【作者】 夏玉坤；

【导师】 祝庆余； 刘伯华；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2009， 博士

【摘要】 禽流感病毒(AIV)属正粘病毒科A型流感病毒,禽流感病毒尤其高致病性的H5N1型禽流感病毒已经跨越种属屏障直接引起人类感染并致感染者死亡。1997年首次发现H5N1流感病毒从禽感染到人并引起死亡,之后不断有散发感染病例出现。特别是2003年底以来,世界上已有15个国家和地区报告有高致病性H5N1型流感病毒感染人的病例,已有423人感染,其中258人死亡,死亡率高达61%。虽然主要集中在亚洲,但欧洲和非洲也有感染病例出现,并且在上述国家和地区同时出现大规模家禽感染死亡,对人类健康和经济发展都造成了巨大影响,引起了各国的高度关注。A型流感病毒颗粒表面存在两种由糖蛋白组成的棘突,即血凝素(HA)和神经氨酸酶(NA),依靠它们可以把A型流感病毒分成很多亚型。同时,由于它们是流感病毒的主要表面抗原蛋白,可以刺激机体诱导产生免疫保护,又由于它们的变异可致流感病毒发生抗原漂移和转换,产生新的亚型和变异株,对这两个蛋白,特别是血凝素(HA)抗原性的研究,不但可以揭示流感病毒的抗原变异规律也可为疫苗的研发提供理论指导。已有研究证明,H5亚型上至少5个中和抗原表位(A、B、C、D和E)均位于HA的重链区HA1,各表位之间存在着一定程度的氨基酸区域重叠,但其区域大小及精细结构尚未确定。为进一步探索H5N1禽流感病毒HA蛋白的抗原表位及其结构功能,深入了解病毒的免疫保护抗原及病毒的致病机制,我们开展了H5N1禽流感病毒HA蛋白的抗原表位及其结构功能的研究。本研究的思路是:利用我们自行从人感染标本中分离的人源H5N1禽流感病毒株A/Beijing/01/03,制备多克隆血清抗体和多株特异性单克隆抗体,同时将该病毒的HA基因片段进行分段表达,利用多克隆和单克隆抗体分析各肽段的免疫反应性来鉴定及定位HA上的相关抗原表位,分析抗原表位的功能,确定单抗识别的氨基酸序列及表位构象。通过用单抗对流感病毒HA的抗原表位进行精细分析,为研发新型重组疫苗和多表位疫苗提供理论指导。此外,为了探索H5N1病毒跨越种属屏障直接从禽类感染人类的分子基础,我们又进行了10株人及野鸟H5N1分离株在人靶细胞上复制能力及拮抗人细胞因子等的差异研究。首先,我们根据人H5N1病毒A/Beijing/01/03株的HA1基因编码区984bp的序列,利用生物分析软件Oligo6.0设计了17条引物(共16对),采用常规PCR方法,利用这些引物分别扩增出HA1的16个不同长度的重叠肽的基因片段。利用限制性内切酶双酶切,将16条不同长度的HA1基因片段克隆到pDisplay载体中,筛选阳性克隆并测序鉴定,构建了16个重组质粒pDisplay-HA1-1~10及pDisplay-HA1-1’~6’。测定16个真核表达重组质粒的浓度及纯度,转染HeLa细胞,各HA1蛋白重叠肽段均获得成功表达。将表达各HA1重组肽段的细胞制备成各HA1肽段抗原片,建立起间接免疫荧光检测方法,用于抗原表位分析。其次,我们制备了抗H5N1(A/Beijing/01/03株)的单克隆抗体和多克隆抗体并对其性质进行研究。用甲醛灭活的病毒鸡胚尿囊免疫动物三次后,对小鼠血清检测出现特异性抗体者,取脾进行细胞融和实验,有限稀释克隆法筛选单克隆抗体细胞株,建立了12株稳定分泌抗AIV抗体的杂交瘤细胞。对自行制备的12株单抗和车小燕研究员惠赠的18株单抗的免疫学特性如抗体效价、血凝抑制活性、中和效价、单抗对HA蛋白特异性的识别等进行了鉴定。结果显示,应用间接免疫荧光方法测定全部30株杂交瘤腹水效价均在320-20480之间;发现13株McAb对病毒有较高的血凝抑制活性;用单抗对表达的HA1全长肽段进行识别检测,发现18株单抗对HA蛋白是特异性的;采用微量细胞培养中和试验测定了18株抗HA1单抗,6株具有中和抗体活性,中和效价在1﹕52~1﹕2032之间。同时,用灭活病毒免疫小鼠,成功地制备出了高效价的鼠抗H5N1多克隆血清抗体。第三,利用H5N1病毒特异性单抗及多克隆血清抗体采用间接免疫荧光法检测表达的禽流感病毒HA蛋白重叠肽抗原,分析HA的抗原表位及优势抗原表位。将每株单抗分别与16个表达的HA1重叠肽段结合,确定单抗识别的肽段区域,并对单抗的识别表位特征进行分析。同样方法,利用制备的多抗与表达的HA1各重叠肽段进行结合反应性分析,确定HA蛋白上的优势抗原区域。初步分析确定3B5、3D1、3B2、M6、M3等18株单抗识别表位区域分别位于氨基酸残基aa133-143,aa155-165,aa166-196,aa166-176,aa34-66及aa296-328之间,其中3B5、3D1、3B2、M6等单抗识别的表位为序列依赖型表位,M3和M7等具有中和活性的单抗所识别表位为构象性中和表位。将18株单抗识别的抗原表位在HA分子一级和三级结构上进行定位,确定了它们在三维HA中的位置。通过分析,初步确定HA1蛋白的优势抗原表位区为aa133-165 ,序列为IPKSSWSNHEASSGVSSACPYLGRPSFFRNVVW。这些结果为进一步了解HA抗原表位相关结构与功能的关系提供了重要数据,对探索流感病毒抗原变异规律和新型疫苗的研发具有一定的理论意义。第四,对A/Beijing/01/03等10株人及野鸟源H5N1病毒在人体靶细胞上复制能力差异的分子机制进行了研究。首先进行了人及野鸟H5N1病毒分离株空斑滴度测定,又进行了10株分离株在人A549细胞上复制能力以及对人IFN和TNF等细胞因子的敏感性检测,并对病毒基因组全序列进行分析。结果显示,人H5N1病毒株跨越种属屏障感染人可能与其在人体靶细胞的高效复制能力有着密切关系;初步确定NS1蛋白上三处氨基酸残基的突变可能与病毒适应人细胞复制有关。成功的构建了人H5N1病毒(A/Vietnam/1194/2004)株NS1基因突变株,为H5N1病毒适应并引起人类严重感染的分子生物学研究奠定了一定的基础。通过上述研究,获得以下结果:1、成功地构建了一批表达HA不同长度肽段的质粒,并转染LeLa细胞,获得了真核细胞表达,制备了一批抗H5N1病毒的单克隆抗体,并对单抗进行了特性研究。这些肽段和单抗对研制甲型流感病毒特异诊断试剂具有重要的应用价值;2、利用单抗和不同长度的HA肽段对HA上抗原表位分析初步证明,HA1上存在着血凝型、非血凝型和中和型等多种抗原表位,血凝型抗原表位主要为序列依赖性表位;3、通过构象分析证明,中和型表位主要为构象表位;4、通过优势抗原表位分析,确定HA1蛋白的优势抗原表位区为aa133-165,序列为IPKSSWSNHEASSGVSSACPYLGRPSFFRNVVW。这些结果为进一步了解HA抗原表位相关结构与功能的关系研究提供了重要数据,对探索流感病毒抗原变异规律和新型疫苗的研发具有一定的理论意义。对A/Beijing/01/03等10株人及野鸟H5N1病毒在人体靶细胞上复制能力差异分子机制的初步分析发现,人源H5N1病毒在人细胞上复制能力强,可能是H5N1病毒感染人类的前提。NS1蛋白效应区上3处氨基酸残基突变可能与病毒适应人体复制相关,这对进一步探索H5N1病毒的分子生物学致病机理提供了可参考的资料。更多还原

【Abstract】 Avian influenza virus (AIV) is type A influenza virus of the family Orthomyxoviridae, and in particular the highly pathogenic H5N1 AIV has crossed the species barrier and caused a direct result of human infection and death of the infected persons. It was first found in 1997 that H5N1 AIV could infect humans and cause deaths and disseminated infections continued to occur. Since the end of 2003, highly pathogenic H5N1 AIV has spreaded rapidly from Southeast Asia to more than 60 countries and regions including China, Africa, Turkey and Siberia, et al. 15 countries and regions have reported human cases infected by the virus. Totally 423 persons were infected and 258 of them died,with the mortality rate as high as 61%. Although the human cases were mainly distributed in Asia, but Europe and Africa also had accasional cases. The epidemic caused a large scale poultry deaths and particularly, caused human infection and death,resulting in huge economic losses and great threatening to public health and social security.Two glycoprotein spikes, hemagglutinin (HA) and neuraminidase (NA), located on surface of influenza A virus. Based on the differences of HA and NA, influenza virus was further divided into many subtypes. HA and NA are not only the major surface antigens of AIV and can induce protective immunity, but also the main reason for antigenic drift and antigenic shift to cause new subtypes and variants by their mutations. The research of their antigenicity, especially HA, would not only disclose the regulations of antigenic variation, but also provide theoretical guidance for the vaccine development. Research has proven that there are at least five neutralizing epitopes (A, B, C, D and E) distributed on HA protein of H5N1, all located in the heavy chain HA1. The eptitopes are overlapped in some extent. However, the size and detailed structure of the epitopes has not yet been determined. It will be helpful for understand the pathogenesis of the virus by exploring the detaied structure of these epitopes.Based on a human strain of AIV A/Beijing/01/03(H5N1) isolated in China, mutiple specific monoclonal antibodies(McAb) and polyclonal serums were utilized to determine the antigenic epitopes (in particular, the neutralizing epitopes) on hemagglutinin of human H5N1 AIV, and to ascertain the amino acid sequences recognized by those McAbs. The detailed analyze of the epitope structures by specialized McAbs could be applied as the first step for further developing of novel influenza epitope vaccines. At the same time, in order to explore the molecular basis of H5N1 virus crossing the species barrier to infect human directly from birds, another 10 strains of H5N1 isolates from human and wild birds in Qinghai, China were employed to explore the differences of the replication capacity in human target cells and the antagonism against human cytokines.Firstly, 17 primers (a total of 16 pairs) were designed by Oligo6.0 software according to 984bp sequences encoding HA1 of H5N1 strain A/Beijing/01/03. 16 overlapped HA1 gene fragments in different length were amplified by RT-PCR and cloned into the mammalian expression vector pDisplay. Then the positive clones were screened and sequenced for subsequent construction of 16 recombinant plasmids pDisplay-HA1-1~10 and pDisplay-HA1-1’~6’. The concentration and purity of the 16 recombinant plasmids were determined and then recombinant plasmids were prepared and transfected into HeLa cells by lipidosome lipofetaineTM2000. The HA1 overlapped peptides were expreed successfully in vitro. Indirect immunofluorescence was established for epitope analysis.Secondly, anti-H5N1 (A/Beijing/01/03 strain) McAbs and polyclonal antibodies were prepared and their properties were analyzed. The BALB/c mice were immunized three times with chick embryo allantoic fluid inactivated by formaldehyde, then the mouse with positive serum antibodies was selected and their spleens were taken for cell fusion experiments. McAbs were screened by limiting dilution cloning assay and 12 hybridoma cell lines which secret anti-H5N1 McAbs were obtained. Totally, 30 McAbs were included in this study. Among them, 18 were kindly supplied by professor Che Xiaoyan. The immunological properties, such as antibody titers, hemagglutination inhibition activity, neutralizing activity, specific recognization of these McAbs to HA protein were investigated and identified. The results showed that the titers of all 30 McAbs were between 400-20480; 13 McAbs exhibited higher hemagglutination inhibition activities; 18 McAbs were specific against HA protein; 6 fo 18 anti-HA McAbs showed neutralizing activities, with titers between 1:52-1:2032. The anti-H5N1 polyclonal serum antibodies were collected from the peripheral blood of those mice after three immunizations.Thirdly, overlapped HA1 peptides expressed on the transfected cell surface and the prepared McAbs and polyclonal antibodies were utilized to analyze the neutralizing epitopes and dominating epitopes on HA by indirect immunological assay (IFA). Each McAb reacted with every peptide expressed on HeLa cells transfected with 16 recombinant plasmids respectively. Similarly, the major immunodominant regions on HA protein were determined according to the reactions of polyclonal antibodies with different HA1 protein fragments. The antigenic epitopes recognized by 3B5、3D1、3B2 and M6 McAbs were located in the region of amino acid residues 133-143,155-165,166-196 and 166-176 of HA1 protein, respectively, and all of them seemed to be linear epitopes. The antigenic epitopes recognized by M3 and M7 were mapped to the regions of amino acid residues 34-66 and 296-328 of HA1 protein, which presumed to be conformational epitopes. The major immunodominant epitope of HA1 protein was located in the region of amino acid residues 133-165 with the sequence of IPKSSWSNHEASSGVSSACPYLGRPSFFRNVVW. These results disclosed the major immunodominant region and epitopes on HA protein recognized by antibodies, and the amino acid sequence recognized by McAb. Through the analysis of the relationship between the key amino acid sites and the epitopes, the detailed structure of epitopes were demonstrated and could be utilized for further study of the function of HA protein. These results provided important data for further understanding of the relationship between the structure and function of HA epitopes, and would be helpful for exploring the regulation of antigenic variations and for development of new vaccines.Fourthly, the molecular basis of the different replicating abilities of A/Beijing/01/03 H5N1 virus and other 9 human and wild birds strains of H5N1 isolates on human target cells were investigated. Plaque titers of all viruses were determined. Then the replication capacities of these isolates in human A549 cells and their sensitivity against human IFN and TNF cytokines were evaluated. The genome sequences of these isolates were analyzed and aligned. The results indicated that the ability of H5N1 virus crossing the species barrier from birds to humans might be closely related to their strong replication capacities in human target cells. Three amino acid mutations on NS1 protein may be associated with the ability of these strains to adapt to and reproduce in human target cells. A/Vietnam/1194/2004 (H5N1) NS1 gene mutant strain was constructed for further molecular basis analysis of H5N1 virus to adapt to and cause serious infections in humans.In summary, by using the prepared McAbs and expressed overlapped HA1 peptides, several linear and conformational epitopes and major immunodominant regions on hemagglutinin protein of H5N1 virus were primarily determined in this study. The epitopes can be divided into hemagglutination, non-hemagglutination-related and neutralizing epitopes, and hemagglutination-related epitopes were mainly linear epitopes, and the neutralizing-related epitopes were mainly conformational epitopes. The McAbs and pepetides prepared were important for further developing of influenza A virus-specific diagnostic reagents. These results would provide theoretical evidence for further developing of novel vaccines and exploring the mechanism of viral antigenic variation. At the same time, it was presumed that the strong replication capacity on human target cells of human-derived H5N1 virus may be a prerequisite for its ability to adapt to and infect humans. It was also presumed that the three amino acid mutations on NS1 protein may be associated with its adaptation to humans and it had great significance for further exploring the pathopoiesis of H5N1 virus.更多还原