【作者】 马千里；

【导师】 王长征；

【作者基本信息】 第三军医大学 ， 内科学， 2009， 博士

【摘要】 背景与目的:肺炎链球菌是引起社区获得性肺炎、中耳炎、菌血症及儿童脑膜炎的主要致病菌,发展中国家每年有超过500万例的5岁以下儿童死于肺炎链球菌感染。目前控制肺炎链球菌感染的手段是抗生素治疗,但是日趋严重的肺炎链球菌耐药问题已成为全球公共卫生领域的重大难题,亦危及肺炎链球菌的治疗效果。应用疫苗防治肺炎链球菌感染是目前WHO提出减少耐药菌传播、缓解抗生素耐药压力,保护高危人群的唯一方法。目前应用的传统肺炎链球菌疫苗(23价荚膜多糖疫苗和7价蛋白-多糖结合疫苗)存在血清型依赖、针对高危人群保护不足、价格高昂等诸多不足,研究新一代肺炎链球菌DNA和蛋白疫苗因其广覆盖、高效能、低成本的优势在发展中国家具有深远的社会价值和经济效益。选择广泛覆盖、高度保守、具备强免疫原性的靶抗原是研究基因疫苗的首要问题。肺炎链球菌的众多毒力蛋白在肺炎链球菌感染的不同阶段发挥不同的毒力作用,虽然在众多国内外研究中发现了许多对肺炎链球菌感染具有一定保护性作用的毒力蛋白,但到目前为止尚无一种蛋白被认为具有激发机体针对所有致病肺炎链球菌菌株携带、定殖、迁移及黏附侵袭全过程的免疫保护效能。因此,评估具有协同激发机体保护性免疫的优势抗原组合及针对优势抗原组分的进一步探索研究具有重要意义。本课题围绕“肺炎链球菌毒力相关蛋白基因工程疫苗优势抗原筛选”进行了以下研究工作:⑴采集肺炎链球菌感染的社区获得性肺炎患者血清,分析其恢复期血清特异性抗体水平升高情况,评价肺炎链球菌表面蛋白A(pneumococcal surface protein A, PspA)、肺炎链球菌黏附素A(pneumococcal surface adhesion A ,PsaA)及肺炎链球菌溶血素(pneumolysin,Ply)三种毒力蛋白在肺炎链球菌经自然途径感染人体过程中的免疫原性;⑵复制肺炎链球菌鼻咽部定植及腹腔感染动物模型,检测三种毒力蛋白单独或联合形式的免疫原性差异及对肺炎链球菌携带及侵袭性感染的免疫保护效能差异,筛选肺炎链球菌新一代疫苗的优势靶抗原组合形式;⑶进一步利用分子遗传学及免疫学技术针对筛选出的优势抗原蛋白进行分子进化及比较基因组学的初步研究。旨在为肺炎链球菌新一代疫苗的研发提供理论基础和实验室依据。方法:1.采集临床确诊为肺炎链球菌感染的社区获得性肺炎患者急性期(第0+3天)及恢复期(第21±3天)血清样本,设立同期临床确诊为其它细菌感染的社区获得性肺炎患者和同期临床确断无细菌侵入性感染的其它患者血清样本为对照;酶联免疫吸附法(ELISA)检测肺炎链球菌三种毒力蛋白PsaA,PspA及Ply激发机体血清特异性抗体升高情况,以期评价这三种毒力蛋白作为新一代肺炎链球菌疫苗候选抗原在肺炎链球菌经自然途径感染人体过程中的免疫原性。2.分子克隆技术构建Ply减毒突变体PdB的重组真核表达质粒pcDNA3.1-PdB,并应用RT-PCR及免疫印迹(Western-blot)方法检测重组质粒在哺乳动物叙利亚仓鼠肾细胞株BHK-21中的表达。三种重组质粒DNA疫苗pcDNA3.1-PdB(本课题构建)和pcDNA3.1-PsaA(本研究组前期构建)和pcDNA3.1-PspA’(本研究组前期构建,PspA’为PspA的N端表位决定簇所在区域基因片段)以单独或不同的组合方式(pcDNA3.1-PsaA+pcDNA3.1-PspA’, pcDNA3.1-PspA’+pcDNA3.1-PdB, pcDNA3.1-PsaA + pcDNA3.1-PdB,pcDNA3.1-PsaA + pcDNA3.1-PspA’+ pcDNA3.1-PdB)肌肉注射免疫BALB/c小鼠,设立空质粒及PBS组为对照组,ELISA检测免疫小鼠血清中三种蛋白特异性抗体IgG水平;复制BALB/c小鼠肺炎链球菌D39株鼻咽部携带模型和腹腔侵袭性感染模型,观察免疫小鼠鼻咽部肺炎链球菌携带改变和腹腔攻击小鼠21天生存情况,以期筛选出优势的靶抗原蛋白及其组合方式。3.采用分子遗传学方法和免疫学技术进一步初步分析肺炎链球菌第三代疫苗优势抗原蛋白的分子进化特点及抗原多样性。⑴采集上呼吸道共生环境中的临床致病轻链球菌家族菌株(包含肺炎链球菌、轻链球菌、口腔链球菌、血链球菌、婴儿链球菌),PCR技术探查临床致病轻链球菌家族菌株基因组中PsaA基因分布情况,应用生物信息学技术比对所有PsaA基因的氨基酸序列及蛋白结构差异;进一步应用分子遗传学技术构建基于看家基因及PsaA基因的系统发生树,并检测PsaA基因内重组情况,以分析PsaA在轻链球菌家族中可能的遗传机制。⑵采集本地区临床致病肺炎链球菌感染菌株及患者恢复期(第21±3天)血清样本,设立同期确诊为非感染性疾病患者血清样本为对照组;应用PspA家族分型特异性引物PCR鉴定本地区临床自然感染肺炎链球菌株PspA家族分布特点, ELISA检测临床肺炎链球菌自然感染情况下各家族亚类间抗原抗体交叉反应情况,分析肺炎链球菌PspA作为新一代肺炎链球菌疫苗优势候选抗原应包含的组成成分。结果:1.临床肺炎链球菌自然感染患者血清PspA、Ply及PsaA特异性抗体IgG水平⑴实验组急性期血清三种肺炎链球菌毒力蛋白特异性IgG抗体水平与对照组间无显著性差异(P>0.05);实验组恢复期血清的三种肺炎链球菌毒力蛋白特异性IgG抗体水平均高于对照组相应的抗体水平(P<0.01)。⑵实验组恢复期血清三种肺炎链球菌毒力蛋白特异性IgG抗体水平明显高于急性期血清抗体水平(P<0.01),其中PspA和Ply蛋白抗原特异性抗体水平升高更显著。2.三种肺炎链球菌毒力蛋白DNA疫苗免疫原性及免疫保护效能⑴三种肺炎链球菌毒力蛋白DNA疫苗免疫小鼠血清中特异性抗体水平①三种肺炎链球菌毒力蛋白DNA疫苗以两种蛋白形式组合的联合免疫组中, PspA’和PsaA的DNA疫苗联合免疫组(pcDNA3.1-PsaA+pcDNA3.1-PspA’组)较单独免疫组(pcDNA3.1-PsaA组,pcDNA3.1-PspA’组)小鼠血清中激发的针对PspA’和PsaA蛋白的特异性抗体水平更高(P<0.01);而PdB分别与PspA’和PsaA联合的DNA疫苗免疫组(pcDNA3.1-PspA’+pcDNA3.1-PdB, pcDNA3.1-PsaA + pcDNA3.1-PdB)与单独免疫组(pcDNA3.1-PdB组)比较小鼠血清中激发的针对PdB蛋白的特异性抗体水平无显著性差异(P>0.05)。②三种肺炎链球菌毒力蛋白DNA疫苗以三种蛋白形式组合的联合免疫组( pcDNA3.1-PsaA + pcDNA3.1-PspA’+ pcDNA3.1-PdB组)较单独免疫组(pcDNA3.1-PsaA组,pcDNA3.1-PspA’组,pcDNA3.1-PdB组)和PdB分别与PspA’和PsaA联合的DNA疫苗免疫组(pcDNA3.1-PspA’+pcDNA3.1-PdB, pcDNA3.1-PsaA + pcDNA3.1-PdB组)小鼠血清中针对PsaA和PsaA蛋白的特异性抗体水平更高(P<0.01),而针对PdB蛋白的特异性抗体水平无显著性差异(P>0.05);但与PsaA和PspA’联合免疫组(pcDNA3.1-PsaA+pcDNA3.1-PspA’)比较小鼠血清中针对PsaA和PspA’蛋白的特异性抗体水平无显著性差异(P>0.05)。⑵三种肺炎链球菌毒力蛋白DNA疫苗免疫保护效能①免疫小鼠鼻咽部定殖7天后鼻腔灌洗液菌落计数提示:PsaA和PspA’联合免疫组( pcDNA3.1-PsaA + pcDNA3.1-PspA’)较单独免疫组( pcDNA3.1-PsaA组,pcDNA3.1-PspA’组,pcDNA3.1-PdB组)和PdB分别与PspA’和PsaA联合的DNA疫苗免疫组(pcDNA3.1-PspA’+pcDNA3.1-PdB, pcDNA3.1-PsaA + pcDNA3.1-PdB)小鼠鼻咽部肺炎链球菌数量较少更明显(P<0.01),并且与三种蛋白联合免疫组(pcDNA3.1-PsaA+pcDNA3.1-PspA’+ pcDNA3.1-PdB组)间无显著差异(P>0.05)。②免疫小鼠28天后致死剂量的肺炎链球菌强毒株D39腹腔攻击21天生存情况分析提示:PsaA和PspA’联合免疫组(pcDNA3.1-PsaA+pcDNA3.1-PspA’)较单独免疫组(pcDNA3.1-PsaA组,pcDNA3.1-PspA’组,pcDNA3.1-PdB组)和PdB分别与PspA’和PsaA联合的DNA疫苗免疫组(pcDNA3.1-PspA’+pcDNA3.1-PdB, pcDNA3.1-PsaA + pcDNA3.1-PdB)小鼠中位生存时间延长更明显(P<0.01),生存曲线明显差异,并且与三种蛋白联合免疫组(pcDNA3.1-PsaA+pcDNA3.1-PspA’+ pcDNA3.1-PdB组)间小鼠中位生存时间相同,生存曲线相似。3.肺炎链球菌PsaA基因的种群基因起源进化分析提示PsaA基因在临床分离的轻链球菌群菌株中的广泛存在,但是仅仅在肺炎链球菌属中PsaA基因是垂直遗传的,其它的轻链球菌群菌株中PsaA基因可能来源于共生环境中基因的水平转移,并且推测PsaA基因之间的差异可能与BOX元件的参与及转移后发生的频繁的基因内重组现象相关。4.肺炎链球菌PspA区域基因组学分析提示本地区PspA家族分布趋势为:PspA-Fam1-Clade2(31.0%)和PspA-Fam2-Clade3(47.6%)的菌株作为优势分布。并且在本地区所有侵入性肺炎链球菌感染患者血清中针对PspA-Fam1特异性抗体水平高于针对PspA-Fam2的特异性抗体水平,血清PspA抗体与PspA蛋白6种亚类抗原的交叉反应发现异家族各亚类间交叉反应弱,但同家族亚类间显著交叉反应以PspA-Fam1-Clade1和PspA-Fam2-Clade3为优势。结论:1.PsaA,PspA及Ply在人体经自然途径的肺炎链球菌感染中作为具有良好免疫原性的肺炎链球菌菌体成分可作为新一代肺炎链球菌疫苗的理想侯选抗原。2.PspA’联合PsaA的抗原组合方式具有等效于PsaA,PspA’及PdB(Ply的减毒突变体)三种抗原组合方式的免疫效能,对肺炎链球菌的鼻咽部定植及侵入性感染均具有高效的协同保护效能,又避免了PdB存在的弱毒性问题,是肺炎链球菌新一代疫苗靶抗原的优势候选抗原组合。3.PsaA作为肺炎链球菌新一代疫苗优势候选抗原组合成分之一,其基因在肺炎链球菌属是垂直遗传的。推论它作为一种重要的黏附因子,在细菌对鼻咽部上皮的黏附中发挥重要作用而容易被共生环境的同种群的其它链球菌通过水平基因转移及基因内重组方式获得。4.PspA作为肺炎链球菌新一代疫苗优势候选抗原组合成分之一,抗原多样性存在地域差异和亚类间抗原抗体交叉反应差异,靶抗原成分应同时包括本区域产生高保护性抗体效价和强交叉反应的Fam1及Fam2的优势Clade亚类成分才能针对性的覆盖临床致病肺炎链球菌菌株感染。更多还原

【Abstract】 Background and ObjectiveStreptococcus pneumoniae is the main pathogen, which causes community-acquired pneumonia, otitis media, bacteremia and meningitis in children. In developing countries, there are more than five million cases of children under the age of 5 died of Streptococcus pneumoniae infection every year. At present, the control way of Streptococcus pneumoniae infections is treatment with antibiotics, but the more and more serious drug- resistances of Streptococcus pneumoniae has became a major global public health problem and endangered the treatment of Streptococcus pneumoniae. Application of vaccines for combating pneumococcal infections is the only way proposed by WHO(World Health Organization), which shoμld be help to reduce the spread of antibiotic resistance strains and alleviat the stress of antibiotics and protect high-risk groups.In view of the serotype-dependent existence and the high cost of current pneumococcal vaccines (23-valent capsμlar polysaccharide vaccine and protein - polysaccharide conjugate vaccine), a new generation DNA and protein vaccines of Streptococcus pneumoniae has far-reaching social and economic benefits in developing countries because of its wide coverage, high-performance, low-cost advantage and the increasingly sophisticated technology of molecμlar biology.The choice of target antigens of vaccines is the most important question. The large number of Streptococcus pneumoniae virμlence proteins play the important and different roles at different stages of Streptococcus pneumoniae infections, although many research resμlts suggested that some virμlence proteins of Streptococcus pneumoniae played protective roles, but so far there was no protein considered to covering the all pathogenic processes of colonization, adhesion and invasion. The use of a wide range of virμlence proteins was thought of more effective to stimμlate protective immune and bringing about protective synergy effect.Therefore, this study“screening of dominant antigen formats of genome-based vaccines of Streptococcus pneumoniae”was as the following works: First, we test the specific antibody levels against Streptococcus pneumoniae surface protein A ( PspA), pneumococcal adhesin A ( PsaA) and Streptococcus pneumoniae pneumolysin(Ply) in the seras from patients with the community-acquired pneumonia, and evaluated the immunogenicities of three proteins in the human body naturally infected with Streptococcus pneumoniae; Second, we detected the immunopotency performance differences of the three virμlence proteins in animal models, which was for screening the dominant antigen formats of genome-based vaccines of Streptococcus pneumoniae; At last, we study preliminary the comparative genomics of dominant antigens by analyzing the sequence characteristics of dominant antigen proteins from molecμlar level and their possible functional differences, which was for learning more about the possible antigen diversity of dominant antigen proteins on the basis of molecμlar genetics.Thus, the resμlts provide the theoretical foundation and laboratory basis for the development of new generation Streptococcus pneumoniae vaccines.Methods1. We characterized antibody responses to PspA, Ply and PsaA proteins in acute serum samples ((0+3)days) and convalescent samples ((21±3) days later) obtained from adμlts hospitalized with CAP(community acquired pneumonia) dued to Streptococcus pneumoniae, with two groups of age-matched controls (group1: patients with invasive disease due to other microorganisms and group2: patients hospitalized for non infectious conditions). Antibody levels to PspA, Ply and PsaA were measured by Enzyme-linked immunosorbent assay(ELISA), and with a view to evaluate the toxicity of these three proteins as a new generation of pneumococcal vaccine candidate antigen immunogenicity.2. The fragement of PdB (the attenuated mutant of Ply) was cloned into the eukaryotic expression vector pcDNA3.1(+) by molecμlar cloning technology. The recombinant vector pcDNA3.1-PdB was transfected into BHK-21 cells, and the expression of PdB protein was detected with RT-PCR and Western- blot. The fours formats of recombinant plasmids (pcDNA3.1-PsaA+pcDNA3.1-PspA’, pcDNA3.1-PspA’+pcDNA3.1-PdB, pcDNA3.1-PsaA + pcDNA3.1-PdB,pcDNA3.1-PsaA+ pcDNA3.1-PspA’+ pcDNA3.1-PdB)(pcDNA3.1-PsaA and pcDNA3.1-PspA’had been pre-constructed )were used to inject intramuscμlarly BALB/c mice , with the vector pcDNA3.1(+) and PBS as controls. The levels of antibodies against PspA’, PsaA and PdB proteins were checked. The nasopharyngeal colony count of Streptococcus pneumoniae D39 strain and the live time of intraperitoneal infection mice by Streptococcus pneumoniae D39 strain were analysis in BALB/c models, and with a view to filter out the dominant antigen and their formats.3. Methods of molecμlar genetics and immunology technology were used for further analyzing the comparative genomics and the diversity of protein antigens as the third generation Streptococcus pneumoniae vaccine antigen proteins.⑴The presences of PsaA gene in streptococcus mitis group isolates of clinical upper respiratory tract pathogens in the symbiotic environment were identified using special PCR approach. The characters of these PsaA sequences were compared, and phylogenies basing on the sequences of housekeeping genes and PsaA genes were producted for analyzing the possible genetic mechanisms of PsaA gene in streptococcus mitis group.⑵Streptococcus pneumoniae strains were collected from patients with invasive Streptococcus pneumoniae infections,and the convalescent serum samples ((21±3) days later) were obtained from these patients.We characterized the serum antibody cross-reactivity to 6 clades of PspA in the adμlts with invasive Streptococcus pneumoniae infections by ELISA, and the patients in non infectious conditions as control group. All pneumococcal isolates were typed for the PspA families and clades.Resμlts1. PspA, Ply and PsaA-specific IgG antibody levels were characterized in the serum of patients with invasive Streptococcus pneumoniae infections.⑴The three Streptococcus pneumoniae virμlence protein-specific IgG antibody levels had no significant differences between acute phase serum of experimental group and that of the control group (P> 0.05); But the three Streptococcus pneumoniae virμlence protein-specific IgG antibody levels were higher in convalescent serum of experimental group than that of the control group (P <0.01).⑵The three Streptococcus pneumoniae virμlence protein-specific IgG antibody levels were significantly higher than that of acute-phase serum antibody levels (P<0.01), The increasing of antigen-specific anti-Ply and anti-PspA antibody levels were more significant .2. Immunogenicity and the effectiveness of immune protection of three Streptococcus pneumoniae virμlence protein DNA vaccines.⑴Specific antibody levels in serum in mice immunized with DNA vaccines of three Streptococcus pneumoniae virμlence proteins.①In the form of combination of the two combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA ’), specific serum antibody levels against PsaA and PspA ’ were higher than immunized with alone (pcDNA3.1-PsaA, pcDNA3.1-PspA ’) (P<0.01); But in the forms of combinations of PdB and PspA ’ (or PsaA) combined immunization groups (pcDNA3.1-PspA’ + pcDNA3.1-PdB, pcDNA3.1-PsaA + pcDNA3.1-PdB), Specific serum antibody levels against PdB had no significant different (P> 0.05).②In the form of combination of the three proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA ’+ pcDNA3.1-PdB), specific serum antibody levels against PsaA and PspA ’ were significantly higher than immunized with alone (pcDNA3.1-PsaA, pcDNA3.1-PspA ’) (P<0.01), and specific serum antibody levels against PdB were were similar with alone (pcDNA3.1-PdB) (P> 0.05); But specific serum antibody levels against PsaA and PspA ’ were similar with the form of combination of the two proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA ’) (P> 0.05).⑵Effectiveness of immune protection of three Streptococcus pneumoniae virμlence protein DNA vaccines①The resμlt of nasopharynx in immunized mice on the seventh day after colonization colony counting tips nasal lavage fluid showed that the pneumococci colony counting of nasopharynx was significantly fewer in the mice immunized with the form of combination of the two proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA ’) than the mice immunized with alone (pcDNA3.1-PsaA, pcDNA3.1-PspA ’) (P<0.01)and the forms of combinations of PdB and PspA ’ or PsaA combined immunization groups (pcDNA3.1-PspA’ + pcDNA3.1-PdB, pcDNA3.1-PsaA + pcDNA3.1-PdB) (P<0.01); And there had no significant differences of the pneumococci colony counting of nasopharynx in the mice immunized with between the two proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA ’) and the three proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA ’+ pcDNA3.1-PdB) (P> 0.05). ②Mice were intraperitoneal infection by the lethal dose of Streptococcus pneumoniae D39 strain after immunized 28 days, and survival analysis of 21 days showed that showed that the median survival time was significantly longer in the mice immunized with the form of combination of the two proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA ’) than the mice immunized with alone (pcDNA3.1-PsaA, pcDNA3.1-PspA ’)(P<0.01)and the forms of combinations of PdB and PspA ’ or PsaA combined immunization groups (pcDNA3.1-PspA’ + pcDNA3.1-PdB, pcDNA3.1-PsaA + pcDNA3.1-PdB) (P<0.01); And there had no significant differences of the median survival time and the survival curves of the mice immunized with between the two proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA ’) and the three proteins combined immunization group (pcDNA3.1-PsaA + pcDNA3.1-PspA ’+ pcDNA3.1-PdB) (P> 0.05).3. High frequency events of HCG and recombination support the prevalent presence of PsaA gene in streptococcus mitis group. These species may acquire transforming PsaA gene from the other species of streptococcus mitis group living in the same ecological niche, and maybe involved in promote the adhesion behavior and subsequent oropharyngeal colonization behavior. But we have no enough evident to confirm the possibility that these PsaA genes all come from Streptococcus pneumoniae, even though PsaA gene is comfirmed vertical inheritance in Streptococcus pneumoniae and BOX element is all absence in the flanking region of PsaA gene in Streptococcus pneumoniae.4. The characteristic of regional distribution of PspA families of Streptococcus pneumoniae was that Fam1-Clade2 (31.0%) and Fam1-Clade3 (47.6%) strains were as the advantages distribution. Serum specific anti-PspA-Fam1 antibody levels in invasive Streptococcus pneumoniae infection patients were higher than that of specific anti-PspA-Fam2 antibody levels. Serum anti-PspA antibodies against 6 clades of PspA protein were found weak cross-reaction amomg the various clades of various families, but a significant cross-reaction to Fam1-Clade1 and Fam1-Clade3 for advantage among the same family I .Conclusion1. PsaA, PspA and Ply, as the Streptococcus pneumoniae cell components, have the immunogenicity, which produced specific antibodies in the human body’s with Streptococcus pneumoniae infections through the natural way. So they can be used as candidate antigens of new generation Streptococcus pneumoniae vaccines.2. United PspA and PsaA had the equivalent and coordinative immune effectiveness compared to combinations of three antigens PsaA, PspA, and PdB (attenuated mutant of Ply) against Streptococcus pneumoniae nasopharyngeal colonization and invasive infections, and avoided the existence of weak toxicity PdB. So the combination of PspA and PsaA was the dominant form of candidate antigens of the new generation vaccines.3. PsaA is one of the forms of the dominant antigen candidates as the new Streptococcus pneumonia generation vaccines, and PsaA gene is vertical inheritance in Streptococcus pneumoniae. As an important adhesion molecμle in the bacteria to nasopharyngeal epithelium, it plays an important role in adhesion and is aquired by other symbiotic streptococcus through horizontal gene transfer and recombinant gene manner.4. PspA is one of the forms of the dominant antigen candidates as the new Streptococcus pneumonia generation vaccines,and PspA gene has the diversity. The target antigens shoμld also include the clades components of Fam1 and Fam2 with high protective and strong cross-reaction against a wide range of clinical pathogenic strains of Streptococcus pneumoniae.更多还原