以肺炎球菌溶血素为载体的中耳炎肺炎球菌结合疫苗的制备及免疫原性研究
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摘要
目前治疗急性中耳炎仍以使用抗生素为主要手段，但由于新型抗生素研发的相对滞后及临床上常出现抗生素运用的不当，耐药现象较为普遍。因此目前急性中耳炎患者存在治疗周期长、效果差、费用偏高等问题，不少病人反复发作，易形成慢性中耳炎。根据急性中耳炎感染的流行病学研究资料，研制针对中耳炎病原菌的疫苗，接种于急性中耳炎的易感人群，以期有效预防中耳炎的发生，将有望解决这一困惑。肺炎球菌是国内外急性中耳炎发病的最主要致病菌。目前国内已研制出肺炎球菌结合疫苗，但其使用的蛋白载体为破伤风类毒素或白喉类毒素，在将来的临床运用中可能会面临与破伤风疫苗或白喉疫苗冲突等问题。肺炎球菌溶血素作为一种新型蛋白载体，具有各亚型肺炎球菌间交叉免疫保护作用等多方面的优势。本研究通过多糖蛋白偶联技术、基因工程技术等成功研制出用于预防急性中耳炎的肺炎球菌多糖和肺炎球菌溶血素结合疫苗，并通过幼龄小鼠的接种试验，证明有较好的免疫原性。
第一部分肺炎球菌荚膜多糖与蛋白质载体CEA结合疫苗的研制
目的探索和建立肺炎球菌荚膜多糖与蛋白质载体结合疫苗的制备的适宜方法和条件，并通过幼龄小鼠的动物接种试验检测其免疫原性，同时为今后检测肺炎球菌多糖与溶血素结合疫苗的免疫原性制备包被抗原。
方法用19F型肺炎球菌多糖(PS)以氨基还原法分别与鸡卵清蛋白(CEA)及牛血清白蛋白(BSA)偶联结合，以SepharoseCL-4B凝胶柱层析纯化。PS-CEA结合物加入氢氧化铝佐剂后制备成疫苗，PS-BSA结合物则用作本实验及以后研制肺炎球菌溶血素为载体的结合疫苗时ELISA法检测抗PS抗体时包被酶标板的包被抗原。分别以每次0.25μg、1.0μg、4.0μg 3种不同多糖含量的PS(19F)-CEA结合疫苗腹腔注射3周龄幼小鼠，每隔1周复种1次，接种第3次后7天采血。多糖疫苗对照组幼小鼠以19F型肺炎球菌多糖疫苗接种(每次4.0μg)，阴性对照组以0.15M PBS注射。用ELISA法检测小鼠血清中抗肺炎球菌多糖抗体水平。
结果经SepharoseCL-4B凝胶柱层析洗脱后肺炎球菌多糖与蛋白CEA或BSA结合物的A_(280)峰值位置均较多糖及载体蛋白的各自峰值位置明显前移。以多糖含量分别为0.25μg、1.0μg、4.0μg的结合疫苗接种的3组幼小鼠血清中抗肺炎球菌多糖抗体IgG的几何平均滴度(GMT)分别为18.05、28.85、36.09，多糖疫苗对照组为7.70，阴性对照组为5.69。多糖疫苗对照组抗体水平无明显增高(P＞0.05)，
3组结合疫苗实验组抗体水平均明显增高(P＜0.001)。
结论已成功建立制备肺炎球菌多精蛋白结合疫苗的适宜方法和条件，该疫苗能在幼龄小鼠体内引起明显的免疫应答。
第二部分基因工程技术制备肺炎球菌溶血素
目的制备肺炎球菌溶血素，为研制肺炎球菌多糖蛋白结合疫苗提供新型的蛋白载体。
方法根据Walker等1987年报道的肺炎球菌溶血索(Ply)基因序列(GenBank accession no．X52474)，设计一对带有两个限制性内切酶酶切位点的引物，运用PCR的方法，从肺炎球菌的染色体DNA中扩增出Ply的基因。对该PCR产物进行酶切、回收，再将其克隆入表达载体pET-28a质粒中，用此基因重组质粒转化大肠杆菌JM109(DE3)宿主细胞，并以IPTG诱导蛋白质的表达。通过超声波破菌收集蛋白，对所得蛋白质用固相Ni-NTA树脂作亲和吸附纯化，再用SDS-PAGE电泳法鉴定表达的纯化基因重组Ply蛋白(rPly)。
结果通过常规克隆方法和PCR技术成功克隆Ply基因，并经琼脂糖凝胶电泳和DNA测序分析证实。成功构建了重组质粒并转化宿主细胞，蛋白表达的产物相对分子量为52KDa，经纯化后的rPly蛋白纯度达到90％以上。
结论可通过基因工程技术克隆Ply基因并进行蛋白表达而获得肺炎球菌溶血素，采用固化镍树脂亲和纯化技术所得Ply蛋白的纯度可满足于用作蛋白质载体和酶标检测包被抗原的要求。
第三部分肺炎球菌荚膜多糖与肺炎球菌溶血素结合疫苗的制备及免疫原性研究
目的研制肺炎球菌19F型荚膜多糖与肺炎球菌溶血素(蛋白)偶联结合疫苗，并检测其在幼龄动物体内的免疫原性。
方法先用福尔马林将通过基因工程技术制备得到的肺炎球菌溶血素脱毒，去除其溶血活性，再用19F型肺炎球菌荚膜多糖(PS)以氨基还原法与之偶联结合，以SepharoseCL-4B凝胶柱层析纯化。将纯化后的结合物与铝佐剂混合制成疫苗。以该疫苗腹腔注射接种3周龄幼小鼠，剂量为3μg多糖含量／次，每周接种1次，接种1～3次。阴性对照组小鼠腹腔注射等量0.15M PBS。接种完成后用ELISA法检测小鼠血清中抗PS及抗Ply的IgG抗体水平。
结果Ply经福尔马林脱毒后失去溶血活性。19F PS-PIy结合物经SepharoseCL-4B凝胶层析柱沈脱、纯化，其洗脱峰位置较19F PS与Ply二者的峰值位置均明显前移。用该结合疫苗免疫的幼龄小鼠接种1次、2次、3次时血清中抗PS(19F)抗体IgG的GMT分别为32.25、42.07、72.41，阴性对照组为8.9(P值均＜0.001)：抗Ply抗体IgG的GMT分别为36.21、80.71、138.3，对照组为22.37(P＜0.01或P＜0.001)。接种1～3次时两种抗体效价均明显高于阴性对照组。
结论用基因工程技术制备的肺炎球菌溶血素以福尔马林脱毒后作为蛋白载体与肺炎球菌荚膜多糖偶联结合，制成的结合疫苗能在幼龄小鼠体内诱导出明显的针对PS和Ply的免疫应答反应。
Currently, acute otitis media (AOM) is one of the most common infections in childhood and a frequent reason for prescribing antibacterials in infancy. The increasing of drug resistant bacteria against antibiotics is a big problem particularly in China. However, the increase in prevalence of respiratory antibiotics-resistant bacterial pathogens has not been matched by the development of new antibacterial agents. It will result in a vicious circle between overuse of antibiotics and resistance of bacteria. The cost for the treatment of AOM has become a heavy burden in many families. Multiple recurrence of AOM usually leads to chronic otitis media (COM). The recent emergence of drug-resistant strains has provided a strong incentive for preventing AOM by vaccination. Many studies showed Streptococcus pneumoniae was the most common AOM pathogen. At present, the protein-polysaccharide conjugate pneumococcal vaccine has been developed in China. However, tetanus toxoid (TT) or diphtheria toxoid (DT) was regular used as protein carrier in general. Because of some disadvantages of TT and DT as carriers of conjugate vaccine against AOM, pneumolysin has become a promising candidate as a carrier for conjugate vaccine. This study is to research and develop the conjugate vaccine against AOM using pneumolysin carrier.
Part 1. The preparation of pneumococcal conjugate vaccine bycoupling 19F polysaccride with BSA or CEA
Objectives To explore the technique of preparing the pneumococcal conjugate vaccine and evaluate its immunogenicity in infant mice, and to prepare the coating antigen for evaluating the immunogenicity of PS-Ply conjugate vaccine by ELISA in the future. Methods Type 19F polysaccharide (19F PS) was covalently coupled directly to protein carrier bovine serum albumin (BSA) or chicken egg albumin (CEA) by reductive amination method. The conjugates was purified by sepharose CL-4B chromatography. Al_2(OH)_3 was used as adjuvant to prepare the conjugate vaccine. The 19F-CEA conjugate vaccine was injected into infant mice i.p. with various PS dose, 0.25μg, 1.0μg, 4.0μ.g, per mouse every time, respectively. Every mouse was vaccinated 3 times at 1-week intervals. 19F PS vaccine was injected into the animals as a PS control (4.0μg every time). The negative controls were injected with 0.15M PBS. The antibody titers to 19F PS in mice serum were measured by ELISA using PS-BSA as antigen coating on the plate.
Results The peak of A_(280) reading of the conjugate 19F-CEA or 19F-BSA eluate shifted remarkably forward compared with that of 19F PS or the proteins, respectively. The antibody titers gradually increased with the increasment of the PS dose. The control group vaccinated with 19F PS vaccine had almost not antibody response against 19F PS compared with the non-treated group (P>0.05). In contrast, the groups immunized with conjugate vaccine had statistically significant response (P<0.001). In the three study groups, the anti-PS IgG titer of the group with dose 1.0μg was significantly higher than that with dose 0.25μg, whereas there was no significant difference between 1.0μg group and 4.0μg group.
Conclusion The procedures to prepare pneumococcal conjugate vaccine have been successfully developed. The conjugate vaccine has effective immunogenicity in infant mice rather than PS vaccine. The titers of anti-PS IgG will increase with the increasement of PS dose of the conjugate vaccine at an extent.
Part 2. Preparation of recombinant pneumolysin by genetic engineering technology
Objective To prepare pneumolysin as a new protein carrier of pneumococcalconjugate vaccine.
Methods According to the reported gene sequence of Pneumolysin (Ply) by Walker in 1987, a pair of primers which included two restriction enzyme sites were designed. Genomic DNA was isolated from Streptococcus pneumoniae. The gene encoding for pneumolysin was amplified from pneumococcal DNA with PCR. The restriction enzyme digested fragment was linked into the cloning vector PET-28a. The recombinant plasmid DNA containing pneumolysin gene was then transformed into host cell E. coli JM109 (DE3) to express pneumolysin. The positive clones should be seeked out and digested by enzyme for identifying and sequence analyzing. The recombinant pneumolysin(rPly) induced by IPTG was expressed in vivo and purified by using Ni-NTA gel-filtration chromatography . The SDS-PAGE was used to identify the rPly.
Results The DNA fragment was amplified from pneumococal chromosomal DNA by PCR and then subcloned into the expression vector pET-28a. The inserted pneumolysin gene sequence was confirmed by DNA sequencing and agarose gel electrophoresis. The pneumolysin protein was successfully expressed by JM109(DE3) with IPTG inducement. The relative molecular mass of the expressed product was 52kDa. The purity grade of rPly purified by Ni-NTA gel-filtration chromatography was above 90%.
Conclusion The rPly could be obtained by genetic engineering technology. The purity and the yield of rPly purified by Ni-NTA gel-filtration chromatograph were good enough for the conjugation with PS.
Part 3. The preparation of pneumococcal conjugate vaccine using pneumolysin as a protein carrier and the immunogenicity ofthe vaccine in infant mice
Objectives To prepare pneumococcal conjugate vaccine using pneumolysin as a protein carrier and evaluate the immunogenicity of the conjugate vaccine in infant mice.
Methods The pneumolysin obtained via genetic engineering technology was detoxified by 0.2% or 0.5% formalin respectively. Pneumococcal polysaccharide (PS,19F serotype) was conjugated with detoxified Ply using amino reductive method. The conjugate was purified via SepharoseCL-4B chromatography. Then the adjuvant Al_2(OH)_3 was mixed with the conjugate before vaccination of the mice. The study groups of infant mice of 3 weeks old were vaccinated with the conjugate vaccine 1 to 3 times at 1-week intervals. The infant mice injected with 0.15M PBS i.p. served as negative control group. The antibody response to 19F PS or Ply was measured by ELISA respectively.
Results Pneumolysin could be detoxified by 0.2% or 0.5% formalin. The peak of A_(280) reading of the conjugate 19F-Ply eluate shifted remarkably forward compared with that of 19F PS or the protein Ply. So the PS(19F serotype) was successfully conjugated with Ply. The GMTs of IgG against 19F PS of three groups which were vaccinated with conjugate vaccine for 1 to 3 times were 32.25, 42.07, 72.41, respectively, and it was only 8.9 in the control group. The GMTs of IgG against Ply were 36.21, 80.71 and 138.3, and it was 22.37 in the control group. There was a significant difference of antibody titer against 19F PS or Ply between vaccinated and non-vaccinated mice (P<0.01), despite of the times of vaccination. Conclusion The 19F PS-Ply conjugate vaccine produced using amino reductive method can induce specific immune response to both 19F PS and Ply in infant mice. It can be considered as preferable vaccine candidate against otitis media for otitis prone children.
第一部分肺炎球菌荚膜多糖与蛋白质载体CEA结合疫苗的研制
目的探索和建立肺炎球菌荚膜多糖与蛋白质载体结合疫苗的制备的适宜方法和条件，并通过幼龄小鼠的动物接种试验检测其免疫原性，同时为今后检测肺炎球菌多糖与溶血素结合疫苗的免疫原性制备包被抗原。
方法用19F型肺炎球菌多糖(PS)以氨基还原法分别与鸡卵清蛋白(CEA)及牛血清白蛋白(BSA)偶联结合，以SepharoseCL-4B凝胶柱层析纯化。PS-CEA结合物加入氢氧化铝佐剂后制备成疫苗，PS-BSA结合物则用作本实验及以后研制肺炎球菌溶血素为载体的结合疫苗时ELISA法检测抗PS抗体时包被酶标板的包被抗原。分别以每次0.25μg、1.0μg、4.0μg 3种不同多糖含量的PS(19F)-CEA结合疫苗腹腔注射3周龄幼小鼠，每隔1周复种1次，接种第3次后7天采血。多糖疫苗对照组幼小鼠以19F型肺炎球菌多糖疫苗接种(每次4.0μg)，阴性对照组以0.15M PBS注射。用ELISA法检测小鼠血清中抗肺炎球菌多糖抗体水平。
结果经SepharoseCL-4B凝胶柱层析洗脱后肺炎球菌多糖与蛋白CEA或BSA结合物的A_(280)峰值位置均较多糖及载体蛋白的各自峰值位置明显前移。以多糖含量分别为0.25μg、1.0μg、4.0μg的结合疫苗接种的3组幼小鼠血清中抗肺炎球菌多糖抗体IgG的几何平均滴度(GMT)分别为18.05、28.85、36.09，多糖疫苗对照组为7.70，阴性对照组为5.69。多糖疫苗对照组抗体水平无明显增高(P＞0.05)，
3组结合疫苗实验组抗体水平均明显增高(P＜0.001)。
结论已成功建立制备肺炎球菌多精蛋白结合疫苗的适宜方法和条件，该疫苗能在幼龄小鼠体内引起明显的免疫应答。
第二部分基因工程技术制备肺炎球菌溶血素
目的制备肺炎球菌溶血素，为研制肺炎球菌多糖蛋白结合疫苗提供新型的蛋白载体。
方法根据Walker等1987年报道的肺炎球菌溶血索(Ply)基因序列(GenBank accession no．X52474)，设计一对带有两个限制性内切酶酶切位点的引物，运用PCR的方法，从肺炎球菌的染色体DNA中扩增出Ply的基因。对该PCR产物进行酶切、回收，再将其克隆入表达载体pET-28a质粒中，用此基因重组质粒转化大肠杆菌JM109(DE3)宿主细胞，并以IPTG诱导蛋白质的表达。通过超声波破菌收集蛋白，对所得蛋白质用固相Ni-NTA树脂作亲和吸附纯化，再用SDS-PAGE电泳法鉴定表达的纯化基因重组Ply蛋白(rPly)。
结果通过常规克隆方法和PCR技术成功克隆Ply基因，并经琼脂糖凝胶电泳和DNA测序分析证实。成功构建了重组质粒并转化宿主细胞，蛋白表达的产物相对分子量为52KDa，经纯化后的rPly蛋白纯度达到90％以上。
结论可通过基因工程技术克隆Ply基因并进行蛋白表达而获得肺炎球菌溶血素，采用固化镍树脂亲和纯化技术所得Ply蛋白的纯度可满足于用作蛋白质载体和酶标检测包被抗原的要求。
第三部分肺炎球菌荚膜多糖与肺炎球菌溶血素结合疫苗的制备及免疫原性研究
目的研制肺炎球菌19F型荚膜多糖与肺炎球菌溶血素(蛋白)偶联结合疫苗，并检测其在幼龄动物体内的免疫原性。
方法先用福尔马林将通过基因工程技术制备得到的肺炎球菌溶血素脱毒，去除其溶血活性，再用19F型肺炎球菌荚膜多糖(PS)以氨基还原法与之偶联结合，以SepharoseCL-4B凝胶柱层析纯化。将纯化后的结合物与铝佐剂混合制成疫苗。以该疫苗腹腔注射接种3周龄幼小鼠，剂量为3μg多糖含量／次，每周接种1次，接种1～3次。阴性对照组小鼠腹腔注射等量0.15M PBS。接种完成后用ELISA法检测小鼠血清中抗PS及抗Ply的IgG抗体水平。
结果Ply经福尔马林脱毒后失去溶血活性。19F PS-PIy结合物经SepharoseCL-4B凝胶层析柱沈脱、纯化，其洗脱峰位置较19F PS与Ply二者的峰值位置均明显前移。用该结合疫苗免疫的幼龄小鼠接种1次、2次、3次时血清中抗PS(19F)抗体IgG的GMT分别为32.25、42.07、72.41，阴性对照组为8.9(P值均＜0.001)：抗Ply抗体IgG的GMT分别为36.21、80.71、138.3，对照组为22.37(P＜0.01或P＜0.001)。接种1～3次时两种抗体效价均明显高于阴性对照组。
结论用基因工程技术制备的肺炎球菌溶血素以福尔马林脱毒后作为蛋白载体与肺炎球菌荚膜多糖偶联结合，制成的结合疫苗能在幼龄小鼠体内诱导出明显的针对PS和Ply的免疫应答反应。
Currently, acute otitis media (AOM) is one of the most common infections in childhood and a frequent reason for prescribing antibacterials in infancy. The increasing of drug resistant bacteria against antibiotics is a big problem particularly in China. However, the increase in prevalence of respiratory antibiotics-resistant bacterial pathogens has not been matched by the development of new antibacterial agents. It will result in a vicious circle between overuse of antibiotics and resistance of bacteria. The cost for the treatment of AOM has become a heavy burden in many families. Multiple recurrence of AOM usually leads to chronic otitis media (COM). The recent emergence of drug-resistant strains has provided a strong incentive for preventing AOM by vaccination. Many studies showed Streptococcus pneumoniae was the most common AOM pathogen. At present, the protein-polysaccharide conjugate pneumococcal vaccine has been developed in China. However, tetanus toxoid (TT) or diphtheria toxoid (DT) was regular used as protein carrier in general. Because of some disadvantages of TT and DT as carriers of conjugate vaccine against AOM, pneumolysin has become a promising candidate as a carrier for conjugate vaccine. This study is to research and develop the conjugate vaccine against AOM using pneumolysin carrier.
Part 1. The preparation of pneumococcal conjugate vaccine bycoupling 19F polysaccride with BSA or CEA
Objectives To explore the technique of preparing the pneumococcal conjugate vaccine and evaluate its immunogenicity in infant mice, and to prepare the coating antigen for evaluating the immunogenicity of PS-Ply conjugate vaccine by ELISA in the future. Methods Type 19F polysaccharide (19F PS) was covalently coupled directly to protein carrier bovine serum albumin (BSA) or chicken egg albumin (CEA) by reductive amination method. The conjugates was purified by sepharose CL-4B chromatography. Al_2(OH)_3 was used as adjuvant to prepare the conjugate vaccine. The 19F-CEA conjugate vaccine was injected into infant mice i.p. with various PS dose, 0.25μg, 1.0μg, 4.0μ.g, per mouse every time, respectively. Every mouse was vaccinated 3 times at 1-week intervals. 19F PS vaccine was injected into the animals as a PS control (4.0μg every time). The negative controls were injected with 0.15M PBS. The antibody titers to 19F PS in mice serum were measured by ELISA using PS-BSA as antigen coating on the plate.
Results The peak of A_(280) reading of the conjugate 19F-CEA or 19F-BSA eluate shifted remarkably forward compared with that of 19F PS or the proteins, respectively. The antibody titers gradually increased with the increasment of the PS dose. The control group vaccinated with 19F PS vaccine had almost not antibody response against 19F PS compared with the non-treated group (P>0.05). In contrast, the groups immunized with conjugate vaccine had statistically significant response (P<0.001). In the three study groups, the anti-PS IgG titer of the group with dose 1.0μg was significantly higher than that with dose 0.25μg, whereas there was no significant difference between 1.0μg group and 4.0μg group.
Conclusion The procedures to prepare pneumococcal conjugate vaccine have been successfully developed. The conjugate vaccine has effective immunogenicity in infant mice rather than PS vaccine. The titers of anti-PS IgG will increase with the increasement of PS dose of the conjugate vaccine at an extent.
Part 2. Preparation of recombinant pneumolysin by genetic engineering technology
Objective To prepare pneumolysin as a new protein carrier of pneumococcalconjugate vaccine.
Methods According to the reported gene sequence of Pneumolysin (Ply) by Walker in 1987, a pair of primers which included two restriction enzyme sites were designed. Genomic DNA was isolated from Streptococcus pneumoniae. The gene encoding for pneumolysin was amplified from pneumococcal DNA with PCR. The restriction enzyme digested fragment was linked into the cloning vector PET-28a. The recombinant plasmid DNA containing pneumolysin gene was then transformed into host cell E. coli JM109 (DE3) to express pneumolysin. The positive clones should be seeked out and digested by enzyme for identifying and sequence analyzing. The recombinant pneumolysin(rPly) induced by IPTG was expressed in vivo and purified by using Ni-NTA gel-filtration chromatography . The SDS-PAGE was used to identify the rPly.
Results The DNA fragment was amplified from pneumococal chromosomal DNA by PCR and then subcloned into the expression vector pET-28a. The inserted pneumolysin gene sequence was confirmed by DNA sequencing and agarose gel electrophoresis. The pneumolysin protein was successfully expressed by JM109(DE3) with IPTG inducement. The relative molecular mass of the expressed product was 52kDa. The purity grade of rPly purified by Ni-NTA gel-filtration chromatography was above 90%.
Conclusion The rPly could be obtained by genetic engineering technology. The purity and the yield of rPly purified by Ni-NTA gel-filtration chromatograph were good enough for the conjugation with PS.
Part 3. The preparation of pneumococcal conjugate vaccine using pneumolysin as a protein carrier and the immunogenicity ofthe vaccine in infant mice
Objectives To prepare pneumococcal conjugate vaccine using pneumolysin as a protein carrier and evaluate the immunogenicity of the conjugate vaccine in infant mice.
Methods The pneumolysin obtained via genetic engineering technology was detoxified by 0.2% or 0.5% formalin respectively. Pneumococcal polysaccharide (PS,19F serotype) was conjugated with detoxified Ply using amino reductive method. The conjugate was purified via SepharoseCL-4B chromatography. Then the adjuvant Al_2(OH)_3 was mixed with the conjugate before vaccination of the mice. The study groups of infant mice of 3 weeks old were vaccinated with the conjugate vaccine 1 to 3 times at 1-week intervals. The infant mice injected with 0.15M PBS i.p. served as negative control group. The antibody response to 19F PS or Ply was measured by ELISA respectively.
Results Pneumolysin could be detoxified by 0.2% or 0.5% formalin. The peak of A_(280) reading of the conjugate 19F-Ply eluate shifted remarkably forward compared with that of 19F PS or the protein Ply. So the PS(19F serotype) was successfully conjugated with Ply. The GMTs of IgG against 19F PS of three groups which were vaccinated with conjugate vaccine for 1 to 3 times were 32.25, 42.07, 72.41, respectively, and it was only 8.9 in the control group. The GMTs of IgG against Ply were 36.21, 80.71 and 138.3, and it was 22.37 in the control group. There was a significant difference of antibody titer against 19F PS or Ply between vaccinated and non-vaccinated mice (P<0.01), despite of the times of vaccination. Conclusion The 19F PS-Ply conjugate vaccine produced using amino reductive method can induce specific immune response to both 19F PS and Ply in infant mice. It can be considered as preferable vaccine candidate against otitis media for otitis prone children.
引文
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27 Michon F, Fusco PC, Minetti CA, et al. Multivalent pneumococcal capsular polysaccharide conjugate vaccines employing genetically detoxified pneumolysin as a carrier protein[J]. Vaccine, 1998, 16(18): 1732-1741.
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41 Michon F, Fusco PC, Minetti CA, et al. Multivalent pneumococcal capsular polysaccharide conjugate vaccines employing genetically detoxified pneumolysin as a carrier protein[J]. Vaccine, 1998, 16(18): 1732-1741.
2. Alho OP, Koivu M, Sorri M. What is an "otitis-prone" child[J]? Int J Pediatr Otorhinolaryngol, 1991, 21 (3): 201-209.
3. Hermansson A, Emgard P, Prellner K, et al. A rat model for pneumococcal otitis media[J]. Am J Otolaryngol, 1988, 9(3): 97-100.
4. Kaplan B, Wandstat TL, Cunningham JR. Overall cost in the treatment of otitis media[J]. Pediatr Infect Dis J, 1997, 16: S9-S11.
5. Teele DW, Klein JO, Rosner B. Epidemiology of otitis media during the first seven years of life in children in greater Boston: a prospective, cohort study[J]. J Infect Dis. 1989, 160(1): 83-94.
6. Gates GA. Cost-effectiveness considerations in otitis media treatment[J]. Otolaryngol Head Neck Surg, 1996, 114: 525-530.
7. Reinert P. The role of pneumococcal and haemophilus type B vaccination in the prevention of acute otitis media[J]. Clin Microbiol Infect. 1997, 3: s59-s61.
8. Karma P. Vaccination and otitis media[J]. ORL J Otorhinolaryngol Relat Spec. 2002, 64[J]: 80-85.
9. Doweil SF, Butler JC, Giebink GS. et ai. Acute otitis media: management and surveillance in an era of pneumococcal resistance-a report from the Drug-resistant Streptococcus pneumoniae Therapeutic Working Group[J]. Pediatr Infect Dis J. 1999, 18(1): 1-9.
10．汪若峰，王鸿，诸小侬，等．婴幼儿急性化脓性中耳炎肺炎双球菌的研究[J]．中华耳鼻咽喉科杂志，1985，20：132-134．
11．王传清，王爱敏，张颖华，等．呼吸道感染患儿肺炎链球菌血清分型及耐药性．中国抗感染化疗杂志[J]．2002，2：218-220．
12. Jacobs MR, Bajaksouzian S, Zilles A, et al. Susceptibilities of Streptococcus pneumoniae and Haemophilus influenzae to 10 oral antimicrobial agents based on pharmacodynamic parameters: 1997 U.S. Surveillance study[J]. Antimicrob Agents Chemother. 1999, 43(8): 1901-1908.
13. American Academy of Pediatrics Committee on Infectious Diseases. Therapy for children with invasive pneumococcal infections. American Academy of Pediatrics Committee on Infectious Diseases[J]. Pediatrics. 1997, 99: 289-299.
14. Jacobs MR. Prevention of otitis media: Role of pneumococcal conjugate vaccines in reducing incidence and antibiotic resistance[J]. The Journal of Pediatrics. 2002, 141(2): 287-293.
15. Dagan R. Treatment of acute otitis media - challenges in the era of antibiotic resistance[J]. Vaccine, 2000, 19(Suppl 1): S9-S16.
16. Dhooge IL, Van Kempen MJ, Sanders LA, et al. Deficient IgA and IgG2 anti-pneumococcal antibody levels and response to vaccination in otitis prone children[J], Int J Pediatr Otorhinolaryngol, 2002, 64(2): 133-141.
17. Douglas RM, Paton JC, Duncan SJ, et al. Antibody response to pneumococcal vaccination in children younger than five years of age[J]. J Infect Dis, 1983, 148: 131-137.
18. Leinonen M, Sakkinen A, Kalliokoski R, et al. Antibody response to 14-valent pneumococcal capsular polysaccharide vaccine in pre-school age children[J]. Pediatr Infect Dis J, 1986, 5: 39-44.
19．顾新星，胡伟钢，陈静．细菌多糖、脂多糖及其蛋白质结合疫苗[A]．见：李忠明主编．当代新疫苗[M]．北京：高等教育出版社，2001：293-294．
20. Overturf GD. Pneumococcal vaccination of children[J]. Semin Pediatr Infect Dis, 2002, 13(3):155-64.
21. Straetemans M, Sanders EA, Veenhoven RH, et al. Pneumococcai vaccines for preventing otitis media[J]. Cochrane Database Syst Rev, 2002(2):CD001480.
22. Sipila M, Pukander J, Karma P. Incidence of acute otitis media up to the age of 1 1/2 years in urban infants[J]. Acta Otolaryngol, 1987, 104:138-145.
23. Jacobson RM, Poland GA. The pneumococcal conjugate vaccine[J]. Minerva Pediatr 2002, 54(4):295-303.
24. Breukels MA, Rijkers GT, Voorhorst-Ogink MM, et al. Pneumococcal conjugate vaccine primes for polysaccharide-inducible IgG2 antibody response in children with recurrent otitis media acuta[J]. J Infect Dis, 1999, 179(5):1152-1156.
25. Snow JB Jr. Progress in the prevention of otitis media through immunization[J]. Otol Neurotol, 2002, 23(1): 1-2.
26. Cheng AT, Young NM. Middle ear effusion in children[J]. Indian J Pediatr, 1997, 64(6):755-761.
27. Le TM, Rovers MM, Veenhoven RH, et al. Effect of pneumococcal vaccination on otitis media with effusion in children older than 1 year[J]. Eur J Pediatr, 2006, Epub ahead of print.
28. van Heerbeek N, Straetemans M, Wiertsema SP, et al. Effect of combined pneumococcal conjugate and polysaccharide vaccination on recurrent otitis media with effusion[J]. Pediatrics, 2006, 117(3):603-608.
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