PNEUMOCOCCAL GLYCOCONJUGATE VACCINES USING PNEUMOCOCCAL

目前的23价肺炎球菌多糖（PS）疫苗在成人中诱导高抗体应答，但大多数PS疫苗在幼儿中的免疫原性较低。增强儿童抗体反应的一种方法是开发针对特定类型的ps蛋白结合疫苗。已经制备了几种肺炎球菌结合疫苗，目前正在进行临床试验。一些研究人员已经研究了使用肺炎球菌细胞表面蛋白（如溶气酶、自溶酶、肺炎球菌表面蛋白A (PspA)）作为载体的可能性。使用细胞表面蛋白作为载体蛋白可以提供交叉保护抗原，以预防肺炎球菌感染。本项目的总体目标是检测与肺炎球菌细胞表面蛋白载体结合的肺炎球菌ps的交叉保护免疫。采用含溶气血素基因（ply）的重组大肠杆菌制备了19A型灭活溶气血素。PCR扩增19A型，插入PCR -blunt载体，转化为感受态细胞。将质粒层DNA插入pET-24b（+）载体，转化为大肠杆菌。通过缺失编码c端7个氨基酸残基的19A型ply基因的核苷酸，使ply基因发生突变。加入IPTG后2-5小时，细胞裂解液中产生溶肺素。细胞裂解液通过树脂亲和柱，用咪唑浓度梯度洗脱。由r-E测定溶血素的溶血活性。含有野生型ply基因的大肠杆菌与从19A型肺炎球菌中分离出来的大肠杆菌相同。突变的溶血素溶血活性降低99%以上。用兔抗溶血素血清免疫印迹法检测到分子量为56000的突变溶血素。通过以下方法制备了肺炎球菌9V型PS-和19F型PS-ply偶联物：(a)希夫碱形成和还原胺化；或(b)偶联剂聚乙二醇和聚乙烯吡咯烷酮的偶联。为了进行偶联表征，分析了PS与蛋白质的比值和分子大小谱。各组幼鼠分别用5ug的9V PS-ply或19F PS-ply偶联物免疫。我们希望不久就能证明，通过补充肺炎球菌细胞表面蛋白作为PS结合物的载体，可以提高肺炎球菌结合疫苗的交叉保护和有效性。

The current 23-valent pneumococcal polysaccharide (PS) vaccine induces high antibody response in adults, but the immunogenicity of most PSs in the vaccine is low in young children. One approach for enhancing the antibody response in children is development of PS-protein conjugate vaccines for selected types. Several pneumococcal conjugate vaccines have been prepared and are currently in clinical trials. Several investigators have examined the possibility of using pneumococcal cell-surface proteins, such as pneumolysin, autolysin, pneumococcal surface protein A (PspA as carriers. Use of a cell-surface proteins as the carrier protein may provide a cross-protective antigen for prevention of pneumococcal infection. The overall objective of the present project is to examine cross-protective immunity of pneumococcal PSs conjugated with pneumococcal cell-surface protein carriers. Inactivated type 19A pneumolysin was prepared using recombinant E. coli containing the pneumolysin gene (ply). Type 19A ply was amplified by PCR reaction, inserted into pCR-blunt vector, and transformed into competent cells. The plasmid ply DNA was then inserted into pET-24b(+) vector, and transformed into competent E. coli cells. The ply gene was mutated by deletion of the nucleotides of type 19A ply gene that encode seven amino acid residues the from C-terminal. Pneumolysin was produced in cell lysate at 2-5 hrs after addition of IPTG. The cell lysate was passed through a resin affinity column and eluted with a concentration gradient of imidazole. The hemolytic activity of pneumolysin obtained from r-E. coli containing wild type ply gene is the same as that isolated from type 19A pneumococci. Hemolytic activity of mutated pneumolysin decreased more than 99%. Mutated pneumolysin having molecular weight of 56,000 was detected by the immunoblot analysis using a rabbit anti-pneumolysin serum. Pneumococcal type 9V PS- and type 19F PS-ply conjugates have been prepared by the following methods: (a) Schiff base formation and reductive amination; or (b) Conjugation with couping reagents of polyethylene glycol and polyvinylpyrrolidone. For conjugate characterization PS to protein ratios and molecular size profiles were analyzed. Groups of young mice are being immunized with 5 ug of 9V PS-ply or 19F PS-ply conjugate. We soon hope to show that cross-protection and effectiveness of pneumococcal conjugate vaccines can be improved by supplementation with pneumococcal cell-surface proteins as carrier for PS conjugates.