摘要
腺病毒(Adenovirus, Adv)感染在全球范围内广泛流行,具有传染性强,宿主范围广的特点。腺病毒不仅是呼吸道感染、结膜感染和肠道感染较为常见的病原体之一,它们还可能引起无菌性脑膜炎、心肌炎、脑炎和肝炎等。对于移植手术患者、HIV感染者等免疫功能低下的人群,腺病毒更是常见感染因素之一。因此,对于腺病毒感染的预防、诊断和治疗一直都是国内外学者关注的热点之一
人类腺病毒属于腺病毒科(Adenoviridae)的哺乳动物腺病毒属(Mastadenovirus)。人类腺病毒根据不同的免疫学、生物和生化特性分为7个亚群(A至G),至今国际病毒分类委员会(ICTV)已经公布57种血清型。在2011年,有科学家报道发现了58型腺病毒,但还未通过ICTV的鉴定。不同血清型的腺病毒宿主嗜性不同,因此表现出不同的临床症状。B组的3型和7型腺病毒以及E组的4型腺病毒是呼吸道感染的常见病毒,常引起下呼吸道感染症状如发热,咳嗽和肺炎等,以及在夏季引起咽结膜性发热的爆发感染。国内在2004-2005年间多地区暴发了3型腺病毒感染,流行范围包括江苏、北京、湖北等地；广州在2004年也报道了一起幼儿园咽结膜性发热感染；山西省2009年暴发了一起7型腺病毒感染,该病毒引起婴儿严重下呼吸道症状；2008-2009年,3型腺病毒在加拿大NewBrunswick省暴发流行,几百人被感染,甚至有3名病人死亡；在美国新兵训练营和老年看护机构都报道有4型腺病毒的感染。
目前,腺病毒感染和其他病毒性感染疾病一样,没有特效药物及疗效显著的治疗方法,主要依靠机体自身免疫系统的抵抗反应,消灭病毒而痊愈。先前的研究已表明,机体产生的腺病毒抗体对同型腺病毒具有长久而稳定的保护性。因此,开发高效、安全低价的腺病毒疫苗是预防腺病毒感染的有效方法。70年代初,美国军队曾在新兵训练时期使用Adv4-Adv7口服疫苗,使腺病毒相关的呼吸道疾病得到了有效的遏制。1999年,疫苗停止供应后,新兵训练中心监测发现呼吸道感染率明显上升。直到2011年经FDA批准启用新的Adv4-Adv7口服疫苗,再次有效降低了呼吸道感染疾病的发生。但由于口服活疫苗经粪便排出后仍具有传染的可能性；同时服用两种或以上不同亚型的腺病毒疫苗可能进行同源重组而产生新型病毒,带来新的感染风险,故国外只限于军队中使用。除此之外,国内外关于腺病毒的疫苗研究及临床应用都还在基础研究阶段。
虽然腺病毒的疫苗研究还在起步,但是以腺病毒为载体的基因工程疫苗已经被广泛研究。腺病毒载体和其他病毒载体相比,具有以下优点：病毒结构和功能背景清楚；可以携带10kb以内的外源基因；感染范围广；结构稳定,不整合到宿主细胞基因组也不易发生突变等。最新的数据表明全球约有1/4的基因治疗和疫苗载体的临床试验应用了Adv2和Adv5载体。但是强烈的宿主排斥反应一直是腺病毒载体在临床上应用的阻碍,而且,近期有学者发现,因为腺病毒受体CAR在许多肿瘤细胞表面并不表达,导致病毒载体无法吸附细胞,对CAR受体依赖的2型和5型腺病毒载体可能并不适合肿瘤治疗使用。人3型腺病毒的受体作用机制虽仍未清楚,但目前研究表明大部分肿瘤细胞表面都有其受体的表达,因而Adv3将可能作为肿瘤治疗的新型载体候选,也有些学者已经在进行4型,35型等其他血清型腺病毒载体相关的研究。
本课题组自2007年开始腺病毒疫苗的研发工作,至今,我们的研究团队已经围绕腺病毒基因工程疫苗的研制做了大量相关研究,并取得阶段性进展。目前,我们已经成功构建了非复制型基因工程Adv3；置换Adv3和Adv7的六邻体区域构建新的重组腺病毒,并鉴定其免疫原性也发生了改变；对3型和7型腺病毒中和表位的进行了预测和鉴定,置换Adv3和Adv7上中和表位,构建新的嵌合病毒,研究证明其在动物实验中可同时诱导抗人3型和7型腺病毒中和抗体；以3型腺病毒为载体,成功构建了带有肠道病毒EV71中和表位的重组3型腺病毒等,其可能为同时抗EV71和Adv3的双价疫苗。
本课题组在腺病毒疫苗研究中取得的成果都已经发表,然而,当疫苗应用于临床,则需要了解目标人群中Adv中和抗体的预存情况,清楚了解普通人群血清中是否存在中和抗体,中和抗体阳性率以及其分布特点,这些结果将指导我们选择合适的免疫人群及最佳免疫时间,对疫苗的开发及使用具有指导意义。因此,本研究的第一部分主要对广州健康人群体内3型,4型和7型腺病毒中和抗体水平进行了检测和分析,了解腺病毒中和抗体分布特点及变化。
本研究的第二部分则是在课题组前期对Adv3中和表位的预测及研究基础上,通过制备和筛选Adv3的中和性单克隆抗体,以此为媒介,对3型腺病毒的中和表位进行鉴定研究。通过体外中和抗体和中和表位的特异性结合,确认预测中和表位的有效序列。
第一章普通人群血清中腺病毒的中和抗体分析
本实验收集了2008年广州市儿童医院职工体检血清标本、广州银行医院体检血清标本和门诊非呼吸道患儿血清标本共452份,通过微孔板血清中和试验检测3型,4型和7型腺病毒中和抗体水平。将标本按照年龄共分为7个组：0-6m组,7-12m组,1-3y组,3-7y组,7-20y组,20-40y组合40-60y组。先进行病毒滴度测定和血清效价预实验,确定微孔板血清中和试验的病毒工作浓度是100TCID50,血清的稀释比例是1：8。然后开始进行血清标本的中和试验,当阳性对照、正常细胞对照,血清毒性对照全部成立时,进行判定结果判定,以被检血清孔出现100%CPE判为阴性,50%以上细胞出现保护者为阳性。
采用SPSS20.0软件分析数据,通过R×C资料卡方检验,普通人群腺病毒中和抗体平均分布具有统计学差异,P<0.05。从7-20y组开始,腺病毒中和抗体阳性率显著上升。除了0-6m组人群,3型,4型和7型腺病毒中和抗体阳性率都随年龄增加而增高。3型腺病毒中和抗体率最低的是6-12m组和1-3y组,之后,随年龄增加,在20-40y组人群中和抗体阳性率已经达到78%,在40-60y组中和抗体阳性率也稳定在78%；4型腺病毒中和抗体率最低是在6-12m组,随年龄增加而升高,在7-20y组人群中和抗体阳性率达到60%,20-40y组人群中和抗体阳性率保持稳定,但在40-60y组人群中和抗体阳性率又开始上升至82%；7型腺病毒中和抗体率最低的是1-3y组,阳性率仅5%,之后随年龄增加而上升,直到40-60y组人群中和抗体阳性率高达76%。研究结果充分显示了人群中和抗体水平的年龄依赖性,而且不同血清型的腺病毒中和抗体水平分布也有所不同。
对0-1y婴幼儿腺病毒中和抗体水平进行分析,结果发现0-1m婴儿群体的3型腺病毒中和抗体阳性率最高,达到58%,之后开始下降,到3-6m年龄组的时阳性率最低,阳性率为13%,之后开始缓慢上升。7型腺病毒中和抗体水平变化趋势和3型腺病毒相同,但是每组的阳性率都要低于3型腺病毒中和抗体水平,尤其是3-6m组,阳性中和抗体率为0。根据卡方分析,Adv3组x2=16.979,P<0.05,结果具有统计学差异,Adv7的P<0.05,其抗体水平变化也有差异,而Adv4组P>0.05,抗体变化无统计学差异。而4型腺病毒中和抗体阳性率变化和以上两组都不同,它也是0-1m组最高,但其他3个年龄组变化不大,都维持在10-13%之间,可能和其感染方式及流行特点有关。该组分析显示母体抗体对0-6m婴儿具有保护作用。
不同血清型腺病毒感染后会诱发型特异型的中和抗体,因此,在人体内可能会同时存在不同型的腺病毒中和抗体,我们对人群中不同型腺病毒中和抗体混合存在的情况进行分析,结果显示：3种血清型病毒的中和抗体都不存在的人数比例在6-12m组和1-3y组最高,都有70%以上,之后,随年龄增大而比例下降,尤其是从3-7y组到7-20y组,比例急剧下降,从54%降至9%,而到40-60y组人群,抗体阳性比例为0,即检测人群中任何人都可能已经携带了1种或多种腺病毒中和抗体；只携带一种腺病毒中和抗体的人群比例最高的是0-6m组和7-20y组,0-6m组人群抗体阳性率为45%,7-20y组人群抗体阳性率为37%。携带两种和三种腺病毒中和抗体人群比例则是随年龄增加而增加,40-60y年龄组人群中,他们的中和抗体阳性率分别是42%和46%。这组研究揭示,每个人在一生中都可能遭遇一次或者多次的腺病毒感染,腺病毒疫苗的使用可能减少每个人被腺病毒感染的几率。
对不同环境中人群的腺病毒中和抗体水平进行分析,从数据来看,医护职工不同型别的腺病毒中和抗体阳性率都要稍高于普通人群,但通过卡方检验,三种腺病毒中和抗体的阳性率分别统计,他们的P>0.05,说明不同腺病毒的两种人群检测结果都无显著性差异。
本章从不同角度对人群腺病毒中和抗体阳性水平进行了分析和比较,结果发现腺病毒中和抗体的阳性率有明显的年龄依赖性,随着年龄增长,抗体携带阳性率也增高,6m-3y之间,不同型腺病毒的中和抗体阳性率都偏低,这段时间不但是腺病毒的易感时期,也可能是接种腺病毒疫苗的最佳时期。而且不同血清型的腺病毒中和抗体的流行分布也有不同,3型和7型腺病毒的流行趋势较为接近,而4型腺病毒散发性更为明显。腺病毒感染在人群中普遍存在,40-60y人群抗体携带的数据显示,每个人都可能携带一种或多种腺病毒中和抗体。本次研究的结果将为腺病毒疫苗的开发及腺病毒基因工程载体的研究都有一定的促进作用。
第二章3型腺病毒单克隆抗体的制备和初步鉴定
本实验将增殖的3型腺病毒用蔗糖梯度密度离心方法进行纯化,然后免疫BALB/c小鼠,通过PEG使小鼠脾细胞和SP2/0细胞融合,并使用HAT选择性培养基筛选融合细胞,用间接ELISA方法测定单克隆抗体的效价,结果获得1株能稳定传代的杂交瘤细胞株1B6。然后对获得的单克隆抗体1B6进行免疫球蛋白亚型,1B6MAb鉴定IgG1亚型,轻链为K链。然后制备腹水,最后用饱和硫酸铵-辛酸沉淀法对单克隆抗体腹水进行纯化,获得高效价的1B6MAb。根据ELISA和Western blotting结果显示,1B6MAb与Adv3病毒粒子和原核表达的六邻体蛋白有良好的反应性和特异性,同时也证明1B6MAb可以识别六邻体的线性表位。并且通过细胞免疫化学法检测,也能直观看出1B6抗体只和Adv3腺病毒感染的细胞有高度特异反应性的结合,1B6MAb对病毒蛋白表位的天然状态也能识别,都具有反应性,而且抗体灵敏度高,效价能达到105以上。
通过本次实验,我们获得了一株能够稳定传代的杂交瘤细胞株,1B6MAb既能识别原核表达的六邻体蛋白,也能识别天然病毒,很可能是一株针对六邻体蛋白线性表位的多肽,而这个表位是否具有中和作用,还需要进一步证实。
第三章单克隆抗体1B6对3型腺病毒中和表位的鉴定和分析
首先,复苏并增殖Ad3EGF和A7HAd3egf两株基因工程构建病毒,Ad3EGF病毒载体是将EGFP蛋白转导进病毒载体,使其带有EGFP标记,易于被检测。而A7HAd3egf病毒则是前期同事将7型腺病毒的六邻体蛋白取代3型腺病毒的六邻体蛋白而形成的基因工程构建病毒,该病毒已经被从分子和免疫学角度证实其合成表达的六邻体蛋白确实是7型腺病毒六邻体蛋白。
我们将1B6MAb与这两株带有绿色荧光蛋白的基因工程构建病毒孵育后,进行体外中和试验,通过荧光测定法,检测抗体和病毒中和后,细胞受感染的情况。采用SPSS两独立样本t检验分析,每组t检验比较的P<0.05,即每组数据都有显著差异,即表明和对照试验相比,Ad3EGF病毒感染能被1B6抗体保护。从荧光检测的结果来看,在1：64抗体稀释作用的时候,病毒的复制未受到影响,Ad3EGF和A7HAd3egf病毒荧光检测相对比值都在70%以上,而A7HAd3egf荧光值更是高达99%。而之后,抗体稀释到1：32时,Ad3EGF的复制开始受到1B6抗体的抑制,荧光值下降到40%,在1：8抗体稀释度时候,相对荧光值已经在小于20%,病毒复制明显被抑制,Ad3EGF荧光值随抗体滴度的增加而明显下降。而病毒A7HAd3egf荧光值则一直稳定在80-100%之间,它的复制几乎未受到抗体中和作用限制。中和试验的结果可以明显看出,1B6抗体具有中和作用,而且特异针对3型腺病毒的六邻体蛋白。
然后,我们通过比对同源性较高的Adv3-gz01和Adv7-gz8腺病毒的六邻体氨基酸序列,选择了6个变异区段进行原核表达。间接ELISA结果可以看出,1B6MAb对R7和R7s都具有高特异性,但是R7s的特异性比R7稍低,而对其他变异区肽段都呈阴性反应。推测可能R7s缺少了几个氨基酸残基,从而亲和力比R7弱,但这个推测还有待进一步的实验证实。
为了对鉴定的表位进行确证,我们又设计了竞争抑制实验。ELISA实验采用3×4析因分析结果,三因素的P<0.05,说明稀释浓度和竞争抗原不同,所检测的OD值都有差异,而且稀释浓度和竞争抗原两因素间存在交互作用,即蛋白浓度的高低也影响了其竞争能力的高低。R7片段上的表位可以完全封闭MAb1B6的对位,令单克隆抗体失去对全长衣壳蛋白的识别能力,R7s片段封闭功能较弱,只能部分封闭单克隆抗体的对位,而作为对照的R1蛋白,没有封闭能力。
最后,我们还把本课题组之前构造的各种腺病毒基因工程载体,都与MAb1B6抗体进行了特异性验证,结果发现,只要是含有3型腺病毒六邻体R7序列的重组病毒都能够被识别,而其他R7序列突变的六邻体蛋白的重组腺病毒则不能被识别。该实验不但再次证实了MAb1B6对3型腺病毒六邻体蛋白的特异性。而且,由于这些腺病毒基因工程载体都保持了表位的天然结构,再次证明1B6抗体可以识别天然表位,也能识别线性表位,而我们所鉴定的R7表位应该是一个连续性的线性的天然表位。
本章不但通过中和试验验证了1B6MAb对3型腺病毒六邻体的中和作用,而且能专一识别六邻体上R7表位,也通过竞争ELISA试验等证实了六邻体上R7表位是一个连续性线性的天然表位。
结论
一、本课题对普通人群血清中腺病毒中和抗体水平从不同角度进行了分析。结果表明,成年人群血清中70%以上都带有一种以上腺病毒中和抗体,尤其是40-60y年龄组,人群中和抗体阳性率为100%。6m以前的婴儿,50%以上都受到母体抗体的保护,而6m-3y年龄组人群抗体阳性率最低,他们是腺病毒感染的易感人群,而该年龄段也是疫苗接种的最佳时期。
二、本课题获得了一株能高特异性、高灵敏性的能识别连续的线性的天然表位的中和性单克隆抗体。
三、3型腺病毒六邻体的R7多肽被鉴定是一个中和表位,抗原部位能被1B6单克隆抗体封闭。
Background
Adenovirus (Adv) infections spread through the whole world. It is strong infective and can cause respiratory infection, acute pharyngitis, epidemic conjunctivitis, gastrointestinal infection and other multi-system organ infections. HIV people and transplantation patient is likely to be infected by Adenovirus due to immunocompromised. Therefore, more and more researchers focus on adenovirus prevention, diagnosis and treatment.
Human adenovirus (HAdVs) belongs to family Adenoviridae, genus Mastadenovirus. The57serotypes of HAdV have been places in seven species (A to G) by International Taxonomy of Viruses Commission (ICTV), depending on the immunological, biological and biochemical characteristics. In2011, the serotype58of adenovirus had been reported which have been submitted to the ICTV. Different serotypes of adenovirus could infect different tissue, which caused different clinical symptoms. HAdV-3and-7of Group B, and HAdV-4of Group E often cause symptoms such as fever, cough, pneumonia, pharyngeal conjunctival fever and other lower respiratory tract infections. In2004-2005, an outbreak and epidemic of HAdV- 3occurred in Jiangsu, Hubei, Jiangsu Province and Beijing; An outbreak of pharyngeal-conjunctival fever induced by HAdV-3occurred in Guangzhou in2004; An outbreak of severe lower respiratory tract symptoms with HAdV-7occurred in Shanxi Province in2009; In2008-2009, an outbreak of HAdV-3occurred in New Brunswick, Canada, with hundreds of people infected. The infections of HAdV-4spread in military trainees and a long-term care facility for elderly in the U.S.
Similar other viral infections, Adenovirus infection cured by the resistance of own immune system, not by the effects of drugs. Previous studies have shown serum antibodies against adenovirus could prevent infection by the same serotype adenovirus. Therefore, the development of efficient, safe and low-cost adenovirus vaccine is the most effective way to prevent adenovirus infection. The introduction in1971of oral vaccines against adenovirus types4and7for use among military trainees in the U.S., dramatically reduced the incidence of adenovirus disease and febrile respiratory illnesses in that population. In1999, the vaccine stopped the supply. In subsequent years, the incidence of febrile respiratory illness increased markedly at basic training centers. In2011, the FDA approved the new oral Adenovirus Type4and Type7Vaccine to market. Subsequently, rates of febrile respiratory illnesses and adenovirus isolations markedly declined. But, the live oral vaccines were limited used in the military for the risks of infection and recombination. Therefore, development of safe and effective adenovirus vaccine need a long time.
On the other hand, Adenoviruses are currently the most widely used vectors in gene therapy fields. In comparison with other viral vectors, adenovirus vectors have many virtues such as the high capacity to genes, not integrating into the genome of target cells, high-titer and stability, reproduction not only in the intestine but also in the respiratory tract, and with the prospects for the development of oral vaccine. Latest data show Adv2and Adv5vectors are a quarter of all vaccine vectors. Adenovirus vectors were limited to application by intensive host rejection. Recently, the research reports of Adv2and Adv5vectors show the two vectors would not to be used in cancer treatment for the adenovirus receptor CAR not expressed in the surface of many tumor cells. While, Adv3receptor could express in the surface of tumor cells. So the Adv3will likely be a new vector used in cancer treatment.
Some researches of engineered vaccine against HAdV-3and-7have been reported by our research group. A replication-competent human Adv3-based vector was successfully constructed; a recombinant virus of Adv3packaged by Adv7hexon was constructed in2011. Recently, an Enterovirus71vaccine was constructed with neutralizing epitope incorporation within Adv3hexon. To the researches of adenovirus vaccine and adenovirus vector, it will be helpful to analysis prevalence of serum neutralizing antibodies against the Adv3and Adv7in the healthy people. Therefore, it is the part I of my work of the survey to the prevalence of serum neutralizing antibodies of Adenovirus in Guangzhou. The second part of my work is the study about preparation and identification monoclonal antibodies against Adv3. The third part of my work is analysis of neutralizing epitope of Adv3.
Part I Seroprevalence of Neutralizing Antibodies to Human Adenovirus in Healthy People
A total of452sera from humans were collected and tested for serum NAb to HAdV-3,4and7. Serum samples were collected in2007, from Guangzhou Children's Hospital and Guangzhou Bank hospital. Sera were obtained from adults who had a medical examination in the Guangzhou Bank Hospital, and children not exhibiting signs of respiratory tract infection in the Guangzhou Children's Hospital. According age of people, the sera were grouped into seven sets:0-6month,7-12month,1-3year,3-7year,7-20year,20-40year and40-60year. By the pre-experiments of virus titer and serum titer, the virus titers were100TCID50and the serum dilution ratio is1:8. Then Micro-serum neutralization (SN) assay were used to analysis serum neutralizations of Adenovirus with positive control, normal cell control, serum toxicity control. The results were assessed according the standards:it is negative if with100%CPE by the sample, and it is positive with less than50%CPE.
The SN assay results showed that the positive rates of neutralizing antibodies of Adv3, Adv4and Adv7increased with age except0-6month group. It was the lowest in Adv3neutralizing antibodies positive rate in the0-12month group and1-3year group. In the20-40year and40-60year group, the Adv3antibody positive was the highest at78%.; The antibody positive rate of Adv4was the lowest in the6-12month group and the higher in the7-20year group(60%). The same positive rate was in the20-40year group, and the highest rate was82%in the40-60year group; the lowest positive rate of Adv7was only5%in the1-3year group, then, the positive rate was increased with age. In the40-60year group, the positive rate was the highest (76%).
The samples of0-1year group were analyzed according the divided group:0-1month,1-3month,3-6month and6-12month. The results showed the highest antibody positive rate of Adv3was58%in the0-1month group. Then the rate decreased to the lowest positive rate13%in the3-6month group. The trends of the positive rate of Adv7were same to Adv3, although Adv7positive rate for each group was lower than the rate of Adv3. According to the chi-square analysis, χ2=12.05, P <0.01between Adv3and Adv7, their antibody positive rates were of a significant difference. The trends of the positive rate of Adv4antibody were different with Adv3and Adv7. The positive rate was also the highest in the0-1month group, but in the other groups, the positive rate was maintained to10-13%.
Type-specific neutralizing antibodies were induced by different serotype adenovirus in vivo. One serum sample could possess several different types of neutralizing antibodies. According the data of three kinds of antibodies, the proportion without any neutralizing antibody against HAdV-3,-4and-7was the highest in the6-12month group and1-3year group, which was more than70%. The proportion decreased from54%to9%in the3-7year group with age. While in the40-60age group population, none was antibody negative. The positive rate with only one kind of adenovirus antibody was highest in the0-6month group and7-20year group. The positive rate with two or three kinds of adenovirus antibodies increased with age. In the40-60year group, their neutralizing antibody positive rate is42%and46%, respectively.
Comparing the antibody positive rate of the group people of different environment, the results showed that:the antibody positive rate of people who work in hospital was slightly higher than the other, but by the chi-square test, x2=0.19, P> 0.05, there was no significant difference between the two sets of data.
In this part, we compared these data from several different contents. The results showed the antibody positive rate increased with age. In the6months to3years old people, neutralizing antibody positive rate was lower than the others. The periods could be the best time to vaccination. The prevalent trends of Adv3antibody was likely to Adv7, while the prevalent of Adv4antibody was characteristic. The results showed that adenovirus infection was widespread in the Guangzhou. In the40-60year group, everyone has been infected by adenovirus once or more. These researches would be helpful to vaccine and vector development.
Part II Preparation and identification of monoclonal antibodies against Adenovirus type3
In this study, the Adv3purified by sucrose density gradient centrifugation was used to immunize BALB/c mice. The immunized mouse spleen cells were fused with the SP2/0cells by PEG3350. Then, the fused cells were selected by HAT medium and cloned by dilution culture. The monoclonal antibody titer was measured by indirect ELISA method. At last, a hybridoma cell strain1B6was obtained and cryopreserved. The immunoglobulin subtype of monoclonal antibody1B6was identified as IgGl subtype, the light chain of antibody was κ chain. Then ascites antibodies were purified by ammonium persulfate-caprylic acid precipitation for high titer. By ELISA and Western blotting, the1B6MAb was specific to Adv3virus particles and the hexon protein from prokaryotic expression. By immunocytochemistry, the MAb could recognize the cells infected by Adv3. So the1B6MAb might to a linear natural epitope.
In the part, we obtained a stable hybridoma cell lines, which was identified by hexon protein of prokaryotic expression and the natural virus. The MAb is likely to complement to a linear natural hexon epitope. Work is needed to be further confirmed whether this epitope could to be a neutralizing effect.
PartⅢ Identification of neutralizing epitopes of Adenovirus type3
The recombinant virus of Ad3EGF which was inserted by EGFP and A7HAd3egf whose hexon of Ad3EGF was substituted by the hexon of Adv7was replication-competent.
In vitro Adv neutralization experiments,1B6MAb was mixed with the two recombinant viruses and incubated for1h. Then, the mixtures were transferred to96-well plates containing85-95%confluent monolayers of HEp-2cells. After72h, infected cells were analyzed with a Varioskan Flash Multimode Reader (Thermo Scientific) to measure the EGFP expression. Results showed the virus replication were decreased in the1:64antibody dilution because the relative fluorescent value of Ad3EGF and A7HAd3egf viruses are more than70. When the antibody was diluted to1:32, replication of Ad3EGF was inhibited by the1B6antibody for the fluorescence value dropped to40. In the1:8antibody dilutions, relative fluorescence value has been less than20, which indicated the virus replication was inhibited significantly. The relative fluorescence value of virus A7HAd3egf was stabilized at80-100, when the replication could not be inhibited. The results of in vitro Adv neutralization experiments showed that the1B6antibody was a neutralizing antibody, and was specific to the hexon protein of Adv3.
By blasting the amino acid sequence of Adv3-gz01and Adv7-GZ8adenovirus hexon, six HVRs were expressed in prokaryotic systems. The results showed1B6MAb was identical to R7and R7s. R7s specificity was lower than the R7which could be caused for several amino acid residues absently.
To indentify the epitopes, we designed a competitive inhibition ELISA assay. The data showed that the R7fragment epitope can be completely closed to the MAb1B6, while the R7s fragment closed function is weak, which only partially enclosed the MAb.
Finally, some Adv vectors which had been constructed by colleagues of the group identified with the MAb by ELISA. Results showed MAb1B6could specifically identify the vectors which had the hexon of Adv3. As R7epitope could be indentified in the natural structure, it is a continuous linear natural epitope, which epitope was consistent with the epitope expressed in prokaryotic systems.
In the part,1B6MAb were identified as a neutralization antibody by vitro Adv neutralization experiments. The R7epitope of Adv3hexon also was identified as a continuous linear natural epitopes by competitive ELISA.
Conclusion
i. The prevalence of neutralizing antibody results show that adult population with Adv antibodies was more than70%. In the40-60years group, none was antibody negative. In the0-6month, more than50%of infants were protected by maternal antibodies. The population of6months to3years was likely to adenovirus infection and vaccination for low antibody positive rate.
ii. MAb1B6was a high specificity, high sensitivity neutralizing antibody to the hexon of Adv3.
iii. R7epitope was a linear natural neutralizing epitopes of Adv3hexon which can be characteristics to MAb1B6.
引文
[1]Rowe W.P., Huebner P.R., Gilrnore L.K., et al., Isolation of a agent from human adenoids undergroing spontaneous degeneration in tissue culture. Proc.Soc Exp Biol Med,1953.84(2):p.570-573.
[2]Hilleman, M.R. and J.H. Werner, Recovery of new agent from patients with acute respiratory illness. Proc Soc Exp Biol Med,1954.85(1):p.183-8.
[3]Hilleman, M.R., Werner, J.H., Dascomb, H. E.& Butler, R. L, (1955). Epidemiology investigations with respiratory disease virus RI-67. Am J Public Health 45,203-210.
[4]张忠信,ICTV第九次报告对病毒分类系统的一些修改.病毒学报,2012.28(5)：p.595-599
[5]殷震,刘景华,《动物病毒学》第二版.北京：科学出版社,1997：p.329-331,343-348,422-428,777-795.
[6]Elizabeth B. Liu, Leonardo Ferreyra, et al., Genetic analysis of a novel human adenovirus with a serologically unique hexon and a recombinant fiber gene. PLOS ONE,2011.6(9):e24491.
[7]金奇,《医学分子病毒学》.北京：科学出版社,2001(2)：p.691-708.
[8]Nia Tatsis, Hildegund C, Ertl J, Adenoviruses as vaccine vectors. Molecular Therapy,2004.10:p.616-629.
[9]Hisatoshi Kaneko.Koki Aoki, Susumu Ishida, et al.,Recombination analysis of intermediate human adenovirus type 53 in Japan by complete genome sequence Journal of General Virology.2011,92:1251-1259.
[10]Qiwei Zhang, Donald Seto, et al., Genome sequence of human adenovirus type 55, a re-emergent acute respiratory disease pathogen in China. Journal of Virology, p.12441-12442.
[11]Satoshi Hiroil, Keiichi Furubayashi, Takuya Kawahatal, et al., A Case of Urethritis Caused by Human Adenovirus Type 56, Jpn. J. Infect. Dis.,2012, 65:273-274.
[12]Toshihide Ariga, Yasushi Shimada, et al., New genome type of adenovirus serotype 4 caused nosocomial infections associated with epidemic conjunctivitis in Japan. Journal of Clinical Microbiology,2004.42(8):3644-3648.
[13]荻野武雄,才超,腺病毒7型复兴感染.日本医学介绍,2001.8：p.365-366.
[14]Li QG, Wadell G, Analysis of 15 different genome types of adenovirus type 7 isolated on five countinents. J Virol,1986.60(1):p.331-335.
[15]Wadell G, Cooney MK, Lindman AC, et al., Molecular epidemiology of adenoviruses:globule distfibution of adenovirus genome type. J Clin Micmbiol, 1985.21(1):p.403-408.
[16]Dean D. Erdman, Wanhong Xu, et al., Molecular epidemioligy of adenovirus type 7 in the United States,1966-2000. Emerging Infectious Diseases,2002. 8(3):p.269-277.
[17]Yae Jean Kim, Jung Yun Hong, et al., Genome type analysis of adenovirus types 3 and 7 isolated during successive outbreaks of lower respiratory tract infections in Children. Journal of Clinical Microbiology,2003.41(10):p. 4594-4599.
[18]Tsugnto Pujirnoto, Itsuki Hamamoto, et al., Molecular epidemiology of adenovirus type 3 detected from 1994 to 2006 in Hyogo Prefecture, Japan. Jpn. J. Infect. Dis.,2008.61:p.143-145.
[19]唐浏英,许文波,腺病毒分子流行病学研究.中华流行病学杂志,2008.29(8)：p.836-839.
[20]Mamoru Noda, Tetsuya Yoshida, et al., Molecular and epidemiological analyses of human adenovirus type 7 strains isolated from the 1995 nationwide outbreak in Japan. Journal of Clinical Microbiology,2002.40(1):p.140-145.
[21]Hierholzer, J.C., Adenovirus. In Diagnostic Procedures for Viral,Rickettsial and Chlamydial Infections,7th edn,169-188
[22]傅文永,梁东,郑永臣,等.长春地区引起的婴幼儿肺炎的3.、7型腺病毒分子流行病学研究.病毒学杂志,1990,2：142-146.
[23]唐浏英,熊英,等,2004年中国四省市腺病毒疫情的流行病学和病原学研究. 疾病监测,2006.21(6)：p.285-287.
[24]居丽雯,蒋露芳,姜仁杰,等.江苏东台市急性上呼吸道腺病毒感染的病毒基因分型.中华传染病杂志,2006,24(3)：194-196
[25]刘维斯,宋铁,等,一起由腺病毒3型引起的儿童咽结合膜热暴发.疾病监测,2005.20(10)：p.176-179.
[26]刘雪林,宋宏彬,等,一起腺病毒感染暴发的分子流行病学调查.解放军预防医学杂志,2004.22(3)：p.199-200.
[27]Stewart, P.L., Fuller, S.D.& Burnett, R.M., Difference imaging of adenovirus: bridging the resolution gap between X-ray crystallography and electron microscopy. EMBO Journal.12,2589-2599.
[28]Rux J J, Burnett RM., Adenovirus structure. Hum Gene Ther,2004.15: p.1167-1176.
[29]Vellinga J, Van Der Heijdt S, Hoeben R C, The adenovirus capsid:major progress in minor proteins [J]. J Gen Virol,2005.86(6):p.1581-1588.
[30]金奇.医学分子病毒学,北京：科学出版社,2001.2：691-708
[31]Russell, W.C., Update on adenovirus and its vectors. J Gen Virol 2000,81: 2573-2604.
[32]Rux J J, Burnett R M,Type-specific epitope locations revealed by X-ray crystallographic study of 150 adenovirus type 5 hexon [J]. Mol Ther,2000.1(1): p.18-30.
[33]Rux J J, Kuser P R, Burnett R M, Structural and phylogenetic analysis of adenovirus hexons by use of high-resolution x-ray crystallographic, molecular modeling, and sequence-based methods [J]. J Virol,2003.77(17):p.9553-9566.
[34]Berman H M, Westbrook J, et al., The protein data bank [J]. Nucleic Acids Res, 2000.28(1):p.155,235-242.
[35]张鸿彦,曲章义,等,腺病毒六邻体蛋白结构、功能及疫苗研究[J].国际免疫学杂志,2006.29(2)：p.126-128.
[36]王鹏,曲章义,等,人腺病毒六邻体蛋白保守区抗原性分析.国际免疫学杂志,2007.30(3)：p.135-138.
[37]金玉霞,腺病毒六邻体蛋白的免疫学研究进展.国外医学免疫学分册,2002.25(4)：p.172-174.
[38]周光炎,免疫学原理[M].上海：科技出版社,2000：p.36-37.
[39]Ichla Gollon S L, Drinker M, et al., Structure-based identification of a major neutralizing site in an adenovirus hexon [J]. J Virol,2007.81(4):p.1680-1689.
[40]Yuan X, Qu Z, Wu X, Wang Y, Liu L, et al. (2009) Molecular modeling and epitopes mapping of human adenovirus type 3 hexon protein. Vaccine 27: 5103-5110.
[41]Doan, M.L., et al., Treatment of adenovirus pneumonia with cidofovir in pediatric lung transplant recipients. J Heart Lung Transplant,2007.26(9):p. 883-9.
[42]Refaat, M., et al., Successful cidofovir treatment in an adult heart transplant recipient with severe adenovirus pneumonia. J Heart Lung Transplant,2008. 27(6):p.699-700.
[43]Arthur Lyonsa, Jenice Longfieldb, Robert Kuschnera, A double-blind, placebo-controlled study of the safety and immunogenicity of live, oral type 4 and type 7 adenovirus vaccines in adults. Vaccine,2008,26:2890-2898.
[44]Charles H. Hoke, Anthony Hawksworth, Cliff ord E. Snyder., Initial Assessment of Impact of Adenovirus Type 4 and Type 7 Vaccine on Febrile Respiratory Illness and Virus Transmission in Military Basic Trainees, Medical Surveillance Monthly Report.2012.19(3):2-5
[45]Xingui Tian, XiaoboSu, et al., Construction and characterization of human adenovirus serotype3 packaged by serotype7 hexon. Virus Research,2011.160: p.214-220
[46]Vorburger SA &Hunt KK. Adenoviral gene therapy. Oncologist,2002,7:46-59
[47]Michael, J. M., et al., Characterization of a permissive epitope insertion site in adenovirus hexon. J Virol.2006.80(11):p.5361-70.
[48]Hongju, W., et al., Identification of sites in adenovirus hexon for foreign peptide incorporation. J virol.2005.79(6):p.3382-90.
[49]Chirstopher, P., et al., Adenovirus particles that display the plasmodium falciparum circumsporozoite protein NANP repeat induce sporozoite-neutrializing antibodies in mice.Vaccine.2011.29(8):1683-9.
[50]Bin Yu, Zhen Wang, Jianing Dong, et al.A serological survey of human adenovirus serotype 2 and 5 circulating pediatric populations in Changchun, China,2011. Virology Journal 2012,9:287
[51]Dan H. Baroucha, Sandra V. Kikc, Gerrit J et al.,International Seroepidemiology of Adenovirus Serotypes 5,26,35, and 48 in Pediatric and Adult Populations. Vaccine.2011,29(32):5203-5209
[52]Qiwei Zhang, Xiaobo Su, et al., Comparative genomic analysis of two strains of human adenovirus type 3 isolated from children with acute respiratory infection in southern China. Journal of General Virology,2006,87:p.1531-1541
[53]Xiaobo Su, Xingui Tian, et al., Complete genome analysis of a novel E3-partial-deleted human adenovirus type7 strain isolated in Southern China. Virology Journal,2011.8:p.91-95
[54]Liuying Tang,. Li Wang, Xiaojuan, et al., Adenovirus serotype 7 associated with a severe lower respiratory tract disease outbreak in infants in Shaanxi Province, China Virology Journal 2011,8:23-30
[55]Gabriel Girouard, Richard Garceau, Louise Thibault, et al., Province-wide adenovirus type 3 outbreak with severe cases in New Brunswick, Can J Infect Dis Med Microbiol,2011,22 (1):e4-e6
[56]Ruth Kandel, Arjun Srinivasan, Erika M. C. D'Agata, et al. Outbreak of Adenovirus Type 4 Infection in a Long-Term Care Facility for the Elderly. Infection control and hospital epidemiology 2010(31);7
[57]苏晓波,马鑫,等.化学发光法中和抗体检测技术对广州地区人5型腺病毒的流行病学研究.南方医科大学学报,2007.27(9)：p.1323-1330.
[58]Bin Yu, Yan Zhou, Hao Wu, et al. Seroprevalence of Neutralizing Antibodies to Human Adenovirus Type 5 in Healthy Adults in China, Journal of Medical Virology,2012,84:1408-1414
[59]Qiwei Zhang, Xiaobo Su, et al., Construction and characterization of a replication-competent human adenovirus type 3-based vector as a live-vaccine candidate and a viral delivery vector. Vaccine.2009.27:p.1145-1153.
[60]Hongling Qiu, Xiao Li, et al., Hexon hypervariable regions (HAdV-3) and HAdV-7 reside in multiple epitopes of human adenovirus type 3. J. Virol,2012. 86(15):7964-7975.
[61]Xingui Tian, Xiaobo Su, et al., Protection against enterovirus 71 with neutralizing epitope incorporation within adenovirus type 3 hexon. PLOS ONE,2012.7(7):e41381.
