,其中有165个编码基因,Varnum等证明HCMV病毒颗粒由59种结构基因编码的产物与众多宿主蛋白构成,是疱疹病毒中最大的,具有严格的种属特异性。其基因由长的独特序列(UL)、短的独特序列(US)及存在于L—S连接部位的重复序列组成,可构成4种同分异构体,至少编码氨基酸残基数100以上的多肽200余种,包括原始基因产物、中间产物及终末产物。HCMV基因的转录及表达有一定的时序性,可分为即刻早期(IE)、早期(E)和晚期(L)基因3种类型。其中,对病灶具有重要监测和检测功能的是IE和L。即刻早期基因是HCMV原发感染或复发感染后最先表达的基因。IE蛋白在HCMV基因表达的后续调控过程中起着主导作用。晚期基因的转录和表达一般发生在感染后36～48h。在HCMV DNA开始复制后,L基因合成病毒的结构蛋白,包括衣壳、被膜和包膜蛋白。因此,L基因表达意味着病毒的装配及成熟,是病毒感染的后期结果。而L基因mRNA可作为特异性的活动感染的诊断指标。HCMV对宿主或培养细胞有高度的种特异性,HCMV只能感染人,及在人纤维细胞中增殖。病毒在细胞培养中增殖缓慢,复制周期长,初次分离培养需30～40天才出现细胞病变,其特点是细胞肿大变圆,核变大,核内出现周围绕有一轮“晕”的大型嗜酸性包涵体。
HCMV几乎感染所有器官,血管系统(内皮细胞和平滑肌细胞)、循环淋巴细胞、单核细胞和多形核白细胞、成纤维细胞是其主要的潜伏部位,一旦病毒进入细胞,病毒衣壳粘附于胞膜,借其包膜蛋白(gB和gH)与细胞表面受体(低结合力的肝素受体和高结合力的蛋白受体)结合后,通过受体介导的机制进入细胞内。也可通过其他途径如直接穿过胞浆膜进入细胞内,然后病毒基因组与宿主细胞核结合。
HCMV有以下方式存在于宿主中,①病毒基因整合到宿主细胞基因组内,不表达病毒抗原和装配病毒颗粒,随着细胞基因复制而转移到子代细胞内,直接处于潜伏状态。②病毒在宿主细胞内复制引起细胞病变,形成核内或/和浆内包涵体,至细胞溶解凋亡,形成产毒型感染。③病毒有少量复制,靶细胞功能发生障碍,但无或极少细胞病变,引起不全感染。④宿主细胞可表达病毒抗原,导致宿主细胞转化和增生,可能参与其致癌机制。⑤HCMV感染除直接导致终末器官病变外,还在统计学上与移植排斥、细菌或真菌的二次感染、动脉粥样硬化加速等发生有密切关系,这些状况被称为HCMV的间接效应。
病毒侵入肺、肝、肾、唾液腺、乳腺其他腺体,可长期或间隙地自唾液、乳汗血液、尿液、精液、子宫分泌物多处排出病毒。通过口腔,生殖道,胎盘,输血或器官移植等多途径传播。HCMV可通过胎盘感染胎儿,影响妊娠,一是病毒在绒毛细胞内增殖后进入胎儿血中并感染之,另一种方式即为病毒在绒毛细胞内不发生增殖而仅是通过或溢出。器官移植患者最常见的感染是HCMV,主要是通过供体器官传播或受体潜伏的HCMV激活。
HCMV的致病机制
1.HCMV潜伏感染形成的机制:受染的细胞为HCMV非容纳细胞,HCMV进入受染细胞首先必须通过细胞表面的病毒相应受体。HCMV进入细胞后能否完整复制则取决于受染细胞的分化成熟程度。凡阻碍或不能使HCMV完整复制成具感染性病毒颗粒的细胞,则为非容纳性细胞。Sinclair等指出,外周血单核细胞之所以成为HCMV潜伏感染的细胞是因为在外周血单核细胞中HCMV即刻早期基因(IE)表达受阻。一旦这些细胞能进一步分化成熟,病毒的基因能正常表达,则就可形成完整的病毒颗粒。Lecoinle等比较了单核细胞衍生的巨噬细胞(MDM)、人胚脑胶质细胞和星状细胞等分化成熟程度以及体外接种HCMV后病毒复制的情况,结果发现HCMV在人胚脑胶质细胞或星状细胞内复制良好,为CPE较高滴度的病毒,相反在MDM细胞中无明显可见的CPE,仅在部分的MDM细胞中检出HCMV早期或晚期病毒抗原,认为HCMV一旦进入非容纳细胞则出现潜伏感染。
有学者运用RT-PCR技术检测并分析了潜伏于粒-巨噬前驱细胞中的HCMV即刻早期基因及其基因产物时发现,在HCMV mRNA的第356和292位碱基的上游分别存在着编码94个氨基酸多肽和152个氨基酸多肽的阅读框架,它的反向mRNA片段为2.1 kb。HCMV抗体阳性的健康携带者血清中(5/7)能检测到这一特异性基因片段,而HCMV抗体阴性的非感染者血清中则无1例可检出这一特异性基因片段(0/7),进一步支持HCMV潜伏感染存在着相关基因或转录产物的观点。
2.HCMV潜伏—再激活的机制:HCMV感染一旦发生即成为终身相伴,绝大多数的受染者是无临床表现的隐性或轻型感染。但当宿主机体内环境受诸方面因素影响,原来不导致临床病理表现的潜伏病毒则可被激活而导致严重的临床表现,甚至是致命性的。其诱因为:①受染细胞的分化成熟:骨髓造血干细胞或外周血单核细胞均为未完全分化成熟的细胞,HCMV在此类细胞中只能以潜伏状态存在。一旦某种诱因使这些受染细胞受到刺激使其进一步分化成熟时,则可能使原处于潜伏状态的病毒再度被激活,产生大量具有感染性的完整的病毒颗粒。Sodrberg等认为只有同源性的单核细胞衍生巨噬细胞刺激才能激发原来潜伏状态的病毒再度活化,并推测在器官或骨髓移植患者中HCMV激活感染的高发频率与之有关。②细胞因子、粘附分子的影响:急性炎症时由于急性炎症细胞大量分泌细胞因子,可能诱发潜伏的HCMV再度激活。γ干扰素(IFN-γ)和肿瘤坏死因子TNF-α可特异性诱导潜伏有HCMV的单核细胞分化为容许HCMV在其内繁殖的巨噬细胞,而不是发挥其本来的抗病毒生物功能。HCMV主要即刻早期基因启动区域存在着cAMP反应功能区,某些炎症因子、细胞因子,如前列腺素E2、IL-1β、TNF-α等都能活化cAMP,从而起到对HCMV的协同上调作用。Steek报道,由于CD3+T细胞的活化能够促进TNF-α的大量释放,而释放的TNF-α又促进了HCMV即刻早期基因的表达。Watandba等证明细胞粘附分子ICAM-1亦可能参与HCMV潜伏再激活。③机体免疫低下:HCMV病常发生于CD4＜0.1×10~9/L的患者,CD4＜0.05×10~9/L时,发病率为30%-40%。免疫功能低下如H1V患者、肿瘤以及器官移植患者中,一方面由于使用激素;另一方面辐射也可能导致潜伏的病毒再度活化。HIV与HCMV二重感染时,两种病毒的相互作用亦影响病毒的潜伏与激活。
HCMV和移植
大量研究显示,HCMV已成为实体器官移植和造血干细胞移植后常见及重要的并发症,是导致骨髓移植后死亡的重要原因。骨髓移植术后应用免疫抑制剂使机体抵抗力下降,体内潜伏的病毒重新激活,引起HCMV活动性感染,是影响造血干细胞移植术后受体生存率和生活质量的重要感染因素。其感染率约为50%～75%,一般于术后1～4个月内发生,早期报道巨细胞病毒感染所致的病死率高达25%。
HCMV是一种免疫调节病毒,病毒感染宿主主要是通过供体器官、细胞传播或受体中潜伏HCMV的激活,其感染的严重程度取决于下列因素:移植的种类、供/受者HCMV血清学状态、免疫抑制方案、移植物排斥情况。HCMV感染可产生免疫抑制,增加机体对严重细菌感染和其他机会性致病微生物(如卡氏肺孢子茵、白色念珠菌)的易感性。HCMV活动性感染对移植受体的影响可分为直接性和间接性,在临床上可见的直接影响是活动性HCMV病的表现,包括发热、白细胞减少、肝炎、肠炎、视网膜炎等,最常见的是巨细胞病毒感染引起的间质性肺炎,国内文献报道造血干细胞移植后HCMV感染引起的间质性肺炎(IP)发病率可达30%,病死率可达80%以上,国外文献也报道骨髓移植患者并发HCMV活动性感染并导致间质性肺炎,其病死率高达87%。间接影响是HCMV可诱导受染细胞表面的MHC-Ⅰ类和Ⅱ类抗原的表达,增加T杀伤淋巴细胞反应而促进宿主的急性排斥反应,降低患者存活率。
将HCMV疫苗注入血清学阴性的器官移植受体,在理论上说是减少HCMV感染和疾病的简单而有效的方法。研究表明,减毒活疫苗和活病毒疫苗在HCMV血清学阴性的受体中使用后,再接受HCMV血清学阳性的肾移植,能降低HCMV感染发生率。虽然HCMV疫苗不能完全预防HCMV感染疾病,但能降低HCMV首次感染后严重的临床症状。但在免疫抑制的状态下,能导致机体对疫苗免疫反应的沉默,在血清学阴性的受体中,接种疫苗后不发生血清学转变的比例是相当高的。尽管应用疫苗不可能完全达到保护性的抑制HCMV感染,但能避免发生严重的HCMV感染性疾病,再者,当与其他治疗方法,如抗病毒药物和/或免疫球蛋白的使用,将达到更好的防治效果。
应用疫苗亚单位可能更为有效,目前研究HCMV亚单位B和H疫苗。Bernstein等研究表明,对HCMV感染阴性的成人分三组,即接种HCMV亚单位糖蛋白B(gB)加MF59(一种含角鲨烯水乳剂)组,同时接种HCMV gB加MF59和金丝雀痘重组疫苗表达的HCMV gB(ALVAC-CMV gB)组,以及先后接种HCMV加MF59和金丝雀痘重组疫苗表达的HCMV gB(ALVAC-CMV gB)组,除有局部的反应外,少有系统副作用,三组均能安全有效的诱导高水平的HCMV抗体表达和细胞免疫的发生,组间无差异。
新的HCMV免疫治疗采用输入HCMV特异的细胞毒性T淋巴细胞(CTL)。在健康和免疫抑制的个体中,Ⅰ类主要组织相容性复合体限制的HCMV特异CTL,在控制HCMV感染中起相当重要的作用。骨髓移植后,增加病毒特异CTL的数量与有功能的CTL,能成功的减少HCMV疾病的发生,骨髓移植后,试验性的应用T细胞免疫治疗HCMV感染表明,使用CD8+T细胞能抑制HCMV疾病。同时输入CD4+HCMV Th细胞和CD8+HCMV CTL的免疫治疗更为有效。Einsele等应用HCMV T细胞(10~7 HCMV T细胞/m~2),其中包括CD4+HCMV Th细胞和CD8+HCMV CTL,有效的抑制接受HCMV阳性供体的受体HCMV感染。Peggs等培养出CD4+和CD8+T细胞,并输入13位同种异体移植受体,移植术后检测6个月以上,6例患者未用抗病毒药物,而有效的清除HCMV,没有病例诊断出患HCMV感染。
HCMV和血液病
HCMV能直接感染造血祖细胞,抑制祖细胞的复制和分化,还可感染骨髓微环境的基质细胞,造成微环境功能紊乱,干扰造血祖细胞成熟和分化,引起轻重不等的骨髓造血功能损害,研究发现体外培养时HCMV可直接或间接影响造血干/祖细胞的增殖和分化,因此HCMV感染与许多血液病有关,如再生障碍性贫血、ITP、自身免疫性溶血性贫血等。HCMV引起造血功能紊乱的机制复杂,可能因素为:(1)直接感染造血祖细胞,利用的是宿主细胞的DNA聚合酶,抑制其增殖与分化。(2)通过损伤骨髓基质细胞导致造血因子网络紊乱。(3)Fas介导的造血干/祖细胞凋亡增加。(4)病毒介导的自身免疫异常而致自身抗体的产生。HCMV感染可激活多克隆B细胞,产生抗血小板、红细胞自身抗体,导致ITP、AIHA、Evans综合征等自身免疫性疾病。
HCMV和免疫
HCMV可引起机体免疫调节障碍或免疫失衡,T淋巴细胞活化,诱发淋巴组织细胞增生。HCMV能直接感染淋巴系造血祖细胞,抑制免疫细胞的增殖和分化,引起免疫功能紊乱。HCMV在与人类共同进化的过程中,形成了多种分子机制逃避机体免疫系统的监视,从而达到在体内长期潜伏的目的。目前发现HCMV的免疫逃避机制主要包括:①HCMV表达的US2、US3、US6、US11等蛋白可通过翻译后机制下调主要组织相容性抗原复合物MHC-Ⅰ和MHC-Ⅱ类分子的表达,从而抑制抗原递呈细胞的抗原递呈功能和CD8+CTL杀伤作用,同时HCMV还可表达MHC-Ⅰ类分子的同源物,与自然杀伤(NK)细胞的杀伤抑制性受体结合后抑制NK细胞对感染细胞的杀伤作用;②HCMV可表达人细胞因子及细胞因子受体的类似物,干扰宿主自身的细胞因子系统,如HCMV表达的人IL-10类似物HCMV IL-10可抑制单核-吞噬细胞系统,抑制细胞增殖,减少炎性细胞因子的生成,降低宿主的抗感染能力;③细胞凋亡是机体的一种抗病毒防御机制,HCMV感染初期可抑制细胞凋亡,以利于在感染细胞内完成病毒复制和生活周期,而在感染晚期又可促进细胞凋亡,干扰机体抗病毒免疫反应,使病毒避免被机体免疫系统清除:④HCMV感染宿主树突状细胞(dendritic cells,DC)后,可通过改变DC表面黏附分子的表达、抑制T细胞活化信号的传递等多种途径影响其功能,从而在人体内长期潜伏。⑤通过动态观测全身播散型MCMV感染小鼠急性和慢性感染期调节性T细胞(regulatory Tcells,Treg)、辅助性T细胞(helper T cells,Th)1型和2型特异性转录因子Foxp3、T-bet和GATA-3 mRNA的表达变化,发现在MCMV感染急性期,Treg未被诱导增殖活化,机体产生有效Th1和Th2免疫反应以清除病毒,但病毒可诱导Th1/Th2应答下降,并逐渐向Th2方向偏移;进入感染慢性期后,病毒诱导Treg细胞增殖活化显著,进一步抑制Th1和Th2免疫反应,且对Th1的抑制效应更强,提示HCMV在感染慢性期诱导Treg细胞增殖活化是其抑制宿主抗病毒免疫而呈持续慢性感染的重要原因。
HCMV与肿瘤
血清流行病学和肿瘤组织中HCMV DNA的存在显示了HCMV感染可能与多种人体肿瘤的发生有关。HCMV DNA和RNA以及抗原可在30%～60%的人体肿瘤中查到。HCMV感染在宫颈癌(34.3%)中比在宫颈炎(5.3%)中更常见。人乳头瘤病毒DNA阴性患者中,HCMV感染是宫颈癌的明显危险因素。HCMV与宫颈细胞转化的相关性在鼠动物模型中也得到证实。此外HCMV感染也与血管肉瘤、Kaposi′s肉瘤、结肠肿瘤、前列腺腺癌、Wilm′s肿瘤以及常发生于儿童的成神经细胞瘤有关。它的感染在某些疾病中还导致类似于肿瘤的表现,如在HCMV结肠炎、肛部病变、胃部病变、直肠病变等。HCMV通过抑制宿主细胞凋亡来逃避免疫攻击,是其参与肿瘤发生发展的重要机制之一。HCMV感染可干扰宿主的内源性和外源性细胞凋亡途径,调控抑癌基因p53和p73的表达,激活抗凋亡的Ras/Raf/MEK/Erk和PI-3K信号转导通路。HCMV还可通过影响宿主细胞的表观遗传状态(epigenetie state)间接影响基因组功能。表观遗传修饰如DNA甲基化、组蛋白乙酰化和甲基化、RNA相关性沉默等,均可造成基因表达、DNA复制和基因组稳定性的改变,这些改变可能触发抑癌基因或DNA修复基因表达沉默,最终导致肿瘤发生。最近,国外学者建议用“肿瘤调控”(oncomodulation)一词来描述HCMV在肿瘤发生过程中的作用机制,并强调了肿瘤细胞提供的不同于正常细胞的基因环境(信号转导通路、转录因子、抑癌蛋白功能紊乱等)更有利于HCMV的持续感染和发挥肿瘤调控作用。
HCMV疫苗
近几年来国内外对HCMV疫苗进行了广泛研究,已先后有减毒活疫苗、亚单位疫苗、DNA疫苗等多种疫苗问世,在国外有的疫苗甚至已完成Ⅱ期临床试验研究。
1、减毒活疫苗:早期的HCMV疫苗主要是灭活疫苗和减毒活疫苗,但在临床上进行应用的是Towne减毒活疫苗。该疫苗是将Towne野毒株反复传代达125次所得,可诱发机体产生与自然感染相似的体液和细胞免疫反应。但由于Towne减毒活疫苗不能彻底清除机体内的病毒,不能阻止移植后病人的感染,也不能阻止病毒由母体向胎儿传播,且存在病毒回复突变甚至致癌的可能,因此未能获得广泛应用。
2、DNA疫苗:是将编码保护性抗原蛋白的基因置于适当的真核启动子下,构建表达目的基因的真核表达质粒,直接导入宿主细胞内进行表达。DNA疫苗具有表达蛋白时间长、可同时诱导体液和细胞免疫、成本低等突出优点,具有较高的实用性。然而,DNA疫苗也存在许多问题有待解决,如外源DNA是否会整合到宿主基因组中诱发肿瘤或导致宿主细胞功能改变等。
3、亚单位疫苗:由于减毒活疫苗可能存在潜在的致肿瘤性、培养困难、生产技术要求高等缺点,人们开始将目光集中到亚单位疫苗研制上。亚单位疫苗是通过提取HCMV中具有免疫功能的蛋白,或利用DNA重组技术表达这类蛋白,或合成蛋白多肽制成。这类蛋白是病毒的一部分。在HCMV蛋白中,gB和pp65分别诱导机体的体液和细胞免疫,是诱导中和抗体和CTL产生的抗原,因此成为HCMV亚单位疫苗的主要候选蛋白。
(1)介导体液免疫的亚单位疫苗:21世纪最有希望成功的HCMV疫苗是gB亚单位疫苗。gB蛋白是HCMV包膜中最丰富的糖蛋白,占包膜蛋白的50%以上,gB是由907个氨基酸组成的跨膜蛋白,成熟gB由116kD和58 kD两部分通过二硫键结合组成。阳性血清中50%以上抗体为gB特异性。Chiron公司将改造后的gB基因转染中国仓鼠卵巢细胞(CHO),获得稳定的重组细胞系,此疫苗与新型佐剂MF259共同使用,在临床受试者中2次免疫后,可明显产生中和抗体,在第3次免疫后,抗体应答水平明显提高,但是Ⅰ期和Ⅱ期临床观察的结果却非常令人失望,在人体产生的抗体应答短暂,几个月后即降低到保护水平之下。
(2)介导细胞免疫的亚单位疫苗:由于HCMV是典型的胞内感染病毒,细胞免疫在抗HCMV感染中较体液免疫具有更关键的作用。近年来认为HCMVpp65具有强且持久的免疫原性,它是HCMV病毒血症的主要抗原,而且是HCMV特异性细胞毒性T细胞的主要靶蛋白,在细胞免疫中可能发挥重要作用。临床观察表明金丝雀痘病毒编码的pp65能在CMV阳性及阴性的志愿者体内能引起CD4和CD8的T细胞反应,且在体内维持26个月。Tanaka等以杆状病毒为载体,在昆虫细胞内表达重组蛋白,能够刺激机体产生体液免疫,更重要的是能够产生细胞免疫。另外调节蛋白IE1(UL123)、磷蛋白pp150(UL32)、pp50/52(UL44)、pp71(UL82)及pp28(UL99)也是CTL应答的最佳诱导者,复合的亚单位疫苗可诱导更强烈的T细胞应答。不久前提出用MVA为载体表达多抗原成分疫苗(pp65、pp150和IE),可能诱导更广泛人群的免疫应答。
4、病毒载体DNA疫苗:病毒载体DNA疫苗即可用于防治,又能用于治疗疾病。研究比较多的两种“非复制型”载体是复制缺陷型金丝雀痘苗载体和复制缺陷型腺病毒载体。两种载体能以可靠的方式表达外源抗原,诱生机体的体液和细胞免疫,具有较广泛的保护性免疫力。Zhongde Wang等根据前人的经验,利用经修饰的安格拉痘苗病毒株MVA表达了可溶性的gB片段gB680(gB680-MVA),免疫小鼠,发现在小鼠体内可产生高滴度的抗gB680特异抗体,抗体滴度基本与人自然感染HCMV相似,抗体在小鼠体内存留的时间为6个月以上。体外实验此抗体能中和多种型别HCMV。为了进一步增强免疫效果,Zhongde Wang等用经过修饰的MVA表达了泛素化的pp65抗原,经泛素化后的抗原在体内产生的特异的CTL的浓度更高。然而这些疫苗只是验证了其初步的安全性,而在体内是否真正能够预防HCMV的感染,还有待进一步的实验验证。
上述所研究的HCMV疫苗通常是在重组疫苗载体中编码一个全长的HCMV抗原基因或显性抗原基因,此类疫苗虽取得了一定的效果,但存在免疫原性弱、免疫范围局限、免疫反应小等缺点,不能非常有效地预防或抑制病毒的感染,主要是因为:(1)抗原分子中能引起免疫应答的部分是与宿主HLA分子特异性结合的抗原表位(epitope),是诱导机体免疫应答的必需的最基本的结构和功能单位;而HCMV基因组全长230kb,抗原表位极其复杂,单一抗原分子所含的局限抗原表位引起的机体免疫应答较弱,无法完全预防或消除HCMV感染;而且由于抗原表位是半抗原,免疫原性弱等特点,单独应用这些抗原表位很难刺激机体产生免疫应答反应,需要将这些表位连接到一定的载体上(2)不同HCMV抗原分子是通过不同HLA基因位点提呈来激活免疫应答的,然而人群中HLA遗传背景复杂,单一抗原分子难以覆盖HLA位点不同的整个人群。虽有学者考虑使用多抗原疫苗,但载体的有限容量及大分子蛋白可能造成的免疫病理反应亦限制了多个抗原在同一载体中的应用。如何在有限的载体容量下大大增强疫苗的免疫原性,而且同时制备的疫苗免疫应答人群覆盖面广,是当前HCMV疫苗研究的难点。
将抗原表位构建于合适的载体中,再辅以必需的侧翼序列或免疫调控序列,可诱导针对所选表位的特异性免疫反应,其最大优势为选用最佳和最具免疫保护潜力的抗原表位来诱导特异性免疫应答,尽量减少无关、干扰或抑制序列可能产生的负面作用。近年来,国外有不少学者利用表位疫苗进行病毒、细菌、寄生虫等疾病的免疫应答研究,取得了一定成绩。Ciernik等将HIV gp120的CTL单一表位疫苗p18ⅢB构建于真核表达载体pRc/CMV中,于耳后和皮下单次免疫BALB/c小鼠后,结果诱生了针对p18ⅢB的特异性免疫应答反应。Meyer等利用针对B细胞表位的HCMV gp116表位疫苗进行免疫效果评价,结果发现,可诱导相应的HCMV的中和抗体,对防治HCMV感染有一定作用。由于HLA基因的高度多态性,受特定HLA基因限制的单表位疫苗通常不能在所有个体中引起预期的免疫反应,因而有学者尝试将多种表位疫苗串联,以期望产生更强的免疫应答。多表位疫苗与单一表位疫苗相比具有以下优势:能克服HLA的遗传限制,精心组合的多表位疫苗可以被多种遗传背景的HLA分子识别并结合,从而得到高效提呈;其次多表位疫苗可以有效避免病原微生物的变异和免疫反应中的某些不利因素;再次多表位疫苗在诱导细胞免疫方面有独特的优势。Rohrlich等选择HCMV的嵌合蛋白IE1-pp65作为表位疫苗进行研究,结果显示,两串联表位表达良好,都可在体内激活和扩增HCMV特异性的CD8+CTL细胞,但由于二者都是被HLA-A2所共同识别的T表位,虽有较好的免疫原性,但相同表位疫苗的串联优势并不明显。
国外HCMV表位疫苗研究大多是单一表位或针对相同HLA位点的同源表位串联,免疫反应单一,协同作用不明显,不能充分发挥接种个体的免疫能力;理想的表位疫苗应具有多能性,诱导机体全面的免疫应答反应,但目前尚未见能同时有效引起细胞免疫和体液免疫应答的HCMV串联表位疫苗。同时国外对HCMV表位疫苗的研究针对的免疫覆盖人群为欧洲及非洲人群,他们的HLA遗传背景与中国人群HLA分布情况不同,其研制的表位疫苗在中国人群很难被HLA分子提呈,不能引起有效的保护性免疫应答。目前国内尚未见有关HCMV表位疫苗的报道,且中国人口和民族的空间分布极度不均匀,人群HLA基因频率和单倍型频率的空间分布呈现高度异质性,因而研制针对中国人群HLA分布特点的免疫效果强大的HCMV串联表位疫苗是我国HCMV防治领域的关键所在。
综上所述,尽管HCMV感染普遍存在,呈无症状感染,但仍有高危人群罹患严重的HCMV疾病,包括器官移植、造血干细胞移植病人,HIV患者和胎儿。由于免疫可以改善HCMV疾病的严重性,已经努力了30年发展HCMV疫苗应用于高危人群,然而,尽管有了这些努力仍然没有可以即将出现的HCMV疫苗获得批准上市。没有批准上市HCMV疫苗的原因是复杂的,但是有几个关键的原因是:(1)宿主的保护性免疫还不清楚;(2)应该纳入HCMV疫苗的病毒蛋白不能确定;(3)临床试验大部分针对免疫缺陷病人,和免疫保护作用低下易患先天性HVMC感染的胎儿;(4)HCMV疫苗接种的目标人群仍然不清楚;(5)最后但最重要的是关于HVMC感染的教育问题很不充分,特别是育龄妇女和下级阶层人群。
设计理念
第一部分覆盖中国人群HLA特异的HCMV串联表位的确定及预测
目的:覆盖中国人群HLA特异的HCMV串联表位的确定及预测。
方法:
1、根据山东大学卫生系统计教研室薛付忠老师建立的中国HLA—Ⅰ积累表型频率(CPF)空间预测系统依次预测A2,A24,A1,A3,A11,A68,B44,B7,A23,A26,B35,B38,B8,B27十四个基因位点在中国人群的覆盖率(CPF值),评价和预测HLA特异的HCMV串联表位核酸疫苗在中国不同地理位置上人群内的免疫效果;
2、利用从1996年以来发表在PubMed上被SCI收录的23篇研究HCMV表位的文献,从中选择能针对HCMV多种蛋白抗原的多个CTL表位、Th表位和B细胞表位,能够激发机体的细胞免疫应答和体液免疫应答,且能与HLA-Ⅰ分子和HLA-Ⅱ分子的基因位点稳定结合的表位;
3、利用SYFPEITHI MHC表型及表位预测系统和MAPPP蛋白酶体降解预测系统对所选表位进行表位肽预测和MAPPP蛋白酶体切割-表位生成预测;
4、从PubMed Nucleotide NC_001347.人巨细胞病病毒AD169株各蛋白的基因序列中找出各表位相对应的核苷酸序列并核对,将各表位的核苷酸序列按等位基因的频率分布顺序排列起来,串联成一条核苷酸序列;
5.根据Kozak规则引入Kozak序列以获得HCMV串联表位核苷酸序列在真核表达载体中的高效表达;
6.将HCMV串联表位的核苷酸序列输入DNAMAN软件,根据输出的酶切位点结果结合所要构建入穿梭载体的多克隆位点,确定串联表位核苷酸序列两端的酶切位点。
结果:
1、HLA特异的HCMV串联表位核酸疫苗在中国人群的覆盖频率可达90%以上;
2、我们选择了HCMV的十五种抗原蛋白pp28,pp50,pp65,pp150,pp71,gH,gB,IE-1,IE-2,US2,US3,US6,US11,UL16,UL18,分别在病毒的黏附、复制、组装、免疫逃逸及再激活过程中表达,在这些抗原中选取了84个表位,其中针对HLA-Ⅰ分子基因位点的CTL表位76个,针对HLA-Ⅱ分子基因位点的Th表位7个,能激发机体产生中和抗体的B细胞表位1个,通过启动针对上述抗原表位的细胞和体液免疫应答,理论上可以在各个阶段控制病毒的复制,防止HCMV疾病的形成;
3、覆盖中国人群HLA特异的HCMV 83个串联表位的SYFPEITHI预测分数均在14分以上,多数在20-30分之间,MAPPP预测分数均在0.500以上;
4、引入Kozak规则,加入6个His鉴定标签,利用PubMedNucleotide NC_001347.Human herpesvirus 5 strain AD169 HCMV基因组得出了覆盖中国人群HLA特异的HCMV串联表位核苷酸序列;
5、输入DNAMAN软件,确定了覆盖中国人群HLA特异的HCMV串联表位核苷酸序列两端的酶切位点是NheⅠ和NotⅠ酶切位点,可以插入穿梭载体pHMCMV5载体的多克隆位点。
结论:
1、根据山东大学卫生系统计教研室薛付忠老师建立的中国HLA—Ⅰ积累表型频率(CPF)空间预测系统,对所选HCMV表位结合的14个HLA基因位点及其组合进行了预测,显示A2,A24,A1,A3,A11五个优势基因位点的CPF值是88.1%,;A2,A24,A1,A3,A11,A68,B44,B7,A23,A26,B35,B38,B8,B27十四个基因位点的CPF值是92.1%,显示HLA特异的HCMV串联表位核酸疫苗在中国人群的覆盖频率达90%以上,预测了HLA特异的HCMV串联表位核酸疫苗在中国不同地理位置上人群内的免疫效果,理论上其免疫效果可达90%以上。
2、我们选择了HCMV的十五种抗原蛋白pp28,pp50,pp65,pp150,pp71,gH,gB,IE-1,IE-2,US2,US3,US6,US11,UL16,UL18,分别在病毒的黏附、复制、组装及再激活过程中表达,在这些抗原中选取了84个表位,其中针对HLA-Ⅰ分子基因位点的CTL表位76个,针对HLA-Ⅱ分子基因位点的Th表位7个,能激发机体产生中和抗体的B细胞表位1个,通过启动针对上述抗原表位的细胞和体液免疫应答,理论上可以在各个阶段控制病毒的复制,防止HCMV疾病的形成。
3、应用DNAMAN软件、Kozak规则及PubMed Nucleotide NC_001347.Humanherpesvirus 5 strain AD169 HCMV基因组得出了覆盖中国人群HLA特异的HCMV串联表位核苷酸序列,在起端加上了Kozak序列,可以获得串联表位核苷酸序列在真核表达载体中的高效表达;末端加上了6个His标签序列,可以通过6个His抗体应用Western blot及细胞免疫组化检测HCMV串联表位的表达;起始两端加入了NheⅠ和NotⅠ酶切位点,可以插入穿梭载体pHMCMV5载体的多克隆位点,为制备中国人群HLA特异的HCMV串联表位腺病毒载体核酸疫苗打下了基础。
第二部分中国人群HLA特异的HCMV串联表位腺病毒核酸疫苗的制备
目的:HCMV感染是引起移植患者和新生儿高发病率和死亡率的原因,世界范围的医学会组织指出应优先发展控制HCMV疾病的疫苗,尽管做出了很大的努力但目前仍无被临床许可的疫苗;嵌合型复制缺陷型腺病毒载体Ad5F35比传统的Ad5型具有更好的趋向性和更高的容纳外源基因的能力,在多种细胞上高效表达外源基因。本实验应用嵌合型Ad5F35构建多重HLA-Ⅰ、HLA-Ⅱ分子限制CTL、Th多表位核酸疫苗Ad5F35-CTL·Th以及针对体液免疫应答的HCMV线性B细胞表位核酸疫苗Ad5F35-AD-1。
方法:
1、采用重叠区扩增基因拼接法(Gene splicing by overlap extension,SOEing)和PCR技术合成HCMV串联T细胞表位全长基因;
2、应用PCR技术从HCMV AD169全基因组扩增线性B细胞表位gB AD-1的基因;
3、利用同源重组技术从穿梭载体,将CTL·Th和AD-1基因分别插入pHMCMV5的NheⅠ/NotⅠ和KpnⅠ/AflⅡ酶切位点,构建穿梭载体pHMCMV5-CTL·Th和pHMCMV5-AD-1,应用NheⅠ/NotⅠ和KpnⅠ/AflⅡ酶切分析和测序分析鉴定目的载体;
4、利用I-CeuⅠ/PI-SceⅠ双酶切和体外重组技术将穿梭载体上包含CMV启动子、目的基因、SV40 polyA尾的表达盒插入腺病毒表达载体pAd5F35,构建重组腺病毒表达载体Ad5F35-CTL·Th和pAd5F35-AD-1,应用XhoⅠ和I-CeuⅠ/PI-SceⅠ酶切分析和测序分析鉴定目的载体;
5、应用PacI线性化重组腺病毒表达载体Ad5F35-CTL·Th和pAd5F35-AD-1,用脂质体2000转染HEK293,包装重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1,待培养10天出现CPE效应时收取病毒;
6、应用HEK293细胞大量扩增重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1,扩增至五代,应用快速腺病毒感染性滴度(TCID50)检测试剂盒测定大量扩增后的的重组腺病毒的滴度;
7、用重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1分别转染HEK293细胞,利用RT-PCR和Western blot技术分析CTL·Th和AD-1在mRNA和蛋白水平的表达;
8、用重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1分别转染PBMC,利用细胞免疫化学和台盼兰染色分析重组腺病毒在PBMC上的表达和对PBMC活性的影响。
结果:
1、采用SOEing和PCR技术成功合成了HCMV串联T-细胞表位全长基因;
2、在HCMV全基因组DNA中成功扩增了线性B细胞表位gB AD-1目的基因;
3、成功构建了穿梭载体pHMCMV5-CTL·Th和pHMCMV5-AD-1,酶切鉴定重组穿梭载体均有目的基因;
4、成功构建了重组腺病毒表达载体Ad5F35-CTL·Th和pAd5F35-AD-1,酶切鉴定重组腺病毒均有目的基因;经测序后目的核苷酸序列无碱基突变。
5、成功包装出重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1,转染HEK293细胞后观察到细胞病变效应(CPE),平行对照Ad5F35-GFP有绿色荧光表达。
6、应用HEK293细胞大量扩增重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1,扩增了五代,每轮扩增只需2-3天。
7、大量扩增后重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1的滴度是2.5×10~9IU/L。
8、应用重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1转染HEK293细胞,可以观测到目的基因CTL·Th和AD-1在mRNA水平均有表达,目的基因CTL·Th和AD-1在蛋白水平亦均有表达,CTL·Th在蛋白水平表达90kda左右的目的带,AD-1在蛋白水平表达55kDa左右的目的带。
9、应用重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1转染外周血单个核细胞(PBMC),分别在转染第1、3、5、7、10天应用台盼兰染色观察PBMC的细胞活性,在转染后10天PBMC仍保持90%以上的活性,说明重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1毒性极低。
10、重组腺病毒Ad5F35-CTL·Th在PBMC的胞膜、胞浆均有表达,重组腺病毒Ad5F35-AD-1在PBMC的胞膜有表达,说明重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1可以在PBMC中高效表达。
结论:
1、我们成功制备出中国人群HLA特异的HCMV串联T细胞表位腺病毒载体核酸疫苗Ad5F35-CTL·Th和针对体液免疫应答的HCMV线性B细胞表位腺病毒载体核酸疫苗Ad5F35-AD-1,为下一步的免疫应答实验打下了基础。
2、重组腺病毒疫苗Ad5F35-CTL·Th和Ad5F35-AD-1可以在PBMC高效表达目的蛋白,且不影响PBMC的活性。
第三部分中国人群HLA特异的HCMV串联表位腺病毒核酸疫苗体外免疫效果评价
目的:过继免疫包括体液免疫和细胞免疫在控制HCMV感染方面起关键作用。本实验评价中国人群HLA特异的HCMV串联表位腺病毒核酸疫苗的体外免疫效果。
方法:
1、用EBV转化PBMC,建立类淋巴母细胞系(lymphoblastoid cell lines-LCL);
2、应用HCMV多表位腺病毒载体疫苗Ad5F35-CTL·Th刺激HCMV健康携带者的外周血单个核细胞(PBMC),体外扩增抗原特异性CTL;
3、应用抗原特异性CTL进行细胞毒试验,检测CTL的杀伤活性;
4、应用ELISPOT实验检测Ad5F35-CTL·Th刺激HCMV健康携带者的PBMC后和抗原特异性CTL分泌IFN-γ的情况;
5、细胞内染色法检测Ad5F35-CTL·Th刺激HCMV健康携带者的PBMC后CD8+T细胞IFN-γ的表达。
结果:
1、利用EBV成功转化了PBMC,建立了2株类淋巴母细胞系(lymphoblastoid celllines-LCL);
2、应用HCMV多表位腺病毒载体疫苗Ad5F35-CTL·Th刺激HCMV健康携带者的PBMC,在体外高效扩增了抗原特异性CTL;
3、Ad5F35-CTL·Th编码的HCMV表位可以被内源性提呈;
4、经Ad5F35-CTL·Th刺激后PBMC大量分泌IFN-γ,体外扩增抗原特异性CTL亦大量表达IFN-γ;
5、细胞内染色法检测Ad5F35-CTL·Th刺激PBMC后CD8+T细胞有IFN-γ的表达。
结论:
1、本实验显示了多表位抗原提呈系统扩增抗原特异性T细胞的有效性。
2、本试验显示了HCMV多表位腺病毒核酸疫苗可以高效扩增免疫显性和亚显性的抗原特异性T细胞。
3、实现了单个表达结构可以扩增免疫显性和亚显性的抗原特异性T细胞,较之以前扩增不同的抗原特异性T细胞需要不同的刺激策略具有强大的优势。
4、我们的多表位重组技术可以有望适用于不同疾病的临床过继免疫治疗。
Background
Human cytomegalovirus(HCMV) is the most complex of the eight human herpesvirus species and has a 235-kb double-stranded DNA that encodes 165 genes.HCMV infects approximately 40-60%of the developed world's population.Primary infection in healthy hosts is usually asymptomatic,and the virus persists in CD33 progenitors expressing markers of dendritic and myeloid lineage as major reservoirs of latent infection virus without any apparent clinical symptoms.HCMV-associated clinical disease has been recognized in three populations:Neonates with immature immune systems,transplant recipients with impaired immune systems due to the use of drugs that suppress rejection and HIV-infected patients with impaired immune systems due to the decline of CD4+T cells
HCMV is the most important viral pathogen affecting transplant recipients,including both solid organ transplant and allogeneic hematopoietic stem cell transplant recipients whose immune systems are suppressed by drugs used to prevent graft rejection.It is noted that HCMV is reported to infect not only stroma and epithelial cells but also hematopoietic cells including CD34+ stem cells,monocytes,and dendritic cells. Late-onset cytomegalovirus disease in hematopoietic stem cell transplant recipients(later than day 100 after transplantation) remains a major cause of morbidity and mortality, despite the introduction of new antiviral agents.Congenital HCMV infection is the most common intrauterine infection and 0.3-2.4%of neonates are born with HCMV infection worldwide.Of the neonates with congenital HCMV infection,10%have symptoms of irreversible CNS involvement in the form of microcephaly,encephalitis,seizures, deafness,upper-motor neuron disorders and psychomotor retardation.
Intensive study over the last 30 years has demonstrated that both innate and adaptive immunity play important roles in controlling latent HCMV infection.The innate immune response,mainly natural killer cells,may shape or augment the adaptive immune response against HCMV infection.NK cells may act by secreting IFN-γ,which can facilitate the expansion of antigen-specific helper T cells to control HCMV infection.The adaptive immunity,including both humoral and cellular immunity,plays a crucial role in controlling HCMV infection.Extensive analysis of humoral immune responses to HCMV has revealed that response is directed toward multiple viral protein and that virus-protection antibodies are directed primarily to the envelope glycoprotein of HCMV with glycoprotein B(gB) as a dominant target antigen.HCMV-specific T-cell immunity is the most important adaptive immune component responsible for suppressing HCMV and keeping it at latency after primary infection.Studies showed that pp65 and IE-1 constituted 40%of the total CD8+ T-cell responses and 60%of these T-cell responses are directed towards other antigens,such as pp28,pp150,pp71 and US proteins.Compared with the CD8+ T cells,CD4+ T cells were generally thought to have an indirect role in controlling HCMV infection by providing helper signals for the generation of antibodies and the maintance of CD8+ T-cell memory.
Taken together,our increasing understanding of immune responses against HCMV infection has firmly established that activation of both humoral and cellular immunity is crucial for successful HCMV vaccine.
PartⅠPrediction and Decision of HLA-specific HCMV Polyepitope in Chinese People
Objective:Prediction and decision of HLA-specific HCMV polyepitope in Chinese people.
Methods:
1.According to the forecasting system on spatial coverage of cumulative phenotypic frequency(CPF) of HLA-I of professor XUE Fu-zhong in Institute of Epidemiology and Medical Statistics of Medical College of Shandong University,the study forcasted the CPF of 14 HLA allel sites including A2,A24,A1,A3,A11,A68,B44,B7,A23,A26, B35,B38,B8,B27 in Chinese people and evaluated immune activity of HLA-specific HCMV polyepitope nucleotide vaccine in different geographical location of Chinese people.
2.Utilizating 23 SCI literature about HCMV epitope which have been published in PubMed since 1996,the study selected multiple CTL epitopes,Th epitopes and B cell epitopes in different HCMV protein antigens which can induce cellullar immunologic response and humoral immunoresponse and stably bind classⅠand classⅡMHC molecules.
3.Through SYFPEITHY combined with MAPPP algorisms,the selected epitopes were predicted for the specific value of peptide and proteasomal cleavage site.
4.The nucleotide sequences of the selected epitopes were found in genome of HCMV AD169 strain and verified in PubMed Nucleotide NC_001347.Furthermore,the nucleotide sequences of these epitope were cascaded a single nucleotide sequence according to frequency distribution of HLA allel.
5.According to Kozak rule,Kozak sequence was put into the nucleotide sequence of HCMV polyepitope which can induce highly efficient expression in eukaryotic expression vector.
6.The nucleotide sequence of HCMV polyepitope was input DNAMAN software. Enzyme cleavage sites of the nucleotide sequence were decided in order to construct shuttle vector in the next experiment.
Results:
1.The coverage of HLA-specific HCMV polyepitope nucleotide vaccine in Chinese people has reached above 90%.
2.The epitopes included in this polyepitope were derived from 15 different viral proteins(pp28,pp50,pp65,pp150,pp71,gH,gB,IE-1,IE-2,US2,US3,US6,US11, UL16 and UL18) involved in virus attachment,replication,assembly,reactivation and immune escape from the latent phase,all of which are crucial stages in the development of HCMV disease.There were 76 CTL epitopes,7 Th epitopes and 1 B cell epitope in selected epitopes.In theory,these epitopes can induce cellular immunologic response and humoral immunoresponse and prevent the development of HCMV disease.
3.Through the SYFPEITHI web site,the predicted values of the selected epitopes have reached above 14 scores,almost between the 20 and 30 scores.Through the MAPPP server,the predicted proteasomal cleavage values of the selected epitope have reached above 0.5 score.
4.After introducing Kozak rule,adding 6×his identify tag and utilizating genome of HCMV AD 169 strain in PubMed Nucleotide NC_001347,the nucleotide sequence of the polyepitope has been decided.
5.In order to construct shuttle vector in the next experiment,the NheⅠand NotⅠenzyme cleavage site,of the nucleotide sequence have been decided after the nucleotide sequence of HCMV polyepitope was input DNAMAN software.
Conclusions:
1.According to the forecasting system on spatial coverage of cumulative phenotypic frequency(CPF) of HLA-Ⅰ,the CPF of 14 HLA allel sites including A2,A24,A1,A3, A11,A65,B44,B7,A23,A26,B35,B35,B8 and B27 in Chinese people has reached 92.1%.It has exhibited the coverage of HLA-specific HCMV polyepitope nucleotide vaccine in Chinese people has reached above 90%.It also has evaluated immune effect of HLA-specific HCMV polyepitope nucleotide vaccine in different geographical location of Chinese people.In theory,immune effect of HLA-specific HCMV polyepitope nucleotide vaccine can reach above 90%.
2.The epitopes included in this polyepitope were derived from 15 different viral proteins(pp28,pp50,pp65,pp150,pp71,gH,gB,IE-1,IE-2,US2,US3,US6,US11, UL16 and UL18) involved in virus attachment,replication,assembly,reactivation and immune escape from the latent phase,all of which are crucial stages in the development of HCMV disease.There were 76 CTL epitopes,7 Th epitopes and 1 B cell epitope in selected epitopes.In theory,these epitope can induce cellular immunologic response and humoral immunoresponse and prevent the development of HCMV disease.
3.After introducing Kozak rule,utilizating genome of HCMV AD169 strain in PubMed Nucleotide NC_001347 and inputting DNAMAN software,the nucleotide sequence of the polyepitope has been decided.Kozak sequence has been added in the upstream that can induce high efficient expression in eukaryotic expression vector.Six×His identify tag has been added in the downstream that can be used in Western blot and cell immunochemistry experiment.The output NheⅠand NotⅠenzyme cleavage site can be used to construct shuttle vector in the next experiment.
PartⅡPreparation of Novel Vaccine against HCMV Based on Ad5F35 Adenovirus Vector Expressing HLA-specific polyepitope nucleotide
Objective:HCMV disease accounted for the high morbidity and mortality in transplant patients and new born babies.The worldwide institute of medicine has assigned the highest priority for a vaccine to control HCMV disease.In spite of numerous attempts, successful licensure of a HCMV vaccine formulation remains elusive.The modified replication-deficient adenoviral vector AdSF35 possesses more expanded tropism and larger foreign DNA package ability than the conventional Ad5 vectors without affecting the viral growth rate and titer.It exhibits high transduction efficiency in many human cells.Here we have developed two novel chimeric vaccine strategy based on AdSF35 which encode multiple HLA classⅠ&Ⅱrestricted CTL and Th epitopes from HCMV as a contiguous polypeptide and encode the high conserved predominant B cell epitope gB AD-1.
Methods:
1.Using SOEing and PCR technology to synthesize the total length CTL·Th gene of HCMV multiple T cell epitope.
2.The AD-1 gene was amplified from AD169 genomic DNA by polymerase chain reaction(PCR) with the oligonucleotide primer.
3.The CTL·Th gene and AD-1 amplicon were ligated the NheⅠ/NotⅠand KpnⅠ/AflⅡsites of shuttle plasmid pHMCMV5 respectively.The desired plasmids were identified by restriction analysis and nucleotide sequence analysis.
4.The expression cassette,comprising the CMV promoter,CTL·Th gene/AD-1 and an SV40 polyA signal,was excised from the shuttle plasmids pHMCMV5-CTL·Th or pHMCMV5-AD-1 with I-CeuI and PI-SceI and ligated into adenoviral backbone vector pAd5F35 cleaved with the same enzymes.The presence of expression cassettes was verified by digestion with XhoⅠandⅠ-CeuⅠ/PI-SceⅠ.
5.pAd5F35-CTL·Th and pAd5F35-AD-1 were linearized with PacI and transfected into HEK293 cells using lipofectamine~(TM) 2000.Transfected HEK293 cells (HEK293-Ad5F35-CTL·Th or HEK293-Ad5F35-AD-1) were incubated at 37℃for 7-10 days until maximal virus cytopathic effect(CPE) was observed.Then recombinant adenovirus was harvested.
6.The recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1 were amplified using HEK293 cells until the 5~(th) passage.The titer of the large-scale recombinant adenovirus was established by plaque assay according to adenovirus TCID50 testing kit protocol.
7.The expression of CTL·Th and AD-1 was identified by using transfected HEK293 cells.To confirm CTL·Th and AD-1 mRNA expression,RT-PCR analysis were conducted.To confirm AD-1 protein expression,western blot analysis was conducted.
8.The expression of CTL·Th and AD-1 in PBMCs infected with Ad5F35-CTL·Th and Ad5F35-AD-1 was analyzed by immunocytochemistry,and cell viability were determined by trypan blue staining.
Results:
1.Using SOEing and PCR technology,the total-length gene of HCMV multiple T cell epitopes has been successful synthesized.
2.AD-lgene has been successfully amplified from the genome of HCMV AD169 strain.
3.Shuttle plasmids pHMCMV5-CTL·Th and pHMCMV5-AD-1 have been successfully constructed.After enzyme digestion identification and DNA sequencing, insertion of the gene was proved correct.
4.Recombinant adenovirus vectors pAd5F35-CTL·Th and pAd5F35-AD-1 have been successfully constructed.After enzyme digestion identification and DNA sequencing,the expression of the gene was proved correct.
5.The recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1 have been successfully packaged.Full CPE was observed for recombinant virus after transfecting HEK293.
6.The recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1 were amplified using HEK293 cells until the 5~(th) passage.Each cycle of amplification only needed 2-3 days.
7.The titer of the large-scale recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1 virus preparation were 2.5×10~9 PFU by plaque assay.
8.After transfecting HEK293 cells using recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1,CTL·Th and AD-1 expression were verified at the level of mRNA protein.
9.After PBMCs were transfected 10 day using Ad5F35-CTL·Th and Ad5F35-AD-1, 90%of cells retained viable.It has shown no any adverse effects of the recombinant adenovirus.
10.Strong expression of CTL·Th polyepitope antigen and AD-1 were recorded in PBMCs infected with Ad5F35-CTL·Th and Ad5F35-AD-1.The typical pattern of staining demonstrates that recombinant CTL·Th polyepitope localizes to the cell membrane and cytoplasm,while the peripheral pattern of staining demonstrates that recombinant AD-1 localizes to the cell membrane.
Conclusion:
1.Two novel vaccines based on a replication-deficient adenovirus that encode HLA-specific HCMV polyepitope nucleotide vaccine(referred to as Ad5F35-CTL·Th) and a single B cell epitope gB AD-1(referred to as Ad5F35-AD-1) were successfully constructed.
2.Ex vivo stimulation of PBMC with Ad5F35-CTL·Th and Ad5F35-AD-1 consistently showed highly efficient expression of CTL·Th polyepitope antigen and epitope AD-1 protein.The viability of PBMC was not affected.It will be useful for HCMV novel vaccine development.
PartⅢImmune Activity Evaluation of Novel Vaccine against HCMV Based on Ad5F35 Adenovirus Vector Expressing HLA-specific polyepitope nucleotide
Objective:The adaptive immunity,including both humoral and cellular immunity, plays a crucial role in controlling HCMV infection.This experiment is to evaluate immune activity of this novel vaccine against HCMV based on a chimeric Ad5F35 adenovirus vector expressing HLA-specific polyepitope nucleotide.
Methods:
1.Lymphoblastoid cell lines-LCL is established as antigen presenting cell by using EBV to transform PBMC.
2.In vitro antigen-specific CTL was generated from a panel of healthy virus carriers by stimulating PBMC with adenoviral chimeric vaccine Ad5F35-CTL·Th.
3.These CTL clone lines were screened for cytotoxic activity on a panel of autologous target cells that were either sensitized with synthetic peptides or infected with Ad5F35-CTL·Th.
4.The ELISPOT assay was used to assess whether adenoviral chimeric vaccine Ad5F35-CTL·Th could stimulate a memory response,as measured by the production of IFN-γ,in PBMC from a large panel of seropositive donors.
5.The Intracellular cytokine staining(ICS) assay was used to measure the levels of specific CD8+T cells elicited by Ad5F35-CTL·Th stimulating PBMC from healthy seropositive individuals.
Results:
1.Two EBV-transformed-LCLs have been successfully established.
2.In vitro stimulation with this adenoviral chimeric vaccine Ad5F35-CTL·Th rapidly expanded multiple antigen-specific human CD8+T-cells from healthy virus carriers.
3.The HCMV epitopes encoded by this polyepitope were endogenously expressed and processed by human cells.
4.The production of IFN-γhas been largely secreted from PBMC stimulated by Ad5F35-CTL·Th or expanded antigen-specific CTL.
5.The levels of specific CD8+T cells have been strongly elicited by Ad5F35-CTL·Th stimulating PBMC from healthy seropositive individuals.
Conclusion:
1.This study shows the effectiveness of a polyepitope antigen presentation system for reproducible expansion of antigen-specific T cells from immunocompetent settings.
2.It also shows that an adenovirus based polyepitope is highly efficient in expanding both immunodominant and subdominant antigen-specific T cells.
3.The comparable expansion of both immunodominant and subdominant T cell responses using a single expression construct was an unexpected result,as previous studies have shown that the different T cell populations require distinct stimulation strategies.
4.The data presented here emphasize the strength and adaptability of the polyepitope-based approach for clinical translation and may lead to significant advances in the application of adoptive immunotherapy to a wide range of diseases.It will be useful for HCMV novel vaccine development.
人巨细胞病毒(Human cytomegalovirus,HCMV)是人类先天性病毒感染的最常见病原体,人是其唯一的宿主。根据社会经济状况和地理位置的不同,人群中抗HCMV血清阳性率可达50%～90%。一般情况下,HCMV原发感染为隐性感染,无明显的临床症状,终生携带,病毒周期性地从黏膜部位释放。在器官和造血干细胞移植、肿瘤、艾滋病等免疫低下人群中,体内潜伏的病毒重新激活,引起HCMV活动性感染,其感染率约为50%～75%,死亡率约是25%,一般于术后1～4个月内发生,是器官移植失败和移植受体死亡的重要原因。对于孕妇来说,HCMV原发或复发感染均可引起胎儿宫内感染或围产期感染,而导致畸形、智力低下和发育迟缓,感染率达0.3-2.4%,其中发病率达15%-30%。在美国每年有8000名HCMV原发感染的婴儿出生,其中有10%～17%的婴儿出现耳聋或神经系统后遗症。在我国因先天HCMV感染造成的先天畸形患儿约4万人左右,占每年我国出生缺陷儿的6.2%。
巨细胞病毒的生物学特征
HCMV属于β-疱疹病毒亚科,为双链线状DNA病毒,直径约为120～300nm,核酸为235kb的双股线性DNA,有213个开放读码框,其中有165个编码基因,Varnum等证明HCMV病毒颗粒由59种结构基因编码的产物与众多宿主蛋白构成,是疱疹病毒中最大的,具有严格的种属特异性。其基因由长的独特序列(UL)、短的独特序列(US)及存在于L—S连接部位的重复序列组成,可构成4种同分异构体,至少编码氨基酸残基数100以上的多肽200余种,包括原始基因产物、中间产物及终末产物。HCMV基因的转录及表达有一定的时序性,可分为即刻早期(IE)、早期(E)和晚期(L)基因3种类型。其中,对病灶具有重要监测和检测功能的是IE和L。即刻早期基因是HCMV原发感染或复发感染后最先表达的基因。IE蛋白在HCMV基因表达的后续调控过程中起着主导作用。晚期基因的转录和表达一般发生在感染后36～48h。在HCMV DNA开始复制后,L基因合成病毒的结构蛋白,包括衣壳、被膜和包膜蛋白。因此,L基因表达意味着病毒的装配及成熟,是病毒感染的后期结果。而L基因mRNA可作为特异性的活动感染的诊断指标。HCMV对宿主或培养细胞有高度的种特异性,HCMV只能感染人,及在人纤维细胞中增殖。病毒在细胞培养中增殖缓慢,复制周期长,初次分离培养需30～40天才出现细胞病变,其特点是细胞肿大变圆,核变大,核内出现周围绕有一轮“晕”的大型嗜酸性包涵体。
HCMV几乎感染所有器官,血管系统(内皮细胞和平滑肌细胞)、循环淋巴细胞、单核细胞和多形核白细胞、成纤维细胞是其主要的潜伏部位,一旦病毒进入细胞,病毒衣壳粘附于胞膜,借其包膜蛋白(gB和gH)与细胞表面受体(低结合力的肝素受体和高结合力的蛋白受体)结合后,通过受体介导的机制进入细胞内。也可通过其他途径如直接穿过胞浆膜进入细胞内,然后病毒基因组与宿主细胞核结合。
HCMV有以下方式存在于宿主中,①病毒基因整合到宿主细胞基因组内,不表达病毒抗原和装配病毒颗粒,随着细胞基因复制而转移到子代细胞内,直接处于潜伏状态。②病毒在宿主细胞内复制引起细胞病变,形成核内或/和浆内包涵体,至细胞溶解凋亡,形成产毒型感染。③病毒有少量复制,靶细胞功能发生障碍,但无或极少细胞病变,引起不全感染。④宿主细胞可表达病毒抗原,导致宿主细胞转化和增生,可能参与其致癌机制。⑤HCMV感染除直接导致终末器官病变外,还在统计学上与移植排斥、细菌或真菌的二次感染、动脉粥样硬化加速等发生有密切关系,这些状况被称为HCMV的间接效应。
病毒侵入肺、肝、肾、唾液腺、乳腺其他腺体,可长期或间隙地自唾液、乳汗血液、尿液、精液、子宫分泌物多处排出病毒。通过口腔,生殖道,胎盘,输血或器官移植等多途径传播。HCMV可通过胎盘感染胎儿,影响妊娠,一是病毒在绒毛细胞内增殖后进入胎儿血中并感染之,另一种方式即为病毒在绒毛细胞内不发生增殖而仅是通过或溢出。器官移植患者最常见的感染是HCMV,主要是通过供体器官传播或受体潜伏的HCMV激活。
HCMV的致病机制
1.HCMV潜伏感染形成的机制:受染的细胞为HCMV非容纳细胞,HCMV进入受染细胞首先必须通过细胞表面的病毒相应受体。HCMV进入细胞后能否完整复制则取决于受染细胞的分化成熟程度。凡阻碍或不能使HCMV完整复制成具感染性病毒颗粒的细胞,则为非容纳性细胞。Sinclair等指出,外周血单核细胞之所以成为HCMV潜伏感染的细胞是因为在外周血单核细胞中HCMV即刻早期基因(IE)表达受阻。一旦这些细胞能进一步分化成熟,病毒的基因能正常表达,则就可形成完整的病毒颗粒。Lecoinle等比较了单核细胞衍生的巨噬细胞(MDM)、人胚脑胶质细胞和星状细胞等分化成熟程度以及体外接种HCMV后病毒复制的情况,结果发现HCMV在人胚脑胶质细胞或星状细胞内复制良好,为CPE较高滴度的病毒,相反在MDM细胞中无明显可见的CPE,仅在部分的MDM细胞中检出HCMV早期或晚期病毒抗原,认为HCMV一旦进入非容纳细胞则出现潜伏感染。
有学者运用RT-PCR技术检测并分析了潜伏于粒-巨噬前驱细胞中的HCMV即刻早期基因及其基因产物时发现,在HCMV mRNA的第356和292位碱基的上游分别存在着编码94个氨基酸多肽和152个氨基酸多肽的阅读框架,它的反向mRNA片段为2.1 kb。HCMV抗体阳性的健康携带者血清中(5/7)能检测到这一特异性基因片段,而HCMV抗体阴性的非感染者血清中则无1例可检出这一特异性基因片段(0/7),进一步支持HCMV潜伏感染存在着相关基因或转录产物的观点。
2.HCMV潜伏—再激活的机制:HCMV感染一旦发生即成为终身相伴,绝大多数的受染者是无临床表现的隐性或轻型感染。但当宿主机体内环境受诸方面因素影响,原来不导致临床病理表现的潜伏病毒则可被激活而导致严重的临床表现,甚至是致命性的。其诱因为:①受染细胞的分化成熟:骨髓造血干细胞或外周血单核细胞均为未完全分化成熟的细胞,HCMV在此类细胞中只能以潜伏状态存在。一旦某种诱因使这些受染细胞受到刺激使其进一步分化成熟时,则可能使原处于潜伏状态的病毒再度被激活,产生大量具有感染性的完整的病毒颗粒。Sodrberg等认为只有同源性的单核细胞衍生巨噬细胞刺激才能激发原来潜伏状态的病毒再度活化,并推测在器官或骨髓移植患者中HCMV激活感染的高发频率与之有关。②细胞因子、粘附分子的影响:急性炎症时由于急性炎症细胞大量分泌细胞因子,可能诱发潜伏的HCMV再度激活。γ干扰素(IFN-γ)和肿瘤坏死因子TNF-α可特异性诱导潜伏有HCMV的单核细胞分化为容许HCMV在其内繁殖的巨噬细胞,而不是发挥其本来的抗病毒生物功能。HCMV主要即刻早期基因启动区域存在着cAMP反应功能区,某些炎症因子、细胞因子,如前列腺素E2、IL-1β、TNF-α等都能活化cAMP,从而起到对HCMV的协同上调作用。Steek报道,由于CD3+T细胞的活化能够促进TNF-α的大量释放,而释放的TNF-α又促进了HCMV即刻早期基因的表达。Watandba等证明细胞粘附分子ICAM-1亦可能参与HCMV潜伏再激活。③机体免疫低下:HCMV病常发生于CD4＜0.1×10~9/L的患者,CD4＜0.05×10~9/L时,发病率为30%-40%。免疫功能低下如H1V患者、肿瘤以及器官移植患者中,一方面由于使用激素;另一方面辐射也可能导致潜伏的病毒再度活化。HIV与HCMV二重感染时,两种病毒的相互作用亦影响病毒的潜伏与激活。
HCMV和移植
大量研究显示,HCMV已成为实体器官移植和造血干细胞移植后常见及重要的并发症,是导致骨髓移植后死亡的重要原因。骨髓移植术后应用免疫抑制剂使机体抵抗力下降,体内潜伏的病毒重新激活,引起HCMV活动性感染,是影响造血干细胞移植术后受体生存率和生活质量的重要感染因素。其感染率约为50%～75%,一般于术后1～4个月内发生,早期报道巨细胞病毒感染所致的病死率高达25%。
HCMV是一种免疫调节病毒,病毒感染宿主主要是通过供体器官、细胞传播或受体中潜伏HCMV的激活,其感染的严重程度取决于下列因素:移植的种类、供/受者HCMV血清学状态、免疫抑制方案、移植物排斥情况。HCMV感染可产生免疫抑制,增加机体对严重细菌感染和其他机会性致病微生物(如卡氏肺孢子茵、白色念珠菌)的易感性。HCMV活动性感染对移植受体的影响可分为直接性和间接性,在临床上可见的直接影响是活动性HCMV病的表现,包括发热、白细胞减少、肝炎、肠炎、视网膜炎等,最常见的是巨细胞病毒感染引起的间质性肺炎,国内文献报道造血干细胞移植后HCMV感染引起的间质性肺炎(IP)发病率可达30%,病死率可达80%以上,国外文献也报道骨髓移植患者并发HCMV活动性感染并导致间质性肺炎,其病死率高达87%。间接影响是HCMV可诱导受染细胞表面的MHC-Ⅰ类和Ⅱ类抗原的表达,增加T杀伤淋巴细胞反应而促进宿主的急性排斥反应,降低患者存活率。
将HCMV疫苗注入血清学阴性的器官移植受体,在理论上说是减少HCMV感染和疾病的简单而有效的方法。研究表明,减毒活疫苗和活病毒疫苗在HCMV血清学阴性的受体中使用后,再接受HCMV血清学阳性的肾移植,能降低HCMV感染发生率。虽然HCMV疫苗不能完全预防HCMV感染疾病,但能降低HCMV首次感染后严重的临床症状。但在免疫抑制的状态下,能导致机体对疫苗免疫反应的沉默,在血清学阴性的受体中,接种疫苗后不发生血清学转变的比例是相当高的。尽管应用疫苗不可能完全达到保护性的抑制HCMV感染,但能避免发生严重的HCMV感染性疾病,再者,当与其他治疗方法,如抗病毒药物和/或免疫球蛋白的使用,将达到更好的防治效果。
应用疫苗亚单位可能更为有效,目前研究HCMV亚单位B和H疫苗。Bernstein等研究表明,对HCMV感染阴性的成人分三组,即接种HCMV亚单位糖蛋白B(gB)加MF59(一种含角鲨烯水乳剂)组,同时接种HCMV gB加MF59和金丝雀痘重组疫苗表达的HCMV gB(ALVAC-CMV gB)组,以及先后接种HCMV加MF59和金丝雀痘重组疫苗表达的HCMV gB(ALVAC-CMV gB)组,除有局部的反应外,少有系统副作用,三组均能安全有效的诱导高水平的HCMV抗体表达和细胞免疫的发生,组间无差异。
新的HCMV免疫治疗采用输入HCMV特异的细胞毒性T淋巴细胞(CTL)。在健康和免疫抑制的个体中,Ⅰ类主要组织相容性复合体限制的HCMV特异CTL,在控制HCMV感染中起相当重要的作用。骨髓移植后,增加病毒特异CTL的数量与有功能的CTL,能成功的减少HCMV疾病的发生,骨髓移植后,试验性的应用T细胞免疫治疗HCMV感染表明,使用CD8+T细胞能抑制HCMV疾病。同时输入CD4+HCMV Th细胞和CD8+HCMV CTL的免疫治疗更为有效。Einsele等应用HCMV T细胞(10~7 HCMV T细胞/m~2),其中包括CD4+HCMV Th细胞和CD8+HCMV CTL,有效的抑制接受HCMV阳性供体的受体HCMV感染。Peggs等培养出CD4+和CD8+T细胞,并输入13位同种异体移植受体,移植术后检测6个月以上,6例患者未用抗病毒药物,而有效的清除HCMV,没有病例诊断出患HCMV感染。
HCMV和血液病
HCMV能直接感染造血祖细胞,抑制祖细胞的复制和分化,还可感染骨髓微环境的基质细胞,造成微环境功能紊乱,干扰造血祖细胞成熟和分化,引起轻重不等的骨髓造血功能损害,研究发现体外培养时HCMV可直接或间接影响造血干/祖细胞的增殖和分化,因此HCMV感染与许多血液病有关,如再生障碍性贫血、ITP、自身免疫性溶血性贫血等。HCMV引起造血功能紊乱的机制复杂,可能因素为:(1)直接感染造血祖细胞,利用的是宿主细胞的DNA聚合酶,抑制其增殖与分化。(2)通过损伤骨髓基质细胞导致造血因子网络紊乱。(3)Fas介导的造血干/祖细胞凋亡增加。(4)病毒介导的自身免疫异常而致自身抗体的产生。HCMV感染可激活多克隆B细胞,产生抗血小板、红细胞自身抗体,导致ITP、AIHA、Evans综合征等自身免疫性疾病。
HCMV和免疫
HCMV可引起机体免疫调节障碍或免疫失衡,T淋巴细胞活化,诱发淋巴组织细胞增生。HCMV能直接感染淋巴系造血祖细胞,抑制免疫细胞的增殖和分化,引起免疫功能紊乱。HCMV在与人类共同进化的过程中,形成了多种分子机制逃避机体免疫系统的监视,从而达到在体内长期潜伏的目的。目前发现HCMV的免疫逃避机制主要包括:①HCMV表达的US2、US3、US6、US11等蛋白可通过翻译后机制下调主要组织相容性抗原复合物MHC-Ⅰ和MHC-Ⅱ类分子的表达,从而抑制抗原递呈细胞的抗原递呈功能和CD8+CTL杀伤作用,同时HCMV还可表达MHC-Ⅰ类分子的同源物,与自然杀伤(NK)细胞的杀伤抑制性受体结合后抑制NK细胞对感染细胞的杀伤作用;②HCMV可表达人细胞因子及细胞因子受体的类似物,干扰宿主自身的细胞因子系统,如HCMV表达的人IL-10类似物HCMV IL-10可抑制单核-吞噬细胞系统,抑制细胞增殖,减少炎性细胞因子的生成,降低宿主的抗感染能力;③细胞凋亡是机体的一种抗病毒防御机制,HCMV感染初期可抑制细胞凋亡,以利于在感染细胞内完成病毒复制和生活周期,而在感染晚期又可促进细胞凋亡,干扰机体抗病毒免疫反应,使病毒避免被机体免疫系统清除:④HCMV感染宿主树突状细胞(dendritic cells,DC)后,可通过改变DC表面黏附分子的表达、抑制T细胞活化信号的传递等多种途径影响其功能,从而在人体内长期潜伏。⑤通过动态观测全身播散型MCMV感染小鼠急性和慢性感染期调节性T细胞(regulatory Tcells,Treg)、辅助性T细胞(helper T cells,Th)1型和2型特异性转录因子Foxp3、T-bet和GATA-3 mRNA的表达变化,发现在MCMV感染急性期,Treg未被诱导增殖活化,机体产生有效Th1和Th2免疫反应以清除病毒,但病毒可诱导Th1/Th2应答下降,并逐渐向Th2方向偏移;进入感染慢性期后,病毒诱导Treg细胞增殖活化显著,进一步抑制Th1和Th2免疫反应,且对Th1的抑制效应更强,提示HCMV在感染慢性期诱导Treg细胞增殖活化是其抑制宿主抗病毒免疫而呈持续慢性感染的重要原因。
HCMV与肿瘤
血清流行病学和肿瘤组织中HCMV DNA的存在显示了HCMV感染可能与多种人体肿瘤的发生有关。HCMV DNA和RNA以及抗原可在30%～60%的人体肿瘤中查到。HCMV感染在宫颈癌(34.3%)中比在宫颈炎(5.3%)中更常见。人乳头瘤病毒DNA阴性患者中,HCMV感染是宫颈癌的明显危险因素。HCMV与宫颈细胞转化的相关性在鼠动物模型中也得到证实。此外HCMV感染也与血管肉瘤、Kaposi′s肉瘤、结肠肿瘤、前列腺腺癌、Wilm′s肿瘤以及常发生于儿童的成神经细胞瘤有关。它的感染在某些疾病中还导致类似于肿瘤的表现,如在HCMV结肠炎、肛部病变、胃部病变、直肠病变等。HCMV通过抑制宿主细胞凋亡来逃避免疫攻击,是其参与肿瘤发生发展的重要机制之一。HCMV感染可干扰宿主的内源性和外源性细胞凋亡途径,调控抑癌基因p53和p73的表达,激活抗凋亡的Ras/Raf/MEK/Erk和PI-3K信号转导通路。HCMV还可通过影响宿主细胞的表观遗传状态(epigenetie state)间接影响基因组功能。表观遗传修饰如DNA甲基化、组蛋白乙酰化和甲基化、RNA相关性沉默等,均可造成基因表达、DNA复制和基因组稳定性的改变,这些改变可能触发抑癌基因或DNA修复基因表达沉默,最终导致肿瘤发生。最近,国外学者建议用“肿瘤调控”(oncomodulation)一词来描述HCMV在肿瘤发生过程中的作用机制,并强调了肿瘤细胞提供的不同于正常细胞的基因环境(信号转导通路、转录因子、抑癌蛋白功能紊乱等)更有利于HCMV的持续感染和发挥肿瘤调控作用。
HCMV疫苗
近几年来国内外对HCMV疫苗进行了广泛研究,已先后有减毒活疫苗、亚单位疫苗、DNA疫苗等多种疫苗问世,在国外有的疫苗甚至已完成Ⅱ期临床试验研究。
1、减毒活疫苗:早期的HCMV疫苗主要是灭活疫苗和减毒活疫苗,但在临床上进行应用的是Towne减毒活疫苗。该疫苗是将Towne野毒株反复传代达125次所得,可诱发机体产生与自然感染相似的体液和细胞免疫反应。但由于Towne减毒活疫苗不能彻底清除机体内的病毒,不能阻止移植后病人的感染,也不能阻止病毒由母体向胎儿传播,且存在病毒回复突变甚至致癌的可能,因此未能获得广泛应用。
2、DNA疫苗:是将编码保护性抗原蛋白的基因置于适当的真核启动子下,构建表达目的基因的真核表达质粒,直接导入宿主细胞内进行表达。DNA疫苗具有表达蛋白时间长、可同时诱导体液和细胞免疫、成本低等突出优点,具有较高的实用性。然而,DNA疫苗也存在许多问题有待解决,如外源DNA是否会整合到宿主基因组中诱发肿瘤或导致宿主细胞功能改变等。
3、亚单位疫苗:由于减毒活疫苗可能存在潜在的致肿瘤性、培养困难、生产技术要求高等缺点,人们开始将目光集中到亚单位疫苗研制上。亚单位疫苗是通过提取HCMV中具有免疫功能的蛋白,或利用DNA重组技术表达这类蛋白,或合成蛋白多肽制成。这类蛋白是病毒的一部分。在HCMV蛋白中,gB和pp65分别诱导机体的体液和细胞免疫,是诱导中和抗体和CTL产生的抗原,因此成为HCMV亚单位疫苗的主要候选蛋白。
(1)介导体液免疫的亚单位疫苗:21世纪最有希望成功的HCMV疫苗是gB亚单位疫苗。gB蛋白是HCMV包膜中最丰富的糖蛋白,占包膜蛋白的50%以上,gB是由907个氨基酸组成的跨膜蛋白,成熟gB由116kD和58 kD两部分通过二硫键结合组成。阳性血清中50%以上抗体为gB特异性。Chiron公司将改造后的gB基因转染中国仓鼠卵巢细胞(CHO),获得稳定的重组细胞系,此疫苗与新型佐剂MF259共同使用,在临床受试者中2次免疫后,可明显产生中和抗体,在第3次免疫后,抗体应答水平明显提高,但是Ⅰ期和Ⅱ期临床观察的结果却非常令人失望,在人体产生的抗体应答短暂,几个月后即降低到保护水平之下。
(2)介导细胞免疫的亚单位疫苗:由于HCMV是典型的胞内感染病毒,细胞免疫在抗HCMV感染中较体液免疫具有更关键的作用。近年来认为HCMVpp65具有强且持久的免疫原性,它是HCMV病毒血症的主要抗原,而且是HCMV特异性细胞毒性T细胞的主要靶蛋白,在细胞免疫中可能发挥重要作用。临床观察表明金丝雀痘病毒编码的pp65能在CMV阳性及阴性的志愿者体内能引起CD4和CD8的T细胞反应,且在体内维持26个月。Tanaka等以杆状病毒为载体,在昆虫细胞内表达重组蛋白,能够刺激机体产生体液免疫,更重要的是能够产生细胞免疫。另外调节蛋白IE1(UL123)、磷蛋白pp150(UL32)、pp50/52(UL44)、pp71(UL82)及pp28(UL99)也是CTL应答的最佳诱导者,复合的亚单位疫苗可诱导更强烈的T细胞应答。不久前提出用MVA为载体表达多抗原成分疫苗(pp65、pp150和IE),可能诱导更广泛人群的免疫应答。
4、病毒载体DNA疫苗:病毒载体DNA疫苗即可用于防治,又能用于治疗疾病。研究比较多的两种“非复制型”载体是复制缺陷型金丝雀痘苗载体和复制缺陷型腺病毒载体。两种载体能以可靠的方式表达外源抗原,诱生机体的体液和细胞免疫,具有较广泛的保护性免疫力。Zhongde Wang等根据前人的经验,利用经修饰的安格拉痘苗病毒株MVA表达了可溶性的gB片段gB680(gB680-MVA),免疫小鼠,发现在小鼠体内可产生高滴度的抗gB680特异抗体,抗体滴度基本与人自然感染HCMV相似,抗体在小鼠体内存留的时间为6个月以上。体外实验此抗体能中和多种型别HCMV。为了进一步增强免疫效果,Zhongde Wang等用经过修饰的MVA表达了泛素化的pp65抗原,经泛素化后的抗原在体内产生的特异的CTL的浓度更高。然而这些疫苗只是验证了其初步的安全性,而在体内是否真正能够预防HCMV的感染,还有待进一步的实验验证。
上述所研究的HCMV疫苗通常是在重组疫苗载体中编码一个全长的HCMV抗原基因或显性抗原基因,此类疫苗虽取得了一定的效果,但存在免疫原性弱、免疫范围局限、免疫反应小等缺点,不能非常有效地预防或抑制病毒的感染,主要是因为:(1)抗原分子中能引起免疫应答的部分是与宿主HLA分子特异性结合的抗原表位(epitope),是诱导机体免疫应答的必需的最基本的结构和功能单位;而HCMV基因组全长230kb,抗原表位极其复杂,单一抗原分子所含的局限抗原表位引起的机体免疫应答较弱,无法完全预防或消除HCMV感染;而且由于抗原表位是半抗原,免疫原性弱等特点,单独应用这些抗原表位很难刺激机体产生免疫应答反应,需要将这些表位连接到一定的载体上(2)不同HCMV抗原分子是通过不同HLA基因位点提呈来激活免疫应答的,然而人群中HLA遗传背景复杂,单一抗原分子难以覆盖HLA位点不同的整个人群。虽有学者考虑使用多抗原疫苗,但载体的有限容量及大分子蛋白可能造成的免疫病理反应亦限制了多个抗原在同一载体中的应用。如何在有限的载体容量下大大增强疫苗的免疫原性,而且同时制备的疫苗免疫应答人群覆盖面广,是当前HCMV疫苗研究的难点。
将抗原表位构建于合适的载体中,再辅以必需的侧翼序列或免疫调控序列,可诱导针对所选表位的特异性免疫反应,其最大优势为选用最佳和最具免疫保护潜力的抗原表位来诱导特异性免疫应答,尽量减少无关、干扰或抑制序列可能产生的负面作用。近年来,国外有不少学者利用表位疫苗进行病毒、细菌、寄生虫等疾病的免疫应答研究,取得了一定成绩。Ciernik等将HIV gp120的CTL单一表位疫苗p18ⅢB构建于真核表达载体pRc/CMV中,于耳后和皮下单次免疫BALB/c小鼠后,结果诱生了针对p18ⅢB的特异性免疫应答反应。Meyer等利用针对B细胞表位的HCMV gp116表位疫苗进行免疫效果评价,结果发现,可诱导相应的HCMV的中和抗体,对防治HCMV感染有一定作用。由于HLA基因的高度多态性,受特定HLA基因限制的单表位疫苗通常不能在所有个体中引起预期的免疫反应,因而有学者尝试将多种表位疫苗串联,以期望产生更强的免疫应答。多表位疫苗与单一表位疫苗相比具有以下优势:能克服HLA的遗传限制,精心组合的多表位疫苗可以被多种遗传背景的HLA分子识别并结合,从而得到高效提呈;其次多表位疫苗可以有效避免病原微生物的变异和免疫反应中的某些不利因素;再次多表位疫苗在诱导细胞免疫方面有独特的优势。Rohrlich等选择HCMV的嵌合蛋白IE1-pp65作为表位疫苗进行研究,结果显示,两串联表位表达良好,都可在体内激活和扩增HCMV特异性的CD8+CTL细胞,但由于二者都是被HLA-A2所共同识别的T表位,虽有较好的免疫原性,但相同表位疫苗的串联优势并不明显。
国外HCMV表位疫苗研究大多是单一表位或针对相同HLA位点的同源表位串联,免疫反应单一,协同作用不明显,不能充分发挥接种个体的免疫能力;理想的表位疫苗应具有多能性,诱导机体全面的免疫应答反应,但目前尚未见能同时有效引起细胞免疫和体液免疫应答的HCMV串联表位疫苗。同时国外对HCMV表位疫苗的研究针对的免疫覆盖人群为欧洲及非洲人群,他们的HLA遗传背景与中国人群HLA分布情况不同,其研制的表位疫苗在中国人群很难被HLA分子提呈,不能引起有效的保护性免疫应答。目前国内尚未见有关HCMV表位疫苗的报道,且中国人口和民族的空间分布极度不均匀,人群HLA基因频率和单倍型频率的空间分布呈现高度异质性,因而研制针对中国人群HLA分布特点的免疫效果强大的HCMV串联表位疫苗是我国HCMV防治领域的关键所在。
综上所述,尽管HCMV感染普遍存在,呈无症状感染,但仍有高危人群罹患严重的HCMV疾病,包括器官移植、造血干细胞移植病人,HIV患者和胎儿。由于免疫可以改善HCMV疾病的严重性,已经努力了30年发展HCMV疫苗应用于高危人群,然而,尽管有了这些努力仍然没有可以即将出现的HCMV疫苗获得批准上市。没有批准上市HCMV疫苗的原因是复杂的,但是有几个关键的原因是:(1)宿主的保护性免疫还不清楚;(2)应该纳入HCMV疫苗的病毒蛋白不能确定;(3)临床试验大部分针对免疫缺陷病人,和免疫保护作用低下易患先天性HVMC感染的胎儿;(4)HCMV疫苗接种的目标人群仍然不清楚;(5)最后但最重要的是关于HVMC感染的教育问题很不充分,特别是育龄妇女和下级阶层人群。
设计理念
第一部分覆盖中国人群HLA特异的HCMV串联表位的确定及预测
目的:覆盖中国人群HLA特异的HCMV串联表位的确定及预测。
方法:
1、根据山东大学卫生系统计教研室薛付忠老师建立的中国HLA—Ⅰ积累表型频率(CPF)空间预测系统依次预测A2,A24,A1,A3,A11,A68,B44,B7,A23,A26,B35,B38,B8,B27十四个基因位点在中国人群的覆盖率(CPF值),评价和预测HLA特异的HCMV串联表位核酸疫苗在中国不同地理位置上人群内的免疫效果;
2、利用从1996年以来发表在PubMed上被SCI收录的23篇研究HCMV表位的文献,从中选择能针对HCMV多种蛋白抗原的多个CTL表位、Th表位和B细胞表位,能够激发机体的细胞免疫应答和体液免疫应答,且能与HLA-Ⅰ分子和HLA-Ⅱ分子的基因位点稳定结合的表位;
3、利用SYFPEITHI MHC表型及表位预测系统和MAPPP蛋白酶体降解预测系统对所选表位进行表位肽预测和MAPPP蛋白酶体切割-表位生成预测;
4、从PubMed Nucleotide NC_001347.人巨细胞病病毒AD169株各蛋白的基因序列中找出各表位相对应的核苷酸序列并核对,将各表位的核苷酸序列按等位基因的频率分布顺序排列起来,串联成一条核苷酸序列;
5.根据Kozak规则引入Kozak序列以获得HCMV串联表位核苷酸序列在真核表达载体中的高效表达;
6.将HCMV串联表位的核苷酸序列输入DNAMAN软件,根据输出的酶切位点结果结合所要构建入穿梭载体的多克隆位点,确定串联表位核苷酸序列两端的酶切位点。
结果:
1、HLA特异的HCMV串联表位核酸疫苗在中国人群的覆盖频率可达90%以上;
2、我们选择了HCMV的十五种抗原蛋白pp28,pp50,pp65,pp150,pp71,gH,gB,IE-1,IE-2,US2,US3,US6,US11,UL16,UL18,分别在病毒的黏附、复制、组装、免疫逃逸及再激活过程中表达,在这些抗原中选取了84个表位,其中针对HLA-Ⅰ分子基因位点的CTL表位76个,针对HLA-Ⅱ分子基因位点的Th表位7个,能激发机体产生中和抗体的B细胞表位1个,通过启动针对上述抗原表位的细胞和体液免疫应答,理论上可以在各个阶段控制病毒的复制,防止HCMV疾病的形成;
3、覆盖中国人群HLA特异的HCMV 83个串联表位的SYFPEITHI预测分数均在14分以上,多数在20-30分之间,MAPPP预测分数均在0.500以上;
4、引入Kozak规则,加入6个His鉴定标签,利用PubMedNucleotide NC_001347.Human herpesvirus 5 strain AD169 HCMV基因组得出了覆盖中国人群HLA特异的HCMV串联表位核苷酸序列;
5、输入DNAMAN软件,确定了覆盖中国人群HLA特异的HCMV串联表位核苷酸序列两端的酶切位点是NheⅠ和NotⅠ酶切位点,可以插入穿梭载体pHMCMV5载体的多克隆位点。
结论:
1、根据山东大学卫生系统计教研室薛付忠老师建立的中国HLA—Ⅰ积累表型频率(CPF)空间预测系统,对所选HCMV表位结合的14个HLA基因位点及其组合进行了预测,显示A2,A24,A1,A3,A11五个优势基因位点的CPF值是88.1%,;A2,A24,A1,A3,A11,A68,B44,B7,A23,A26,B35,B38,B8,B27十四个基因位点的CPF值是92.1%,显示HLA特异的HCMV串联表位核酸疫苗在中国人群的覆盖频率达90%以上,预测了HLA特异的HCMV串联表位核酸疫苗在中国不同地理位置上人群内的免疫效果,理论上其免疫效果可达90%以上。
2、我们选择了HCMV的十五种抗原蛋白pp28,pp50,pp65,pp150,pp71,gH,gB,IE-1,IE-2,US2,US3,US6,US11,UL16,UL18,分别在病毒的黏附、复制、组装及再激活过程中表达,在这些抗原中选取了84个表位,其中针对HLA-Ⅰ分子基因位点的CTL表位76个,针对HLA-Ⅱ分子基因位点的Th表位7个,能激发机体产生中和抗体的B细胞表位1个,通过启动针对上述抗原表位的细胞和体液免疫应答,理论上可以在各个阶段控制病毒的复制,防止HCMV疾病的形成。
3、应用DNAMAN软件、Kozak规则及PubMed Nucleotide NC_001347.Humanherpesvirus 5 strain AD169 HCMV基因组得出了覆盖中国人群HLA特异的HCMV串联表位核苷酸序列,在起端加上了Kozak序列,可以获得串联表位核苷酸序列在真核表达载体中的高效表达;末端加上了6个His标签序列,可以通过6个His抗体应用Western blot及细胞免疫组化检测HCMV串联表位的表达;起始两端加入了NheⅠ和NotⅠ酶切位点,可以插入穿梭载体pHMCMV5载体的多克隆位点,为制备中国人群HLA特异的HCMV串联表位腺病毒载体核酸疫苗打下了基础。
第二部分中国人群HLA特异的HCMV串联表位腺病毒核酸疫苗的制备
目的:HCMV感染是引起移植患者和新生儿高发病率和死亡率的原因,世界范围的医学会组织指出应优先发展控制HCMV疾病的疫苗,尽管做出了很大的努力但目前仍无被临床许可的疫苗;嵌合型复制缺陷型腺病毒载体Ad5F35比传统的Ad5型具有更好的趋向性和更高的容纳外源基因的能力,在多种细胞上高效表达外源基因。本实验应用嵌合型Ad5F35构建多重HLA-Ⅰ、HLA-Ⅱ分子限制CTL、Th多表位核酸疫苗Ad5F35-CTL·Th以及针对体液免疫应答的HCMV线性B细胞表位核酸疫苗Ad5F35-AD-1。
方法:
1、采用重叠区扩增基因拼接法(Gene splicing by overlap extension,SOEing)和PCR技术合成HCMV串联T细胞表位全长基因;
2、应用PCR技术从HCMV AD169全基因组扩增线性B细胞表位gB AD-1的基因;
3、利用同源重组技术从穿梭载体,将CTL·Th和AD-1基因分别插入pHMCMV5的NheⅠ/NotⅠ和KpnⅠ/AflⅡ酶切位点,构建穿梭载体pHMCMV5-CTL·Th和pHMCMV5-AD-1,应用NheⅠ/NotⅠ和KpnⅠ/AflⅡ酶切分析和测序分析鉴定目的载体;
4、利用I-CeuⅠ/PI-SceⅠ双酶切和体外重组技术将穿梭载体上包含CMV启动子、目的基因、SV40 polyA尾的表达盒插入腺病毒表达载体pAd5F35,构建重组腺病毒表达载体Ad5F35-CTL·Th和pAd5F35-AD-1,应用XhoⅠ和I-CeuⅠ/PI-SceⅠ酶切分析和测序分析鉴定目的载体;
5、应用PacI线性化重组腺病毒表达载体Ad5F35-CTL·Th和pAd5F35-AD-1,用脂质体2000转染HEK293,包装重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1,待培养10天出现CPE效应时收取病毒;
6、应用HEK293细胞大量扩增重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1,扩增至五代,应用快速腺病毒感染性滴度(TCID50)检测试剂盒测定大量扩增后的的重组腺病毒的滴度;
7、用重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1分别转染HEK293细胞,利用RT-PCR和Western blot技术分析CTL·Th和AD-1在mRNA和蛋白水平的表达;
8、用重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1分别转染PBMC,利用细胞免疫化学和台盼兰染色分析重组腺病毒在PBMC上的表达和对PBMC活性的影响。
结果:
1、采用SOEing和PCR技术成功合成了HCMV串联T-细胞表位全长基因;
2、在HCMV全基因组DNA中成功扩增了线性B细胞表位gB AD-1目的基因;
3、成功构建了穿梭载体pHMCMV5-CTL·Th和pHMCMV5-AD-1,酶切鉴定重组穿梭载体均有目的基因;
4、成功构建了重组腺病毒表达载体Ad5F35-CTL·Th和pAd5F35-AD-1,酶切鉴定重组腺病毒均有目的基因;经测序后目的核苷酸序列无碱基突变。
5、成功包装出重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1,转染HEK293细胞后观察到细胞病变效应(CPE),平行对照Ad5F35-GFP有绿色荧光表达。
6、应用HEK293细胞大量扩增重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1,扩增了五代,每轮扩增只需2-3天。
7、大量扩增后重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1的滴度是2.5×10~9IU/L。
8、应用重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1转染HEK293细胞,可以观测到目的基因CTL·Th和AD-1在mRNA水平均有表达,目的基因CTL·Th和AD-1在蛋白水平亦均有表达,CTL·Th在蛋白水平表达90kda左右的目的带,AD-1在蛋白水平表达55kDa左右的目的带。
9、应用重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1转染外周血单个核细胞(PBMC),分别在转染第1、3、5、7、10天应用台盼兰染色观察PBMC的细胞活性,在转染后10天PBMC仍保持90%以上的活性,说明重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1毒性极低。
10、重组腺病毒Ad5F35-CTL·Th在PBMC的胞膜、胞浆均有表达,重组腺病毒Ad5F35-AD-1在PBMC的胞膜有表达,说明重组腺病毒Ad5F35-CTL·Th和Ad5F35-AD-1可以在PBMC中高效表达。
结论:
1、我们成功制备出中国人群HLA特异的HCMV串联T细胞表位腺病毒载体核酸疫苗Ad5F35-CTL·Th和针对体液免疫应答的HCMV线性B细胞表位腺病毒载体核酸疫苗Ad5F35-AD-1,为下一步的免疫应答实验打下了基础。
2、重组腺病毒疫苗Ad5F35-CTL·Th和Ad5F35-AD-1可以在PBMC高效表达目的蛋白,且不影响PBMC的活性。
第三部分中国人群HLA特异的HCMV串联表位腺病毒核酸疫苗体外免疫效果评价
目的:过继免疫包括体液免疫和细胞免疫在控制HCMV感染方面起关键作用。本实验评价中国人群HLA特异的HCMV串联表位腺病毒核酸疫苗的体外免疫效果。
方法:
1、用EBV转化PBMC,建立类淋巴母细胞系(lymphoblastoid cell lines-LCL);
2、应用HCMV多表位腺病毒载体疫苗Ad5F35-CTL·Th刺激HCMV健康携带者的外周血单个核细胞(PBMC),体外扩增抗原特异性CTL;
3、应用抗原特异性CTL进行细胞毒试验,检测CTL的杀伤活性;
4、应用ELISPOT实验检测Ad5F35-CTL·Th刺激HCMV健康携带者的PBMC后和抗原特异性CTL分泌IFN-γ的情况;
5、细胞内染色法检测Ad5F35-CTL·Th刺激HCMV健康携带者的PBMC后CD8+T细胞IFN-γ的表达。
结果:
1、利用EBV成功转化了PBMC,建立了2株类淋巴母细胞系(lymphoblastoid celllines-LCL);
2、应用HCMV多表位腺病毒载体疫苗Ad5F35-CTL·Th刺激HCMV健康携带者的PBMC,在体外高效扩增了抗原特异性CTL;
3、Ad5F35-CTL·Th编码的HCMV表位可以被内源性提呈;
4、经Ad5F35-CTL·Th刺激后PBMC大量分泌IFN-γ,体外扩增抗原特异性CTL亦大量表达IFN-γ;
5、细胞内染色法检测Ad5F35-CTL·Th刺激PBMC后CD8+T细胞有IFN-γ的表达。
结论:
1、本实验显示了多表位抗原提呈系统扩增抗原特异性T细胞的有效性。
2、本试验显示了HCMV多表位腺病毒核酸疫苗可以高效扩增免疫显性和亚显性的抗原特异性T细胞。
3、实现了单个表达结构可以扩增免疫显性和亚显性的抗原特异性T细胞,较之以前扩增不同的抗原特异性T细胞需要不同的刺激策略具有强大的优势。
4、我们的多表位重组技术可以有望适用于不同疾病的临床过继免疫治疗。
Background
Human cytomegalovirus(HCMV) is the most complex of the eight human herpesvirus species and has a 235-kb double-stranded DNA that encodes 165 genes.HCMV infects approximately 40-60%of the developed world's population.Primary infection in healthy hosts is usually asymptomatic,and the virus persists in CD33 progenitors expressing markers of dendritic and myeloid lineage as major reservoirs of latent infection virus without any apparent clinical symptoms.HCMV-associated clinical disease has been recognized in three populations:Neonates with immature immune systems,transplant recipients with impaired immune systems due to the use of drugs that suppress rejection and HIV-infected patients with impaired immune systems due to the decline of CD4+T cells
HCMV is the most important viral pathogen affecting transplant recipients,including both solid organ transplant and allogeneic hematopoietic stem cell transplant recipients whose immune systems are suppressed by drugs used to prevent graft rejection.It is noted that HCMV is reported to infect not only stroma and epithelial cells but also hematopoietic cells including CD34+ stem cells,monocytes,and dendritic cells. Late-onset cytomegalovirus disease in hematopoietic stem cell transplant recipients(later than day 100 after transplantation) remains a major cause of morbidity and mortality, despite the introduction of new antiviral agents.Congenital HCMV infection is the most common intrauterine infection and 0.3-2.4%of neonates are born with HCMV infection worldwide.Of the neonates with congenital HCMV infection,10%have symptoms of irreversible CNS involvement in the form of microcephaly,encephalitis,seizures, deafness,upper-motor neuron disorders and psychomotor retardation.
Intensive study over the last 30 years has demonstrated that both innate and adaptive immunity play important roles in controlling latent HCMV infection.The innate immune response,mainly natural killer cells,may shape or augment the adaptive immune response against HCMV infection.NK cells may act by secreting IFN-γ,which can facilitate the expansion of antigen-specific helper T cells to control HCMV infection.The adaptive immunity,including both humoral and cellular immunity,plays a crucial role in controlling HCMV infection.Extensive analysis of humoral immune responses to HCMV has revealed that response is directed toward multiple viral protein and that virus-protection antibodies are directed primarily to the envelope glycoprotein of HCMV with glycoprotein B(gB) as a dominant target antigen.HCMV-specific T-cell immunity is the most important adaptive immune component responsible for suppressing HCMV and keeping it at latency after primary infection.Studies showed that pp65 and IE-1 constituted 40%of the total CD8+ T-cell responses and 60%of these T-cell responses are directed towards other antigens,such as pp28,pp150,pp71 and US proteins.Compared with the CD8+ T cells,CD4+ T cells were generally thought to have an indirect role in controlling HCMV infection by providing helper signals for the generation of antibodies and the maintance of CD8+ T-cell memory.
Taken together,our increasing understanding of immune responses against HCMV infection has firmly established that activation of both humoral and cellular immunity is crucial for successful HCMV vaccine.
PartⅠPrediction and Decision of HLA-specific HCMV Polyepitope in Chinese People
Objective:Prediction and decision of HLA-specific HCMV polyepitope in Chinese people.
Methods:
1.According to the forecasting system on spatial coverage of cumulative phenotypic frequency(CPF) of HLA-I of professor XUE Fu-zhong in Institute of Epidemiology and Medical Statistics of Medical College of Shandong University,the study forcasted the CPF of 14 HLA allel sites including A2,A24,A1,A3,A11,A68,B44,B7,A23,A26, B35,B38,B8,B27 in Chinese people and evaluated immune activity of HLA-specific HCMV polyepitope nucleotide vaccine in different geographical location of Chinese people.
2.Utilizating 23 SCI literature about HCMV epitope which have been published in PubMed since 1996,the study selected multiple CTL epitopes,Th epitopes and B cell epitopes in different HCMV protein antigens which can induce cellullar immunologic response and humoral immunoresponse and stably bind classⅠand classⅡMHC molecules.
3.Through SYFPEITHY combined with MAPPP algorisms,the selected epitopes were predicted for the specific value of peptide and proteasomal cleavage site.
4.The nucleotide sequences of the selected epitopes were found in genome of HCMV AD169 strain and verified in PubMed Nucleotide NC_001347.Furthermore,the nucleotide sequences of these epitope were cascaded a single nucleotide sequence according to frequency distribution of HLA allel.
5.According to Kozak rule,Kozak sequence was put into the nucleotide sequence of HCMV polyepitope which can induce highly efficient expression in eukaryotic expression vector.
6.The nucleotide sequence of HCMV polyepitope was input DNAMAN software. Enzyme cleavage sites of the nucleotide sequence were decided in order to construct shuttle vector in the next experiment.
Results:
1.The coverage of HLA-specific HCMV polyepitope nucleotide vaccine in Chinese people has reached above 90%.
2.The epitopes included in this polyepitope were derived from 15 different viral proteins(pp28,pp50,pp65,pp150,pp71,gH,gB,IE-1,IE-2,US2,US3,US6,US11, UL16 and UL18) involved in virus attachment,replication,assembly,reactivation and immune escape from the latent phase,all of which are crucial stages in the development of HCMV disease.There were 76 CTL epitopes,7 Th epitopes and 1 B cell epitope in selected epitopes.In theory,these epitopes can induce cellular immunologic response and humoral immunoresponse and prevent the development of HCMV disease.
3.Through the SYFPEITHI web site,the predicted values of the selected epitopes have reached above 14 scores,almost between the 20 and 30 scores.Through the MAPPP server,the predicted proteasomal cleavage values of the selected epitope have reached above 0.5 score.
4.After introducing Kozak rule,adding 6×his identify tag and utilizating genome of HCMV AD 169 strain in PubMed Nucleotide NC_001347,the nucleotide sequence of the polyepitope has been decided.
5.In order to construct shuttle vector in the next experiment,the NheⅠand NotⅠenzyme cleavage site,of the nucleotide sequence have been decided after the nucleotide sequence of HCMV polyepitope was input DNAMAN software.
Conclusions:
1.According to the forecasting system on spatial coverage of cumulative phenotypic frequency(CPF) of HLA-Ⅰ,the CPF of 14 HLA allel sites including A2,A24,A1,A3, A11,A65,B44,B7,A23,A26,B35,B35,B8 and B27 in Chinese people has reached 92.1%.It has exhibited the coverage of HLA-specific HCMV polyepitope nucleotide vaccine in Chinese people has reached above 90%.It also has evaluated immune effect of HLA-specific HCMV polyepitope nucleotide vaccine in different geographical location of Chinese people.In theory,immune effect of HLA-specific HCMV polyepitope nucleotide vaccine can reach above 90%.
2.The epitopes included in this polyepitope were derived from 15 different viral proteins(pp28,pp50,pp65,pp150,pp71,gH,gB,IE-1,IE-2,US2,US3,US6,US11, UL16 and UL18) involved in virus attachment,replication,assembly,reactivation and immune escape from the latent phase,all of which are crucial stages in the development of HCMV disease.There were 76 CTL epitopes,7 Th epitopes and 1 B cell epitope in selected epitopes.In theory,these epitope can induce cellular immunologic response and humoral immunoresponse and prevent the development of HCMV disease.
3.After introducing Kozak rule,utilizating genome of HCMV AD169 strain in PubMed Nucleotide NC_001347 and inputting DNAMAN software,the nucleotide sequence of the polyepitope has been decided.Kozak sequence has been added in the upstream that can induce high efficient expression in eukaryotic expression vector.Six×His identify tag has been added in the downstream that can be used in Western blot and cell immunochemistry experiment.The output NheⅠand NotⅠenzyme cleavage site can be used to construct shuttle vector in the next experiment.
PartⅡPreparation of Novel Vaccine against HCMV Based on Ad5F35 Adenovirus Vector Expressing HLA-specific polyepitope nucleotide
Objective:HCMV disease accounted for the high morbidity and mortality in transplant patients and new born babies.The worldwide institute of medicine has assigned the highest priority for a vaccine to control HCMV disease.In spite of numerous attempts, successful licensure of a HCMV vaccine formulation remains elusive.The modified replication-deficient adenoviral vector AdSF35 possesses more expanded tropism and larger foreign DNA package ability than the conventional Ad5 vectors without affecting the viral growth rate and titer.It exhibits high transduction efficiency in many human cells.Here we have developed two novel chimeric vaccine strategy based on AdSF35 which encode multiple HLA classⅠ&Ⅱrestricted CTL and Th epitopes from HCMV as a contiguous polypeptide and encode the high conserved predominant B cell epitope gB AD-1.
Methods:
1.Using SOEing and PCR technology to synthesize the total length CTL·Th gene of HCMV multiple T cell epitope.
2.The AD-1 gene was amplified from AD169 genomic DNA by polymerase chain reaction(PCR) with the oligonucleotide primer.
3.The CTL·Th gene and AD-1 amplicon were ligated the NheⅠ/NotⅠand KpnⅠ/AflⅡsites of shuttle plasmid pHMCMV5 respectively.The desired plasmids were identified by restriction analysis and nucleotide sequence analysis.
4.The expression cassette,comprising the CMV promoter,CTL·Th gene/AD-1 and an SV40 polyA signal,was excised from the shuttle plasmids pHMCMV5-CTL·Th or pHMCMV5-AD-1 with I-CeuI and PI-SceI and ligated into adenoviral backbone vector pAd5F35 cleaved with the same enzymes.The presence of expression cassettes was verified by digestion with XhoⅠandⅠ-CeuⅠ/PI-SceⅠ.
5.pAd5F35-CTL·Th and pAd5F35-AD-1 were linearized with PacI and transfected into HEK293 cells using lipofectamine~(TM) 2000.Transfected HEK293 cells (HEK293-Ad5F35-CTL·Th or HEK293-Ad5F35-AD-1) were incubated at 37℃for 7-10 days until maximal virus cytopathic effect(CPE) was observed.Then recombinant adenovirus was harvested.
6.The recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1 were amplified using HEK293 cells until the 5~(th) passage.The titer of the large-scale recombinant adenovirus was established by plaque assay according to adenovirus TCID50 testing kit protocol.
7.The expression of CTL·Th and AD-1 was identified by using transfected HEK293 cells.To confirm CTL·Th and AD-1 mRNA expression,RT-PCR analysis were conducted.To confirm AD-1 protein expression,western blot analysis was conducted.
8.The expression of CTL·Th and AD-1 in PBMCs infected with Ad5F35-CTL·Th and Ad5F35-AD-1 was analyzed by immunocytochemistry,and cell viability were determined by trypan blue staining.
Results:
1.Using SOEing and PCR technology,the total-length gene of HCMV multiple T cell epitopes has been successful synthesized.
2.AD-lgene has been successfully amplified from the genome of HCMV AD169 strain.
3.Shuttle plasmids pHMCMV5-CTL·Th and pHMCMV5-AD-1 have been successfully constructed.After enzyme digestion identification and DNA sequencing, insertion of the gene was proved correct.
4.Recombinant adenovirus vectors pAd5F35-CTL·Th and pAd5F35-AD-1 have been successfully constructed.After enzyme digestion identification and DNA sequencing,the expression of the gene was proved correct.
5.The recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1 have been successfully packaged.Full CPE was observed for recombinant virus after transfecting HEK293.
6.The recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1 were amplified using HEK293 cells until the 5~(th) passage.Each cycle of amplification only needed 2-3 days.
7.The titer of the large-scale recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1 virus preparation were 2.5×10~9 PFU by plaque assay.
8.After transfecting HEK293 cells using recombinant adenovirus Ad5F35-CTL·Th and Ad5F35-AD-1,CTL·Th and AD-1 expression were verified at the level of mRNA protein.
9.After PBMCs were transfected 10 day using Ad5F35-CTL·Th and Ad5F35-AD-1, 90%of cells retained viable.It has shown no any adverse effects of the recombinant adenovirus.
10.Strong expression of CTL·Th polyepitope antigen and AD-1 were recorded in PBMCs infected with Ad5F35-CTL·Th and Ad5F35-AD-1.The typical pattern of staining demonstrates that recombinant CTL·Th polyepitope localizes to the cell membrane and cytoplasm,while the peripheral pattern of staining demonstrates that recombinant AD-1 localizes to the cell membrane.
Conclusion:
1.Two novel vaccines based on a replication-deficient adenovirus that encode HLA-specific HCMV polyepitope nucleotide vaccine(referred to as Ad5F35-CTL·Th) and a single B cell epitope gB AD-1(referred to as Ad5F35-AD-1) were successfully constructed.
2.Ex vivo stimulation of PBMC with Ad5F35-CTL·Th and Ad5F35-AD-1 consistently showed highly efficient expression of CTL·Th polyepitope antigen and epitope AD-1 protein.The viability of PBMC was not affected.It will be useful for HCMV novel vaccine development.
PartⅢImmune Activity Evaluation of Novel Vaccine against HCMV Based on Ad5F35 Adenovirus Vector Expressing HLA-specific polyepitope nucleotide
Objective:The adaptive immunity,including both humoral and cellular immunity, plays a crucial role in controlling HCMV infection.This experiment is to evaluate immune activity of this novel vaccine against HCMV based on a chimeric Ad5F35 adenovirus vector expressing HLA-specific polyepitope nucleotide.
Methods:
1.Lymphoblastoid cell lines-LCL is established as antigen presenting cell by using EBV to transform PBMC.
2.In vitro antigen-specific CTL was generated from a panel of healthy virus carriers by stimulating PBMC with adenoviral chimeric vaccine Ad5F35-CTL·Th.
3.These CTL clone lines were screened for cytotoxic activity on a panel of autologous target cells that were either sensitized with synthetic peptides or infected with Ad5F35-CTL·Th.
4.The ELISPOT assay was used to assess whether adenoviral chimeric vaccine Ad5F35-CTL·Th could stimulate a memory response,as measured by the production of IFN-γ,in PBMC from a large panel of seropositive donors.
5.The Intracellular cytokine staining(ICS) assay was used to measure the levels of specific CD8+T cells elicited by Ad5F35-CTL·Th stimulating PBMC from healthy seropositive individuals.
Results:
1.Two EBV-transformed-LCLs have been successfully established.
2.In vitro stimulation with this adenoviral chimeric vaccine Ad5F35-CTL·Th rapidly expanded multiple antigen-specific human CD8+T-cells from healthy virus carriers.
3.The HCMV epitopes encoded by this polyepitope were endogenously expressed and processed by human cells.
4.The production of IFN-γhas been largely secreted from PBMC stimulated by Ad5F35-CTL·Th or expanded antigen-specific CTL.
5.The levels of specific CD8+T cells have been strongly elicited by Ad5F35-CTL·Th stimulating PBMC from healthy seropositive individuals.
Conclusion:
1.This study shows the effectiveness of a polyepitope antigen presentation system for reproducible expansion of antigen-specific T cells from immunocompetent settings.
2.It also shows that an adenovirus based polyepitope is highly efficient in expanding both immunodominant and subdominant antigen-specific T cells.
3.The comparable expansion of both immunodominant and subdominant T cell responses using a single expression construct was an unexpected result,as previous studies have shown that the different T cell populations require distinct stimulation strategies.
4.The data presented here emphasize the strength and adaptability of the polyepitope-based approach for clinical translation and may lead to significant advances in the application of adoptive immunotherapy to a wide range of diseases.It will be useful for HCMV novel vaccine development.
引文
[1]章咏裳.肾移植术后巨细胞病毒感染.中华器官移植杂志 2000;21(4):248-249.
[2]Gaytant MA,Steegers EA,Semmekrot BA,Merkus HM,Galama JM.Congenital cytomegalovirus infection:review of the epidemiology and outcome.Obstet Gynecol Surv 2002;57:245-256.
[3]Arvin AM,Fast P,Myers M,Plotkin S,Rabinovich R.Vaccine development to prevent cytomegalovirus disease:report from the National Vaccine Advisory Committee.Clin Infect Dis 2004;39:233-239.
[4]Varnum SM,Streblow DN,Monroe ME,Smith P,Auberry KJ,Pasa-Tolic L,et al.Identification of proteins in human cytomegalovirus(HCMV) particles:the HCMV proteome.J Virol 2004;78(20):10960-10966.
[5]Melnick JL,Hu C,Burek J,Adam E,DeBakey ME.Cytomegalovirus DNA in arterial walls of patients with atherosclerosis.J Med Virol 1994;42:170-174.
[6]Sinclair J,Sissons P.Latent and persistent infections of monocytes and macrophages.Intervirology.1996;39(5-6):293-301.
[7]Lecointe D,Hery C,Janabi N,Dussaix E,Tardieu M.Differences in kinetics of human cytomegalovirus cell-free viral release after in vitro infection of human microglial cells,astrocytes and monocyte-derived macrophages.J Neurovirol 1999;5(3):308-317.
[8]Kline JN,Hunninghake GM,He B,Monick MM,Hunninghake GW.Synergistic activation of the human cytomegalovirus major immediate early promoter by prostaglandin E2 and cytokines.Exp Lung Res1995;24(1):3-16.
[9]Walmsley S,O'Rourke K,Mortimer C,Rachlis A,Fong I,Mazzulli T.Predictive value of cytomegalovirus(CMV) antigenemia and digene hybrid capture DNA assays for CMV disease in human immunodefieiency virus-infected patients.Clin Infect Dis 1998;27(3):573-581.
[10]Nguyen Q,Champlin R,Giralt S,Rolston K,Raad I,Jacobson K,et al.Late cytomegalovirus pneumonia in adult allogeneic blood and marrow transplant recipients.Clin Infect Dis 1999;28(3):618-623.
[11]王霞,张蓓,罗长缨,陈静,沈立松.儿童异基因造血干细胞移植术后感染的实验观察.中国小儿血液与肿瘤杂志 2006;11(3):107-108.
[12]秦茂权,谢正德,王彬,周翱,朱光华,冯涛,吴敏嫒.儿童造血干细胞移植后巨细胞病毒感染的临床研究.临床儿科杂志 2007;25(7):541-543.
[13]Kantrow SP,Hackman RC,Boeckh M,Myerson D,Crawford SW.Idiopathic pnemonia syndrome:changing spectrum of lung injury after marrow transplantation.Transplantation 1997;63(8):1079-1086.
[14]Wagner JE,Eapen M,MacMillan ML,Harris RE,Pasquini R,Boulad F,et al.Unrelated donor bone marrow transplantation for the treatment of Fanconi anemia.Blood 2007;109(5):2256-2262.
[15]Pellegrin I,Garrigue I,Ekouevi D,Couzi L,Merville P,Merel P,et al.New molecular assay to Predict occurrence of cytomegalovirus disease in renal transplant recipients.J Infect Dis 2000;182:36-42.
[16]Temperton NJ.DNA vaccines against cytomagalovirus:current progress.Int J Antimicrob Agents 2002;19(3):169-172.
[17]Bernstein DI,Schleiss MR,Berencsi K,Gonczol E,Dickey M,Khoury P,et al.Effect of previous or simultaneous immunization with canarypox expressing cytomegalovirus(CMV)glycoprotein B(gB)on response to subunit gB vaccine plus MF59in healthy CMV-seronegative adults.J Infect Dis 2002;185(5):686-690.
[18]Meijer E,Boland GJ,Verdonck LF.Prevention of cytomegalovirus disease in recipients of allogeneic stem cell transplants.Clin Microbiol Rev 2003;16(4):647-657.
[19]Li CR,Greenberg PD,Gilbert MJ,Goodrich JM,Riddell SR.Recovery of HLA-restricted cytomegalovirus(CMV)-specific T-cell responses after allogeneic bone marrow transplant:correlation with CMV disease and effect of ganciclovir prophylaxis.Blood 1994;83(7):1971-1979.
[20]Peggs KS,Verfuerth S,Pizzey A,Khan N,Moss P,Goldstone AH,et al.Reconstitution of T-cell repertoire after autologous stem cell transplantation:influence of CD34 selection and cytomegalovirus infection.Biol Blood Marrow Transplant 2003;9(3):198-205.
[21]张迎春,刘文君.巨细胞病毒感染与骨髓衰竭.中华儿科杂志 2002;40(7):423-425.
[22]Goodrum F,Jordan CT,Terhune SS,High K,Shenk T.Differential outcomes of human cytomegalovirus infection in primitive hematopoietic cell subpopulations.Blood 2004;104(3):687-695.
[23]Smirnov SV,Harbacheuski R,Lewis-Antes A,Zhu H,Rameshwar P,Kotenko SV.Bone-marrow-derived mesenchymal stem cells as a target for cytomegalovirus infection:Implications for hematopoiesis,self-renewal and differentiation potential.Virology 2007;360(1):6-16.
[24]Lagneaux L,Delforge A,Snoeck R,Bosmans E,Schols D,De Clereq E,et al.Imbalance in production of cytokines by bone marrow stromal cells fllowing cytomegalovirus infection.J Infect Dis 1996;174(5):913-919.
[25]Mori T,Ando K,Tanaka K,Ikeda Y,Koga Y.Fas-mediated apoptosis of the hematopoietic progenitor cells in mice infected with murine cytomegalovirus.Blood 1997;15;89(10):3565-73.
[26]Minton EJ,Tysoe C,Sinclair JH,Sissons JG.Human cytomegalovirus infection of the monocyte/macrophage lineage in bone marrow.J Virol 1994;68(6):4017-4021.
[27]Mocarski ES Jr.Immune escape and exploitation strategies of cytomegaioviruses:Impact on and imitation of the major histocompatibility system.Cell Microbiol 2004;6(8):707-717.
[28]Spencer JV,Lockridge KM,Barry PA,Lin G,Tsang M,Penfold ME,et al.Potent immunosuppressive activities of cytomegalovirus-encoded interleukin-10.J Virol 2002;76(3):1285-1292.
[29]Goldmacher VS.Cell death suppression by cytomegalovirus.Apoptosis 2005;10(2):251-265.
[30]Beck K,Meyer-K(o|¨)nig U,Weidmann M,Nern C,Hufert FT.Human cytomegalovirus impairs dendritic cell function:a novel mechanism of human cytomegalovirus immune escape.Eur J Immunol 2003;33(6):1528-1538.
[31]Cosmi L,Liotta F,Angeli R,Mazzinghi B,Santarlasci V,Manetti R,et al.Th2 cells are less susceptible than Th1 cells to the suppressive activity of CD25+ regulatory thymocytes because of their responsiveness to different cytokines.Blood 2004;103(8):3117-3121.
[32]朱单丹,方峰,舒赛男,向稚丹,李亚男,杨助锋.巨细胞病毒感染对调节性T 细胞和Ⅰ型、Ⅱ型辅助性T细胞分化增殖的影响.临床儿科杂志 2007;25(7): 537-540,546.
[33]Koffa M, Koumantakis E, Ergazaki M, Tsatsanis C, Spandidos DA.Association of herpesvirus infection with the development of genital cancer.Int J Cancer 1995;63 (1):58-62.
[34]Michaelis M, Kotchetkov R, Vogel JU, Doerr HW, Cinatl J Jr.Cytomegalovirus infection blocks apoptosis in cancer cell.Cell Mol Life Sci 2004;61(11):1307-1316.
[35]Soderberg-Naucler C.Does cytomegalovirus play a causative role in the development of various inflammatory diseases and cancer? J Intern Med 2006;259(3):219-246.
[36]Arvin AM, Fast P, Myers M, Plotkin S, Rabinovich R.Vaccine development to prevent cytomegalovirus disease: report from the National Vaccine Advisory Committee. Clin Infect Dis 2004;39(2):233-239.
[37]Marshall BC, Adler SP.Avidity maturation following immunization with two human cytomegalovirus vaccines : a live attenuated vaccine(Towne)and a recombinant glycoprotein vaccine(gB/MF59).Viral Immunol 2003;16(4):491-500.
[38]Khanna R, Diamond DJ.Human cytomegalovirus vaccine: Time to look for alternative options.Trends Mol Med 2006;12(1):26-33.
[39]Temperton NJ. DNA vaccines against cytomegalovirus: Current progress .Int J Antimicrob Agents 2002; 19(3): 169-172.
[40]Pass RF, Duliege AM, Boppana S, Sekulovich R, Percell S, Britt W, et al. A subunit cytomegalovirus vaccine based on recombinant envelope glycoprotein B and a new adjuvant. J Infect Dis 1999; 180 (4):970-975.
[41]Berencsi K, Gyulai Z, Gonczol E, Pincus S, Cox WI, Michelson S,et al. A canarypox vector-expressing cytomegalovirus ( CMV ) phosphoprotein 65 induces long lasting cytotoxic T cell responses in human CMV-seronegative subjects. Infect Dis 2001; 183(8): 1171-1179.
[42]Tanaka N, Kimura H, Hoshino Y, Nishikawa K, Kojima S, Nishiyama Y,et al. Expression of tegument protein pp65 of human cytomegalovirus(CMV) and its application to the analysis of viral specific cellular immunity in CMV infected individuals. Arch Virol 2002;147(12):2405-2417.
[43]Wang Z, La Rosa C, Maas R, Ly H, Brewer J, Mekhoubad S, et al. Recombinant modified vaccinia virus ankara expressing a soluble form of glycop rotein B causes durable immunity and neutralizing antibodies against multiple strains of human cytomegalovirus.; Virol.2004;78(8): 3965-3976.
[44]Wang Z, La Rosa C, Lacey SF, Maas R, Mekhoubad S, Britt WJ, et al.Attenuated poxvirus expressing three immunodominant CMV antigens as a vaccine strategy for CMV infection. J Clin Virol 2006;35(3):324-31.
[45]Wang Z, La Rosa C, Mekhoubad S, Lacey SF, Villacres MC, Markel S, et al.Attenuated pox viruses generate clinically relevant frequencies of CMV specific T cells.Blood 2004;104(3):847- 856.
[46]Elkington R, Walker S, Crough T, Menzies M, Tellam J, Bharadwaj M, et al. Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers. J Virol 2003,77 (9):5226-5240.
[47]Akiyama Y, Maruyama K, Mochizuki T, Sasaki K, Takaue Y, Yamaguchi K.Identification of HLA-A24-restricted CTL epitope encoded by the matrix protein pp65 of human cytomegalovirus. Immunol Lett 2002;83(1):21-30.
[48]Diamond DJ, York J, Sun JY, Wright CL, Forman SJ. Development of a candidate HLA A*0201 restricted peptide-based vaccine against human cytomegalovirus infection. Blood 1997;90(5): 1751-1767.
[49]Frankenberg N, Pepperl-Klindworth S, Meyer RG, Plachter B. Identification of a conserved HLA-A2-restricted decapeptide from the IE1 protein (pUL123) of human cytomegalovirus. Virology 2002;295(2):208-216.
[50]Kern F, Bunde T, Faulhaber N, Kiecker F, Khatamzas E, Rudawski IM, et al. Cytomegalovirus (CMV) phosphoprotein 65 makes a large contribution to shaping the T cell repertoire in CMV-exposed individuals. J Infect Dis 2002; 185(12): 1709-1716.
[51]Kern F, Faulhaber N, Khatamzas E, Frommel C, Ewert R, Prosch S, et al.Measurement of anti-human cytomegalovirus T cell reactivity in transplant recipients and its potential clinical use: a mini-review. Intervirology 1999;42(5-6):322-324.
[52]Kuzushima K, Hayashi N, Kimura H, Tsurumi T. Efficient identification of HLA-A*2402-restricted cytomegalovirus-specific CD8(+) T-cell epitopes by a computer algorithm and an enzyme-linked immunospot assay.Blood 2001;98 (6): 1872-1881.
[53]Masuoka M, Yoshimuta T, Hamada M, Okamoto M, Fumimori T, Honda J, et al. Identification of the HLA-A24 peptide epitope within cytomegalovirus protein pp65 recognized by CMV-specific cytotoxic T lymphocytes. Viral Immunol 2001; 14 (4):369-377.
[54]Reddehase MJ.The immunogenicity of human and murine cytomegaloviruses. Curr Opin Immunol 2000; 12 (4):390-396.
[55]Reddehase MJ.Antigens and immunoevasins: opponents in cytomegalovirus immune surveillance. Nat Rev Immunol 2002;2(11):831-844.
[56]Retiere C, Prod'homme V, Imbert-Marcille BM, Bonneville M, Vie H, Hallet MM.Generation of cytomegalovirus-specific human T-lymphocyte clones by using autologous B-lymphoblastoid cells with stable expression of pp65 or IE1 proteins: a tool to study the fine specificity of the antiviral response. J Virol 2000;74 (9):3948-3952.
[57]Saulquin X, Ibisch C, Peyrat MA, Scotet E, Hourmant M, Vie H, et al. A global appraisal of immunodominant CD8 T cell responses to Epstein-Barr virus and cytomegalovirus by bulk screening. Eur J Immunol 2000;30(9):2531-2539.
[58]Weekes MP, Wills MR, Mynard K, Carmichael AJ, Sissons JG. The memory cytotoxic T-lymphocyte (CTL) response to human cytomegalovirus infection contains individual peptide-specific CTL clones that have undergone extensive expansion in vivo. J Virol 1999;73(3):2099-2108.
[59]Wills MR, Carmichael AJ, Mynard K, Jin X, Weekes MP, Plachter B, et al. The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL. J Virol 1996;70 (11):7569-7579.
[60]Rist M, Cooper L, Elkington R, Walker S, Fazou C, Tellam J, et al. Ex vivo expansion of human cytomegalovirus-specific cytotoxic T cells by recombinant polyepitope: implications for HCMV immunotherapy. Eur J Immunol 2005;35(3):996-1007.
[61]Hebart H, Daginik S, Stevanovic S, Grigoleit U, Dobler A, Baur M, et al. Sensitive detection of human cytomegalovirus peptide-specific cytotoxic T-lymphocyte responses interferon-gamma-enzyme-linked immunospot assay and flow cytometry in healthy individuals and in patients after allogeneic stem cell transplantation. Blood 2002; 99 (10):3830-3837.
[62]Longmate J, York J, La Rosa C, Krishnan R, Zhang M, et al. Population coverage by HLA class-I restricted cytotoxic T-lymphocyte epitopes. Immunogenetics 2001;52(3-4): 165-173.
[63]Kondo E, Akatsuka Y, Kuzushima K, Tsujimura K, Asakura S, Tajima K,et al. Identification of novel CTL epitopes of CMV-pp65 presented by a variety of HLA alleles. Blood 2004; 103 (2): 630-638.
[64]Hebart H, Rauser G, Stevanovic S, Haenle C, Nussbaum AK, Meisner C, et al. A CTL epitope from human cytomegalovirus IE1 defined by combining prediction of HLA binding and proteasomal processing is the target of dominant immune responses in patients after allogeneic stem cell transplantation. Exp Hematol 2003;31(10):966-973.
[65]Nastke MD, Herrgen L, Walter S, Wernet D, Rammensee HG, Stevanovi6 S. Major contribution of codominant CD8 and CD4 T cell epitopes to the human cytomegalovirus-specific T cell repertoire. Cell Mol Life Sci 2005;62(1):77-86.
[66]Li Pira G, Bottone L, Ivaldi F, Pelizzoli R, Del Galdo F, Lozzi L,et al. Identification of new Th peptides from the cytomegalovirus protein pp65 to design a peptide library for generation of CD4 T cell lines for cellular immunoreconstitution. Int Immunol 2004;16(5):635-642.
[67]Khattab BA, Lindenmaier W, Frank R, Link H. Three T-cell epitopes within the C-terminal 265 amino acids of the matrix protein pp65 of human cytomegalovirus recognized by human lymphocytes. J Med Virol 1997;52(1):68-76.
[68]Gallot G, Vivien R, Ibisch C, Lute J, Davrinche C, Gaschet J, et al. Purification of Ag-specific T lymphocytes after direct peripheral blood mononuclear cell stimulation followed by CD25 selection. I. Application to CD4(+) or CD8(+) cytomegalovirus phosphoprotein pp65 epitope determination. J Immunol 2001;167(8):4196-4206.
[69]Dawson DV, Ozgur M, Sari K, Ghanayem M, Kostyu DD. Ramifications of HLA class I polymorphism and population genetics for vaccine development. Genet Epidemiol 2001 ;20(1):87-106.
[70]XUE Fu-zhong , WANGJie-zhen, HU Ping, et al.Forecasting system on spatial coverage of cumulative phenotypic frequency of HLA class I for designing HLA-based vaccines in China. Immunological Journal 2005;21(2):136-141.
[71]Thomas PG, Keating R, Hulse-Post DJ, Doherty PC.Cell-mediated Protection in Influenza Infection. Emerg Infect Dis 2006;12(1):48-54.
[72]Andersen MH, Schrama D, Thor Straten P, Becker JC.Cytotoxic T cells. J Invest Dermatol 2006;126(1):32-41.
[73]Falk K, Rotzschke O, Stevanovic S, Jung G, Rammensee HG.Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature. 1991 ;351 (6324):290-296.
[74]Peters B, Bui HH, Sidney J, Weng Z, Loffredo JT, Watkins DI, et al. A computational resource for the prediction of peptide binding to Indian rhesus macaque MHC class I molecules. Vaccine 2005;23(45):5212-5224.
[75]Ruppert J, Sidney J, Celis E, Kubo RT, Grey HM, Sette A. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1molecules. Cell 1993;74(5):929-937.
[76]Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 1999;50(3-4):213-219.
[77]Hakenberg J, Nussbaum AK, Schild H, Rammensee HG, Kuttler C, Holzhutter HG,et al. MAPPP: MHC class I antigenic peptide processing prediction. Appl Bioinformatics 2003;2(3):155-158.
[78]Ghei M, Stroncek DF, Provenzano M. Analysis of memory T lymphocyte activity following stimulation with overlapping HLA-A*2402, A*0101 and Cw*0402 restricted CMV pp65 peptides. J Transl Med, 2005;3:23.
[79]Browne EP, Shenk T. Human cytomegalovirus UL83-coded pp65 virion protein inhibits antiviral gene expression in infected cells. Proc Natl Acad Sci USA 2003; 100(20): 11439-11444.
[80]Gyulai Z, Endresz V, Burian K, Pincus S, Toldy J, Cox WI, el al.Cytotoxic T lymphocyte(CTL)responses to human cytomegalovirus pp65, IE 1-exon 4, gB, pp150, and pp28 in healthy individuals; reevaluation of prevalence of 1El-pecific CTLs. J Infect Dis 2000;181(5):1537-1546.
[81]Schleiss MR, Heineman TC. Progress toward an elusive goal: current status of cytomegalovirus vaccines. Expert Rev Vaccines 2005;4(3):381-406.
[82]Bernstein DI, Schleiss MR, Berencsi K, Gonczol E, Dickey M, Khoury P, et al. Effect of previous or simultaneous immunization with canarypox expressing cytomegalovirus (CMV) glycoprotein B (gB) on response to subunit gB vaccine plus MF59 in healthy CMV-seronegative adults.J Infect Dis 2002; 185 (5):686-690.
[83]Furman MH, Loureiro J, Ploegh HL, Tortorella D. Ubiquitinylation of the Cytosolic domain of a type I membrane protein is not required to initiate its dislocation from the endoplasmic reticulum. J Biol Chem 2003;278(37):34804-34811.
[84]Hegde NR, Johnson DC. Human cytomegalovirus US2 causes similar effects on both major histocompatibility complex class I and II proteins in epithelial and glial cells. J Virol 2003;77(17):9287-9294.
[85]Popkin DL,Virgin HW. Murine cytomegalovirus infection inhibits tumor necrosis factor alpha responses in primary macrophages J Virol 2003;77(18):10125-10130.
[86]Gonczol E, Plotkin S.Development of a cytomegalovirus vaccine: lessons from recent clinical trials. Expert Opin Biol Ther 2001;1(3):401-412.
[1]Chrisp P, Clissold SP. Foscarnet.A review of its antiviral activity, pharmacokinetic properties and therapeutic use in immunocompromised patients with cytomegalovirus retinitis. Drugs 1991 ;41(1): 104-129.
[2]Faulds D,Heel RC.Ganciclovir. A review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy in cytomegalovirus infections.Drugs1990; 39 (4): 597-638.
[3]Griffiths PD, McLean A, Emery VC.Encouraging prospects for immunization against primary cytomegalovirus infection.Vaccine2001;19 (11-12):1356-1362
[4]Arvin AM, Fast P, Myers M, Plotkin S, Rabinovich R.Vaccine development to prevent cytomegalovirus disease:report from the National Vaccine Advisory Committee. Clin Infect Dis 2004;39(2):233-239.
[5]Xin K.Q, Jounai N, Someya K, Honma K, Mizuguchi H, Naganawa S, et al.Prime-boost vaccination with plasmid DNA and a chimeric adenovirus type 5 vector with type 35 fiber induces protective immunity against HIV.Gene Ther.2005;12 (24): 1769-1777.
[6]Thammanichanond D, Moneer S, Yotnda P, Aitken C, Earnest-Silveira L, Jackson D,et al.Fiber-modified recombinant adenoviral constructs encoding hepatitis C virus proteins induce potent HCV-specific T cell response. Clin Immunol 2008; 128(3): 329-339
[7]Xin KQ, Sekimoto Y, Takahashi T, Mizuguchi H, Ichino M, Yoshida A, et al.Chimeric adenovirus 5/35 vector containing the clade C HIV gag gene induces a cross-reactive immune response against HIV.Vaccine 2007;25 (19):3809-3815
[8]Hoffrnann D, Bayer W, Heim A, Potthoff A, Nettelbeck DM, Wildner O.Evaluation of twenty-one human adenovirus types and one infectivity-enhanced adenovirus for the treatment of malignant melanoma. J Invest Dermatol 2008;128(4):988-998.
[9]Hoffmann D, Meyer B, Wildner O.Improved glioblastoma treatment with Ad5/35 fiber chimeric conditionally replicating adenoviruses. J Gene Med 2007;9(9):764-778.
[10]Yotnda P, Onishi H, Heslop HE, Shayakhmetov D, Lieber A, Brenner M, et al. Efficient infection of primitive hematopoietic stem cells by modified adenovirus. Gene Ther 2001 ;8(12):930-937.
[11]Mizuguchi H, Hayakawa T. Adenovirus vectors containing chimeric type 5 and type 35 fiber proteins exhibit altered and expanded tropism and increase the size limit of foreign genes. Gene 2002;285(1-2):69-77.
[12]Mizuguchi H, Hayakawa T. Targeted adenovirus vectors. Hum Gene Ther 2004; 15(11):1034-1044
[13]Gaggar A, Shayakhmetov DM, Lieber A. CD46 is a cellular receptor for group B adenoviruses. Nat Med 2003;9 (11):1408-1412
[1]Nguyen Q,Champlin R,Giralt S,Rolston K,Raad I,Jacobson K,et al.Late cytomegalovirus pneumonia in adult allogeneic blood and marrow transplant recipients.Clin Infect Dis 1999;28(3):618-623.
[2]章咏裳.肾移植术后巨细胞病毒感染.中华器官移植杂志 2000;21(4):248-249.
[3]Temperton NJ.DNA vaccines against cytomagalovirus:current progress.Inter J Antimicrobial Angents 2002;19(3):169-172.
[4]Meijer E,Boland GJ,Verdonck LF.Prevention of cytomegalovirus disease in recipients of allogeneic stem cell transplants.Clin Microbiol Rev 2003;16(4):647-657.
[5]Micklethwaite KP,Clancy L,Sandher U,Hansen AM,Blyth E,Antonenas V,et al.Prophylactic infusion of cytomegalovirus-specific cytotoxic T lymphocytes stimulated with Ad5f35pp65 gene-modified dendritic cells after allogeneic hemopoietic stem cell transplantation.Blood 2008;112(10):3974-3981.
[6]Peggs KS,Verfuerth S,Pizzey A,Khan N,Moss P,Goldstone AH,et al.Reconstitution of T-cell repertoire after autologous stem cell transplantation:influence of CD34 selection and cytomegalovirus infection.Biol Blood Marrow Transplant 2003;9(3):198-205.
[7]Gandhi MK,Khanna R.Human cytomegalovirus:clinical aspects,immune regulation,and emerging treatments.Lancet Infect Dis 2004;4(12):725-738.
[8]Boeckh M,Leisenring W,Riddell SR,Bowden RA,Huang ML,et al.Late cytomegalovirus disease and mortality in recipients of allogeneic hematopoietic stem cell transplants:importance of viral load and T-cell immunity.Blood 2003;101:407-414.
[9]Crough T,Fazou C,Weiss J,Campbell S,Davenport MP,et al.Symptomatic and asymptomatic viral recrudescence in solid-organ transplant recipients and its relationship with the antigen-specific CD8(+) T-cell response.J Virol 2007;81:11538-11542.
[10]Engstrand M,Tournay C,Peyrat MA,Eriksson BM,Wadstrom J,et al.Characterization of CMVpp65-specific CD8+T lymphocytes using MHC tetramers in kidney transplant patients and healthy participants.Transplantation 2000;69(11):2243-2250.
[11]Riddell SR,Greenberg PD.Cellular adoptive immunotherapy after bone marrow transplantation.Cancer Treat Res 1995;76:337-369.
[12]Walter EA,Greenberg PD,Gilbert MJ,Finch RJ,Watanabe KS,Thomas ED,et al.Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.N Engl J Med 1995;333(16):1038-1044.
[13]Elkington R,Shoukry NH,Walker S,Crough T,Fazou C,Kaur A,et al.Cross-reactive recognition of human and primate cytomegalovirus sequences by human CD4 cytotoxic T lymphocytes specific for glycoprotein B and H.Eur J Immunol 2004;34(11):3216-3226.
[14]Elkington R,Walker S,Crough T,Menzies M,Tellam J,Bharadwaj M,et al.Ex vivoprofiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecifie reactivities in healthy virus carriers.J Virol 2003;77(9):5226-5240.
[15]Sylwester AW,Mitchell BL,Edgar JB,Taormina C,Pelte C,Ruchti F,et al.Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects.J Exp Med 2005;202(5):673-685.
[16]Manley TJ,Luy L,Jones T,Boeckh M,Mutimer H,Riddell SR.Immune evasion proteins of human cytomegalovirus do not prevent a diverse CD8+cytotoxic T-cell response in natural infection.Blood 2004;104(4):1075-1082.
[17]Zhong J,Khanna R.Vaccine strategies against human cytomegalovirus infection.Expert Rev Anti Infect Ther 2007;5(3):449-459.
[18]Rist M,Cooper L,Elkington R,Walker S,Fazou C,Tellam J,et al.Ex vivo expansion of human cytomegalovirus-specific cytotoxic T cells by recombinant polyepitope:implications for HCMV immunotherapy.Eur J Immunol 2005;35(3):996-1007.
[19]Berencsi K,Gyulai Z,G6ncz(o|¨)l E,Pincus S,Cox WI,Michelson S,et al.A canarypox vector-expressing cytomegalovirus(CMV) phosphoprotein 65 induces long-lasting cytotoxic T cell responses in human CMV-seronegative subjects.J Infect Dis 2001;183(8):1171-1179.
[20]Morello CS,Ye M,Hung S,Kelley LA,Spector DH.Systemic Priming-Boosting Immunization with a Trivalent Plasmid DNA and Inactivated Murine Cytomegalovirus(MCMV) Vaccine Provides Long-Term Protection against Viral Replication following Systemic or Mucosal MCMV Challenge.J Virol 2005;79(1):159-175.
[21]Wang Z,La Rosa C,Li Z,Ly H,Krishnan A,Martinez J,et al.Vaccine properties of a novel marker gene-free recombinant modified vaccinia Ankara expressing immunodominant CMV antigens pp65 and IE1. Vaccine 2007;25 (6): 1132-1141.
[22]Dawson DV, Ozgur M, Sari K, Ghanayem M, Kostyu DD. Ramifications of HLA class I polymorphism and population genetics for vaccine development. Genet Epidemiol 2001;20(1):87-106.
[23]Stewart AK, Lassam NJ, Quirt IC, Bailey DJ, Rotstein LE, Krajden M, et al. Adenovector-mediated gene delivery of interleukin-2 in metastatic breast cancer and melanoma: results of a phase 1 clinical trial. Gene Ther 1999;6(3):350-363.
[24]Yotnda P, Onishi H, Heslop HE, Shayakhmetov D, Lieber A, Brenner M, et al. Efficient infection of primitive hematopoietic stem cells by modified adenovirus. Gene Ther 2001;8(12):930-937.
[25]Thomson SA, Khanna R, Gardner J, Burrows SR, Coupar B, Moss DJ,et al. Minimal epitopes expressed in a recombinant polyepitope protein are processed and presented to CD8+ cytotoxic T cells: implications for vaccine design. Proc Natl Acad Sci U S A 1995;92(13):5845-5849.
[26]Mocarski ES Jr.Immunomodulation by cytomegaloviruses: manipulative strategies beyond evasion. Trends Microbiol 2002;10(7):332-339.
[27]Farrell HE, Davis-Poynter NJ, Andrews DM, Degli-Esposti MA.Function of CMV-encoded MHC class I homologues. Curr Top Microbiol Immunol 2002;269: 131-151.
[28]Zhong J, Rist M, Cooper L, Smith C, Khanna R.Induction of pluripotent protective immunity following immunisation with a chimeric vaccine against human cytomegalovirus. PLOS ONE 2008;3(9):3256
[29]Khanna R, Diamond DJ.Human cytomegalovirus vaccine: time to look for alternative options. Trends Mol Med 2006;12(1):26-33.
[30]Horn B, Bao L, Dunham K, Stamer M, Adler S, Cowan M, et al. Infusion of cytomegalovirus specific cytotoxic T lymphocytes from a sero-negative donor can facilitate resolution of infection and immune reconstitution. Pediatr Infect Dis J 2009;28(1):65-67.
[2]Gaytant MA,Steegers EA,Semmekrot BA,Merkus HM,Galama JM.Congenital cytomegalovirus infection:review of the epidemiology and outcome.Obstet Gynecol Surv 2002;57:245-256.
[3]Arvin AM,Fast P,Myers M,Plotkin S,Rabinovich R.Vaccine development to prevent cytomegalovirus disease:report from the National Vaccine Advisory Committee.Clin Infect Dis 2004;39:233-239.
[4]Varnum SM,Streblow DN,Monroe ME,Smith P,Auberry KJ,Pasa-Tolic L,et al.Identification of proteins in human cytomegalovirus(HCMV) particles:the HCMV proteome.J Virol 2004;78(20):10960-10966.
[5]Melnick JL,Hu C,Burek J,Adam E,DeBakey ME.Cytomegalovirus DNA in arterial walls of patients with atherosclerosis.J Med Virol 1994;42:170-174.
[6]Sinclair J,Sissons P.Latent and persistent infections of monocytes and macrophages.Intervirology.1996;39(5-6):293-301.
[7]Lecointe D,Hery C,Janabi N,Dussaix E,Tardieu M.Differences in kinetics of human cytomegalovirus cell-free viral release after in vitro infection of human microglial cells,astrocytes and monocyte-derived macrophages.J Neurovirol 1999;5(3):308-317.
[8]Kline JN,Hunninghake GM,He B,Monick MM,Hunninghake GW.Synergistic activation of the human cytomegalovirus major immediate early promoter by prostaglandin E2 and cytokines.Exp Lung Res1995;24(1):3-16.
[9]Walmsley S,O'Rourke K,Mortimer C,Rachlis A,Fong I,Mazzulli T.Predictive value of cytomegalovirus(CMV) antigenemia and digene hybrid capture DNA assays for CMV disease in human immunodefieiency virus-infected patients.Clin Infect Dis 1998;27(3):573-581.
[10]Nguyen Q,Champlin R,Giralt S,Rolston K,Raad I,Jacobson K,et al.Late cytomegalovirus pneumonia in adult allogeneic blood and marrow transplant recipients.Clin Infect Dis 1999;28(3):618-623.
[11]王霞,张蓓,罗长缨,陈静,沈立松.儿童异基因造血干细胞移植术后感染的实验观察.中国小儿血液与肿瘤杂志 2006;11(3):107-108.
[12]秦茂权,谢正德,王彬,周翱,朱光华,冯涛,吴敏嫒.儿童造血干细胞移植后巨细胞病毒感染的临床研究.临床儿科杂志 2007;25(7):541-543.
[13]Kantrow SP,Hackman RC,Boeckh M,Myerson D,Crawford SW.Idiopathic pnemonia syndrome:changing spectrum of lung injury after marrow transplantation.Transplantation 1997;63(8):1079-1086.
[14]Wagner JE,Eapen M,MacMillan ML,Harris RE,Pasquini R,Boulad F,et al.Unrelated donor bone marrow transplantation for the treatment of Fanconi anemia.Blood 2007;109(5):2256-2262.
[15]Pellegrin I,Garrigue I,Ekouevi D,Couzi L,Merville P,Merel P,et al.New molecular assay to Predict occurrence of cytomegalovirus disease in renal transplant recipients.J Infect Dis 2000;182:36-42.
[16]Temperton NJ.DNA vaccines against cytomagalovirus:current progress.Int J Antimicrob Agents 2002;19(3):169-172.
[17]Bernstein DI,Schleiss MR,Berencsi K,Gonczol E,Dickey M,Khoury P,et al.Effect of previous or simultaneous immunization with canarypox expressing cytomegalovirus(CMV)glycoprotein B(gB)on response to subunit gB vaccine plus MF59in healthy CMV-seronegative adults.J Infect Dis 2002;185(5):686-690.
[18]Meijer E,Boland GJ,Verdonck LF.Prevention of cytomegalovirus disease in recipients of allogeneic stem cell transplants.Clin Microbiol Rev 2003;16(4):647-657.
[19]Li CR,Greenberg PD,Gilbert MJ,Goodrich JM,Riddell SR.Recovery of HLA-restricted cytomegalovirus(CMV)-specific T-cell responses after allogeneic bone marrow transplant:correlation with CMV disease and effect of ganciclovir prophylaxis.Blood 1994;83(7):1971-1979.
[20]Peggs KS,Verfuerth S,Pizzey A,Khan N,Moss P,Goldstone AH,et al.Reconstitution of T-cell repertoire after autologous stem cell transplantation:influence of CD34 selection and cytomegalovirus infection.Biol Blood Marrow Transplant 2003;9(3):198-205.
[21]张迎春,刘文君.巨细胞病毒感染与骨髓衰竭.中华儿科杂志 2002;40(7):423-425.
[22]Goodrum F,Jordan CT,Terhune SS,High K,Shenk T.Differential outcomes of human cytomegalovirus infection in primitive hematopoietic cell subpopulations.Blood 2004;104(3):687-695.
[23]Smirnov SV,Harbacheuski R,Lewis-Antes A,Zhu H,Rameshwar P,Kotenko SV.Bone-marrow-derived mesenchymal stem cells as a target for cytomegalovirus infection:Implications for hematopoiesis,self-renewal and differentiation potential.Virology 2007;360(1):6-16.
[24]Lagneaux L,Delforge A,Snoeck R,Bosmans E,Schols D,De Clereq E,et al.Imbalance in production of cytokines by bone marrow stromal cells fllowing cytomegalovirus infection.J Infect Dis 1996;174(5):913-919.
[25]Mori T,Ando K,Tanaka K,Ikeda Y,Koga Y.Fas-mediated apoptosis of the hematopoietic progenitor cells in mice infected with murine cytomegalovirus.Blood 1997;15;89(10):3565-73.
[26]Minton EJ,Tysoe C,Sinclair JH,Sissons JG.Human cytomegalovirus infection of the monocyte/macrophage lineage in bone marrow.J Virol 1994;68(6):4017-4021.
[27]Mocarski ES Jr.Immune escape and exploitation strategies of cytomegaioviruses:Impact on and imitation of the major histocompatibility system.Cell Microbiol 2004;6(8):707-717.
[28]Spencer JV,Lockridge KM,Barry PA,Lin G,Tsang M,Penfold ME,et al.Potent immunosuppressive activities of cytomegalovirus-encoded interleukin-10.J Virol 2002;76(3):1285-1292.
[29]Goldmacher VS.Cell death suppression by cytomegalovirus.Apoptosis 2005;10(2):251-265.
[30]Beck K,Meyer-K(o|¨)nig U,Weidmann M,Nern C,Hufert FT.Human cytomegalovirus impairs dendritic cell function:a novel mechanism of human cytomegalovirus immune escape.Eur J Immunol 2003;33(6):1528-1538.
[31]Cosmi L,Liotta F,Angeli R,Mazzinghi B,Santarlasci V,Manetti R,et al.Th2 cells are less susceptible than Th1 cells to the suppressive activity of CD25+ regulatory thymocytes because of their responsiveness to different cytokines.Blood 2004;103(8):3117-3121.
[32]朱单丹,方峰,舒赛男,向稚丹,李亚男,杨助锋.巨细胞病毒感染对调节性T 细胞和Ⅰ型、Ⅱ型辅助性T细胞分化增殖的影响.临床儿科杂志 2007;25(7): 537-540,546.
[33]Koffa M, Koumantakis E, Ergazaki M, Tsatsanis C, Spandidos DA.Association of herpesvirus infection with the development of genital cancer.Int J Cancer 1995;63 (1):58-62.
[34]Michaelis M, Kotchetkov R, Vogel JU, Doerr HW, Cinatl J Jr.Cytomegalovirus infection blocks apoptosis in cancer cell.Cell Mol Life Sci 2004;61(11):1307-1316.
[35]Soderberg-Naucler C.Does cytomegalovirus play a causative role in the development of various inflammatory diseases and cancer? J Intern Med 2006;259(3):219-246.
[36]Arvin AM, Fast P, Myers M, Plotkin S, Rabinovich R.Vaccine development to prevent cytomegalovirus disease: report from the National Vaccine Advisory Committee. Clin Infect Dis 2004;39(2):233-239.
[37]Marshall BC, Adler SP.Avidity maturation following immunization with two human cytomegalovirus vaccines : a live attenuated vaccine(Towne)and a recombinant glycoprotein vaccine(gB/MF59).Viral Immunol 2003;16(4):491-500.
[38]Khanna R, Diamond DJ.Human cytomegalovirus vaccine: Time to look for alternative options.Trends Mol Med 2006;12(1):26-33.
[39]Temperton NJ. DNA vaccines against cytomegalovirus: Current progress .Int J Antimicrob Agents 2002; 19(3): 169-172.
[40]Pass RF, Duliege AM, Boppana S, Sekulovich R, Percell S, Britt W, et al. A subunit cytomegalovirus vaccine based on recombinant envelope glycoprotein B and a new adjuvant. J Infect Dis 1999; 180 (4):970-975.
[41]Berencsi K, Gyulai Z, Gonczol E, Pincus S, Cox WI, Michelson S,et al. A canarypox vector-expressing cytomegalovirus ( CMV ) phosphoprotein 65 induces long lasting cytotoxic T cell responses in human CMV-seronegative subjects. Infect Dis 2001; 183(8): 1171-1179.
[42]Tanaka N, Kimura H, Hoshino Y, Nishikawa K, Kojima S, Nishiyama Y,et al. Expression of tegument protein pp65 of human cytomegalovirus(CMV) and its application to the analysis of viral specific cellular immunity in CMV infected individuals. Arch Virol 2002;147(12):2405-2417.
[43]Wang Z, La Rosa C, Maas R, Ly H, Brewer J, Mekhoubad S, et al. Recombinant modified vaccinia virus ankara expressing a soluble form of glycop rotein B causes durable immunity and neutralizing antibodies against multiple strains of human cytomegalovirus.; Virol.2004;78(8): 3965-3976.
[44]Wang Z, La Rosa C, Lacey SF, Maas R, Mekhoubad S, Britt WJ, et al.Attenuated poxvirus expressing three immunodominant CMV antigens as a vaccine strategy for CMV infection. J Clin Virol 2006;35(3):324-31.
[45]Wang Z, La Rosa C, Mekhoubad S, Lacey SF, Villacres MC, Markel S, et al.Attenuated pox viruses generate clinically relevant frequencies of CMV specific T cells.Blood 2004;104(3):847- 856.
[46]Elkington R, Walker S, Crough T, Menzies M, Tellam J, Bharadwaj M, et al. Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers. J Virol 2003,77 (9):5226-5240.
[47]Akiyama Y, Maruyama K, Mochizuki T, Sasaki K, Takaue Y, Yamaguchi K.Identification of HLA-A24-restricted CTL epitope encoded by the matrix protein pp65 of human cytomegalovirus. Immunol Lett 2002;83(1):21-30.
[48]Diamond DJ, York J, Sun JY, Wright CL, Forman SJ. Development of a candidate HLA A*0201 restricted peptide-based vaccine against human cytomegalovirus infection. Blood 1997;90(5): 1751-1767.
[49]Frankenberg N, Pepperl-Klindworth S, Meyer RG, Plachter B. Identification of a conserved HLA-A2-restricted decapeptide from the IE1 protein (pUL123) of human cytomegalovirus. Virology 2002;295(2):208-216.
[50]Kern F, Bunde T, Faulhaber N, Kiecker F, Khatamzas E, Rudawski IM, et al. Cytomegalovirus (CMV) phosphoprotein 65 makes a large contribution to shaping the T cell repertoire in CMV-exposed individuals. J Infect Dis 2002; 185(12): 1709-1716.
[51]Kern F, Faulhaber N, Khatamzas E, Frommel C, Ewert R, Prosch S, et al.Measurement of anti-human cytomegalovirus T cell reactivity in transplant recipients and its potential clinical use: a mini-review. Intervirology 1999;42(5-6):322-324.
[52]Kuzushima K, Hayashi N, Kimura H, Tsurumi T. Efficient identification of HLA-A*2402-restricted cytomegalovirus-specific CD8(+) T-cell epitopes by a computer algorithm and an enzyme-linked immunospot assay.Blood 2001;98 (6): 1872-1881.
[53]Masuoka M, Yoshimuta T, Hamada M, Okamoto M, Fumimori T, Honda J, et al. Identification of the HLA-A24 peptide epitope within cytomegalovirus protein pp65 recognized by CMV-specific cytotoxic T lymphocytes. Viral Immunol 2001; 14 (4):369-377.
[54]Reddehase MJ.The immunogenicity of human and murine cytomegaloviruses. Curr Opin Immunol 2000; 12 (4):390-396.
[55]Reddehase MJ.Antigens and immunoevasins: opponents in cytomegalovirus immune surveillance. Nat Rev Immunol 2002;2(11):831-844.
[56]Retiere C, Prod'homme V, Imbert-Marcille BM, Bonneville M, Vie H, Hallet MM.Generation of cytomegalovirus-specific human T-lymphocyte clones by using autologous B-lymphoblastoid cells with stable expression of pp65 or IE1 proteins: a tool to study the fine specificity of the antiviral response. J Virol 2000;74 (9):3948-3952.
[57]Saulquin X, Ibisch C, Peyrat MA, Scotet E, Hourmant M, Vie H, et al. A global appraisal of immunodominant CD8 T cell responses to Epstein-Barr virus and cytomegalovirus by bulk screening. Eur J Immunol 2000;30(9):2531-2539.
[58]Weekes MP, Wills MR, Mynard K, Carmichael AJ, Sissons JG. The memory cytotoxic T-lymphocyte (CTL) response to human cytomegalovirus infection contains individual peptide-specific CTL clones that have undergone extensive expansion in vivo. J Virol 1999;73(3):2099-2108.
[59]Wills MR, Carmichael AJ, Mynard K, Jin X, Weekes MP, Plachter B, et al. The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL. J Virol 1996;70 (11):7569-7579.
[60]Rist M, Cooper L, Elkington R, Walker S, Fazou C, Tellam J, et al. Ex vivo expansion of human cytomegalovirus-specific cytotoxic T cells by recombinant polyepitope: implications for HCMV immunotherapy. Eur J Immunol 2005;35(3):996-1007.
[61]Hebart H, Daginik S, Stevanovic S, Grigoleit U, Dobler A, Baur M, et al. Sensitive detection of human cytomegalovirus peptide-specific cytotoxic T-lymphocyte responses interferon-gamma-enzyme-linked immunospot assay and flow cytometry in healthy individuals and in patients after allogeneic stem cell transplantation. Blood 2002; 99 (10):3830-3837.
[62]Longmate J, York J, La Rosa C, Krishnan R, Zhang M, et al. Population coverage by HLA class-I restricted cytotoxic T-lymphocyte epitopes. Immunogenetics 2001;52(3-4): 165-173.
[63]Kondo E, Akatsuka Y, Kuzushima K, Tsujimura K, Asakura S, Tajima K,et al. Identification of novel CTL epitopes of CMV-pp65 presented by a variety of HLA alleles. Blood 2004; 103 (2): 630-638.
[64]Hebart H, Rauser G, Stevanovic S, Haenle C, Nussbaum AK, Meisner C, et al. A CTL epitope from human cytomegalovirus IE1 defined by combining prediction of HLA binding and proteasomal processing is the target of dominant immune responses in patients after allogeneic stem cell transplantation. Exp Hematol 2003;31(10):966-973.
[65]Nastke MD, Herrgen L, Walter S, Wernet D, Rammensee HG, Stevanovi6 S. Major contribution of codominant CD8 and CD4 T cell epitopes to the human cytomegalovirus-specific T cell repertoire. Cell Mol Life Sci 2005;62(1):77-86.
[66]Li Pira G, Bottone L, Ivaldi F, Pelizzoli R, Del Galdo F, Lozzi L,et al. Identification of new Th peptides from the cytomegalovirus protein pp65 to design a peptide library for generation of CD4 T cell lines for cellular immunoreconstitution. Int Immunol 2004;16(5):635-642.
[67]Khattab BA, Lindenmaier W, Frank R, Link H. Three T-cell epitopes within the C-terminal 265 amino acids of the matrix protein pp65 of human cytomegalovirus recognized by human lymphocytes. J Med Virol 1997;52(1):68-76.
[68]Gallot G, Vivien R, Ibisch C, Lute J, Davrinche C, Gaschet J, et al. Purification of Ag-specific T lymphocytes after direct peripheral blood mononuclear cell stimulation followed by CD25 selection. I. Application to CD4(+) or CD8(+) cytomegalovirus phosphoprotein pp65 epitope determination. J Immunol 2001;167(8):4196-4206.
[69]Dawson DV, Ozgur M, Sari K, Ghanayem M, Kostyu DD. Ramifications of HLA class I polymorphism and population genetics for vaccine development. Genet Epidemiol 2001 ;20(1):87-106.
[70]XUE Fu-zhong , WANGJie-zhen, HU Ping, et al.Forecasting system on spatial coverage of cumulative phenotypic frequency of HLA class I for designing HLA-based vaccines in China. Immunological Journal 2005;21(2):136-141.
[71]Thomas PG, Keating R, Hulse-Post DJ, Doherty PC.Cell-mediated Protection in Influenza Infection. Emerg Infect Dis 2006;12(1):48-54.
[72]Andersen MH, Schrama D, Thor Straten P, Becker JC.Cytotoxic T cells. J Invest Dermatol 2006;126(1):32-41.
[73]Falk K, Rotzschke O, Stevanovic S, Jung G, Rammensee HG.Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature. 1991 ;351 (6324):290-296.
[74]Peters B, Bui HH, Sidney J, Weng Z, Loffredo JT, Watkins DI, et al. A computational resource for the prediction of peptide binding to Indian rhesus macaque MHC class I molecules. Vaccine 2005;23(45):5212-5224.
[75]Ruppert J, Sidney J, Celis E, Kubo RT, Grey HM, Sette A. Prominent role of secondary anchor residues in peptide binding to HLA-A2.1molecules. Cell 1993;74(5):929-937.
[76]Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 1999;50(3-4):213-219.
[77]Hakenberg J, Nussbaum AK, Schild H, Rammensee HG, Kuttler C, Holzhutter HG,et al. MAPPP: MHC class I antigenic peptide processing prediction. Appl Bioinformatics 2003;2(3):155-158.
[78]Ghei M, Stroncek DF, Provenzano M. Analysis of memory T lymphocyte activity following stimulation with overlapping HLA-A*2402, A*0101 and Cw*0402 restricted CMV pp65 peptides. J Transl Med, 2005;3:23.
[79]Browne EP, Shenk T. Human cytomegalovirus UL83-coded pp65 virion protein inhibits antiviral gene expression in infected cells. Proc Natl Acad Sci USA 2003; 100(20): 11439-11444.
[80]Gyulai Z, Endresz V, Burian K, Pincus S, Toldy J, Cox WI, el al.Cytotoxic T lymphocyte(CTL)responses to human cytomegalovirus pp65, IE 1-exon 4, gB, pp150, and pp28 in healthy individuals; reevaluation of prevalence of 1El-pecific CTLs. J Infect Dis 2000;181(5):1537-1546.
[81]Schleiss MR, Heineman TC. Progress toward an elusive goal: current status of cytomegalovirus vaccines. Expert Rev Vaccines 2005;4(3):381-406.
[82]Bernstein DI, Schleiss MR, Berencsi K, Gonczol E, Dickey M, Khoury P, et al. Effect of previous or simultaneous immunization with canarypox expressing cytomegalovirus (CMV) glycoprotein B (gB) on response to subunit gB vaccine plus MF59 in healthy CMV-seronegative adults.J Infect Dis 2002; 185 (5):686-690.
[83]Furman MH, Loureiro J, Ploegh HL, Tortorella D. Ubiquitinylation of the Cytosolic domain of a type I membrane protein is not required to initiate its dislocation from the endoplasmic reticulum. J Biol Chem 2003;278(37):34804-34811.
[84]Hegde NR, Johnson DC. Human cytomegalovirus US2 causes similar effects on both major histocompatibility complex class I and II proteins in epithelial and glial cells. J Virol 2003;77(17):9287-9294.
[85]Popkin DL,Virgin HW. Murine cytomegalovirus infection inhibits tumor necrosis factor alpha responses in primary macrophages J Virol 2003;77(18):10125-10130.
[86]Gonczol E, Plotkin S.Development of a cytomegalovirus vaccine: lessons from recent clinical trials. Expert Opin Biol Ther 2001;1(3):401-412.
[1]Chrisp P, Clissold SP. Foscarnet.A review of its antiviral activity, pharmacokinetic properties and therapeutic use in immunocompromised patients with cytomegalovirus retinitis. Drugs 1991 ;41(1): 104-129.
[2]Faulds D,Heel RC.Ganciclovir. A review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy in cytomegalovirus infections.Drugs1990; 39 (4): 597-638.
[3]Griffiths PD, McLean A, Emery VC.Encouraging prospects for immunization against primary cytomegalovirus infection.Vaccine2001;19 (11-12):1356-1362
[4]Arvin AM, Fast P, Myers M, Plotkin S, Rabinovich R.Vaccine development to prevent cytomegalovirus disease:report from the National Vaccine Advisory Committee. Clin Infect Dis 2004;39(2):233-239.
[5]Xin K.Q, Jounai N, Someya K, Honma K, Mizuguchi H, Naganawa S, et al.Prime-boost vaccination with plasmid DNA and a chimeric adenovirus type 5 vector with type 35 fiber induces protective immunity against HIV.Gene Ther.2005;12 (24): 1769-1777.
[6]Thammanichanond D, Moneer S, Yotnda P, Aitken C, Earnest-Silveira L, Jackson D,et al.Fiber-modified recombinant adenoviral constructs encoding hepatitis C virus proteins induce potent HCV-specific T cell response. Clin Immunol 2008; 128(3): 329-339
[7]Xin KQ, Sekimoto Y, Takahashi T, Mizuguchi H, Ichino M, Yoshida A, et al.Chimeric adenovirus 5/35 vector containing the clade C HIV gag gene induces a cross-reactive immune response against HIV.Vaccine 2007;25 (19):3809-3815
[8]Hoffrnann D, Bayer W, Heim A, Potthoff A, Nettelbeck DM, Wildner O.Evaluation of twenty-one human adenovirus types and one infectivity-enhanced adenovirus for the treatment of malignant melanoma. J Invest Dermatol 2008;128(4):988-998.
[9]Hoffmann D, Meyer B, Wildner O.Improved glioblastoma treatment with Ad5/35 fiber chimeric conditionally replicating adenoviruses. J Gene Med 2007;9(9):764-778.
[10]Yotnda P, Onishi H, Heslop HE, Shayakhmetov D, Lieber A, Brenner M, et al. Efficient infection of primitive hematopoietic stem cells by modified adenovirus. Gene Ther 2001 ;8(12):930-937.
[11]Mizuguchi H, Hayakawa T. Adenovirus vectors containing chimeric type 5 and type 35 fiber proteins exhibit altered and expanded tropism and increase the size limit of foreign genes. Gene 2002;285(1-2):69-77.
[12]Mizuguchi H, Hayakawa T. Targeted adenovirus vectors. Hum Gene Ther 2004; 15(11):1034-1044
[13]Gaggar A, Shayakhmetov DM, Lieber A. CD46 is a cellular receptor for group B adenoviruses. Nat Med 2003;9 (11):1408-1412
[1]Nguyen Q,Champlin R,Giralt S,Rolston K,Raad I,Jacobson K,et al.Late cytomegalovirus pneumonia in adult allogeneic blood and marrow transplant recipients.Clin Infect Dis 1999;28(3):618-623.
[2]章咏裳.肾移植术后巨细胞病毒感染.中华器官移植杂志 2000;21(4):248-249.
[3]Temperton NJ.DNA vaccines against cytomagalovirus:current progress.Inter J Antimicrobial Angents 2002;19(3):169-172.
[4]Meijer E,Boland GJ,Verdonck LF.Prevention of cytomegalovirus disease in recipients of allogeneic stem cell transplants.Clin Microbiol Rev 2003;16(4):647-657.
[5]Micklethwaite KP,Clancy L,Sandher U,Hansen AM,Blyth E,Antonenas V,et al.Prophylactic infusion of cytomegalovirus-specific cytotoxic T lymphocytes stimulated with Ad5f35pp65 gene-modified dendritic cells after allogeneic hemopoietic stem cell transplantation.Blood 2008;112(10):3974-3981.
[6]Peggs KS,Verfuerth S,Pizzey A,Khan N,Moss P,Goldstone AH,et al.Reconstitution of T-cell repertoire after autologous stem cell transplantation:influence of CD34 selection and cytomegalovirus infection.Biol Blood Marrow Transplant 2003;9(3):198-205.
[7]Gandhi MK,Khanna R.Human cytomegalovirus:clinical aspects,immune regulation,and emerging treatments.Lancet Infect Dis 2004;4(12):725-738.
[8]Boeckh M,Leisenring W,Riddell SR,Bowden RA,Huang ML,et al.Late cytomegalovirus disease and mortality in recipients of allogeneic hematopoietic stem cell transplants:importance of viral load and T-cell immunity.Blood 2003;101:407-414.
[9]Crough T,Fazou C,Weiss J,Campbell S,Davenport MP,et al.Symptomatic and asymptomatic viral recrudescence in solid-organ transplant recipients and its relationship with the antigen-specific CD8(+) T-cell response.J Virol 2007;81:11538-11542.
[10]Engstrand M,Tournay C,Peyrat MA,Eriksson BM,Wadstrom J,et al.Characterization of CMVpp65-specific CD8+T lymphocytes using MHC tetramers in kidney transplant patients and healthy participants.Transplantation 2000;69(11):2243-2250.
[11]Riddell SR,Greenberg PD.Cellular adoptive immunotherapy after bone marrow transplantation.Cancer Treat Res 1995;76:337-369.
[12]Walter EA,Greenberg PD,Gilbert MJ,Finch RJ,Watanabe KS,Thomas ED,et al.Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.N Engl J Med 1995;333(16):1038-1044.
[13]Elkington R,Shoukry NH,Walker S,Crough T,Fazou C,Kaur A,et al.Cross-reactive recognition of human and primate cytomegalovirus sequences by human CD4 cytotoxic T lymphocytes specific for glycoprotein B and H.Eur J Immunol 2004;34(11):3216-3226.
[14]Elkington R,Walker S,Crough T,Menzies M,Tellam J,Bharadwaj M,et al.Ex vivoprofiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecifie reactivities in healthy virus carriers.J Virol 2003;77(9):5226-5240.
[15]Sylwester AW,Mitchell BL,Edgar JB,Taormina C,Pelte C,Ruchti F,et al.Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects.J Exp Med 2005;202(5):673-685.
[16]Manley TJ,Luy L,Jones T,Boeckh M,Mutimer H,Riddell SR.Immune evasion proteins of human cytomegalovirus do not prevent a diverse CD8+cytotoxic T-cell response in natural infection.Blood 2004;104(4):1075-1082.
[17]Zhong J,Khanna R.Vaccine strategies against human cytomegalovirus infection.Expert Rev Anti Infect Ther 2007;5(3):449-459.
[18]Rist M,Cooper L,Elkington R,Walker S,Fazou C,Tellam J,et al.Ex vivo expansion of human cytomegalovirus-specific cytotoxic T cells by recombinant polyepitope:implications for HCMV immunotherapy.Eur J Immunol 2005;35(3):996-1007.
[19]Berencsi K,Gyulai Z,G6ncz(o|¨)l E,Pincus S,Cox WI,Michelson S,et al.A canarypox vector-expressing cytomegalovirus(CMV) phosphoprotein 65 induces long-lasting cytotoxic T cell responses in human CMV-seronegative subjects.J Infect Dis 2001;183(8):1171-1179.
[20]Morello CS,Ye M,Hung S,Kelley LA,Spector DH.Systemic Priming-Boosting Immunization with a Trivalent Plasmid DNA and Inactivated Murine Cytomegalovirus(MCMV) Vaccine Provides Long-Term Protection against Viral Replication following Systemic or Mucosal MCMV Challenge.J Virol 2005;79(1):159-175.
[21]Wang Z,La Rosa C,Li Z,Ly H,Krishnan A,Martinez J,et al.Vaccine properties of a novel marker gene-free recombinant modified vaccinia Ankara expressing immunodominant CMV antigens pp65 and IE1. Vaccine 2007;25 (6): 1132-1141.
[22]Dawson DV, Ozgur M, Sari K, Ghanayem M, Kostyu DD. Ramifications of HLA class I polymorphism and population genetics for vaccine development. Genet Epidemiol 2001;20(1):87-106.
[23]Stewart AK, Lassam NJ, Quirt IC, Bailey DJ, Rotstein LE, Krajden M, et al. Adenovector-mediated gene delivery of interleukin-2 in metastatic breast cancer and melanoma: results of a phase 1 clinical trial. Gene Ther 1999;6(3):350-363.
[24]Yotnda P, Onishi H, Heslop HE, Shayakhmetov D, Lieber A, Brenner M, et al. Efficient infection of primitive hematopoietic stem cells by modified adenovirus. Gene Ther 2001;8(12):930-937.
[25]Thomson SA, Khanna R, Gardner J, Burrows SR, Coupar B, Moss DJ,et al. Minimal epitopes expressed in a recombinant polyepitope protein are processed and presented to CD8+ cytotoxic T cells: implications for vaccine design. Proc Natl Acad Sci U S A 1995;92(13):5845-5849.
[26]Mocarski ES Jr.Immunomodulation by cytomegaloviruses: manipulative strategies beyond evasion. Trends Microbiol 2002;10(7):332-339.
[27]Farrell HE, Davis-Poynter NJ, Andrews DM, Degli-Esposti MA.Function of CMV-encoded MHC class I homologues. Curr Top Microbiol Immunol 2002;269: 131-151.
[28]Zhong J, Rist M, Cooper L, Smith C, Khanna R.Induction of pluripotent protective immunity following immunisation with a chimeric vaccine against human cytomegalovirus. PLOS ONE 2008;3(9):3256
[29]Khanna R, Diamond DJ.Human cytomegalovirus vaccine: time to look for alternative options. Trends Mol Med 2006;12(1):26-33.
[30]Horn B, Bao L, Dunham K, Stamer M, Adler S, Cowan M, et al. Infusion of cytomegalovirus specific cytotoxic T lymphocytes from a sero-negative donor can facilitate resolution of infection and immune reconstitution. Pediatr Infect Dis J 2009;28(1):65-67.