A群脑膜炎球菌多糖结合物的免疫原性研究

目的：制备A群脑膜炎球菌多糖（PS）结合物，观察其免疫原性。方法：A群脑膜炎球菌多糖经溴化氰活化后共价接上己二酰肼手臂，在碳二亚胺催化下与精制破伤风类毒素（TT）偶联剂备结合物。单独多糖、多糖结合物和TT按相同程序免疫NIH小鼠，用ELISA法检测小鼠血清抗多糖抗体滴度和特异性及抗TT抗体滴度。结果：用上述实验方法制备的结合物具有多糖和蛋白质的抗原性，免疫小鼠可诱导高滴度的血清抗多糖IgG抗体，免疫2针后高滴度抗体至少持续存在3周。血清抗体可中和多糖。加强免疫提示有免疫记忆性。结合物还可诱导产生一定水平的TT抗体。结论：A群脑膜炎球菌多糖结合到TT上可明显增强多糖在小鼠中的免疫原性，为制备结合疫苗并研究其对婴幼儿的免疫原性提供了实验基础。     

rCTB和TT为蛋白载体的A群流脑多糖黏膜免疫初步研究

目的 对重组霍乱毒素B亚单位(rCTB)作为多糖蛋白结合疫苗候选载体的可行性进行分析,并对以破伤风类毒素(TT)与rCTB为蛋白载体的黏膜投递型疫茸的免疫效果进行初步探讨.方法 首先通过基因工程手段获得具有五聚体结构的rCTB.再将rCTB五聚体蛋白利用化学方法(ADH方法)与A群脑膜炎球菌多糖(GAMP)耦联,获得多糖蛋白结合物GAMP-rCTB,并将其与TT为蛋白载体的A群流脑多糖蛋白结合物(GAMP-TT)以滴鼻和注射途径免疫BALB/c小鼠,并对其进行免疫学评价.结果 以rCTB和TT为载体的A群流脑多糖蛋白结合物,通过黏膜投递途径均可在血清中产生相对较高的多糖特异性IgG抗体,在肺部盥洗液和小肠黏膜也产生了相应的特异性IgA抗体.结论 rCTB和TT均可作为黏膜投递型多糖结合疫苗的候选蛋白载体.以rCTB为载体的多糖蛋白结合物,黏膜途径可能在免疫功能方面优于注射途径.     

rCTB和TT为蛋白载体的A群流脑多糖黏膜免疫初步研究

目的 对重组霍乱毒素B亚单位(rCTB)作为多糖蛋白结合疫苗候选载体的可行性进行分析,并对以破伤风类毒素(TT)与rCTB为蛋白载体的黏膜投递型疫茸的免疫效果进行初步探讨.方法 首先通过基因工程手段获得具有五聚体结构的rCTB.再将rCTB五聚体蛋白利用化学方法(ADH方法)与A群脑膜炎球菌多糖(GAMP)耦联,获得多糖蛋白结合物GAMP-rCTB,并将其与TT为蛋白载体的A群流脑多糖蛋白结合物(GAMP-TT)以滴鼻和注射途径免疫BALB/c小鼠,并对其进行免疫学评价.结果 以rCTB和TT为载体的A群流脑多糖蛋白结合物,通过黏膜投递途径均可在血清中产生相对较高的多糖特异性IgG抗体,在肺部盥洗液和小肠黏膜也产生了相应的特异性IgA抗体.结论 rCTB和TT均可作为黏膜投递型多糖结合疫苗的候选蛋白载体.以rCTB为载体的多糖蛋白结合物,黏膜途径可能在免疫功能方面优于注射途径.     

rCTB和TT为蛋白载体的A群流脑多糖黏膜免疫初步研究

目的 对重组霍乱毒素B亚单位(rCTB)作为多糖蛋白结合疫苗候选载体的可行性进行分析,并对以破伤风类毒素(TT)与rCTB为蛋白载体的黏膜投递型疫茸的免疫效果进行初步探讨.方法 首先通过基因工程手段获得具有五聚体结构的rCTB.再将rCTB五聚体蛋白利用化学方法(ADH方法)与A群脑膜炎球菌多糖(GAMP)耦联,获得多糖蛋白结合物GAMP-rCTB,并将其与TT为蛋白载体的A群流脑多糖蛋白结合物(GAMP-TT)以滴鼻和注射途径免疫BALB/c小鼠,并对其进行免疫学评价.结果 以rCTB和TT为载体的A群流脑多糖蛋白结合物,通过黏膜投递途径均可在血清中产生相对较高的多糖特异性IgG抗体,在肺部盥洗液和小肠黏膜也产生了相应的特异性IgA抗体.结论 rCTB和TT均可作为黏膜投递型多糖结合疫苗的候选蛋白载体.以rCTB为载体的多糖蛋白结合物,黏膜途径可能在免疫功能方面优于注射途径.     

rCTB和TT为蛋白载体的A群流脑多糖黏膜免疫初步研究

目的 对重组霍乱毒素B亚单位(rCTB)作为多糖蛋白结合疫苗候选载体的可行性进行分析,并对以破伤风类毒素(TT)与rCTB为蛋白载体的黏膜投递型疫茸的免疫效果进行初步探讨.方法 首先通过基因工程手段获得具有五聚体结构的rCTB.再将rCTB五聚体蛋白利用化学方法(ADH方法)与A群脑膜炎球菌多糖(GAMP)耦联,获得多糖蛋白结合物GAMP-rCTB,并将其与TT为蛋白载体的A群流脑多糖蛋白结合物(GAMP-TT)以滴鼻和注射途径免疫BALB/c小鼠,并对其进行免疫学评价.结果 以rCTB和TT为载体的A群流脑多糖蛋白结合物,通过黏膜投递途径均可在血清中产生相对较高的多糖特异性IgG抗体,在肺部盥洗液和小肠黏膜也产生了相应的特异性IgA抗体.结论 rCTB和TT均可作为黏膜投递型多糖结合疫苗的候选蛋白载体.以rCTB为载体的多糖蛋白结合物,黏膜途径可能在免疫功能方面优于注射途径.     

rCTB和TT为蛋白载体的A群流脑多糖黏膜免疫初步研究

目的 对重组霍乱毒素B亚单位(rCTB)作为多糖蛋白结合疫苗候选载体的可行性进行分析,并对以破伤风类毒素(TT)与rCTB为蛋白载体的黏膜投递型疫茸的免疫效果进行初步探讨.方法 首先通过基因工程手段获得具有五聚体结构的rCTB.再将rCTB五聚体蛋白利用化学方法(ADH方法)与A群脑膜炎球菌多糖(GAMP)耦联,获得多糖蛋白结合物GAMP-rCTB,并将其与TT为蛋白载体的A群流脑多糖蛋白结合物(GAMP-TT)以滴鼻和注射途径免疫BALB/c小鼠,并对其进行免疫学评价.结果 以rCTB和TT为载体的A群流脑多糖蛋白结合物,通过黏膜投递途径均可在血清中产生相对较高的多糖特异性IgG抗体,在肺部盥洗液和小肠黏膜也产生了相应的特异性IgA抗体.结论 rCTB和TT均可作为黏膜投递型多糖结合疫苗的候选蛋白载体.以rCTB为载体的多糖蛋白结合物,黏膜途径可能在免疫功能方面优于注射途径.     

rCTB和TT为蛋白载体的A群流脑多糖黏膜免疫初步研究

目的 对重组霍乱毒素B亚单位(rCTB)作为多糖蛋白结合疫苗候选载体的可行性进行分析,并对以破伤风类毒素(TT)与rCTB为蛋白载体的黏膜投递型疫茸的免疫效果进行初步探讨.方法 首先通过基因工程手段获得具有五聚体结构的rCTB.再将rCTB五聚体蛋白利用化学方法(ADH方法)与A群脑膜炎球菌多糖(GAMP)耦联,获得多糖蛋白结合物GAMP-rCTB,并将其与TT为蛋白载体的A群流脑多糖蛋白结合物(GAMP-TT)以滴鼻和注射途径免疫BALB/c小鼠,并对其进行免疫学评价.结果 以rCTB和TT为载体的A群流脑多糖蛋白结合物,通过黏膜投递途径均可在血清中产生相对较高的多糖特异性IgG抗体,在肺部盥洗液和小肠黏膜也产生了相应的特异性IgA抗体.结论 rCTB和TT均可作为黏膜投递型多糖结合疫苗的候选蛋白载体.以rCTB为载体的多糖蛋白结合物,黏膜途径可能在免疫功能方面优于注射途径.     

rCTB和TT为蛋白载体的A群流脑多糖黏膜免疫初步研究

目的 对重组霍乱毒素B亚单位(rCTB)作为多糖蛋白结合疫苗候选载体的可行性进行分析,并对以破伤风类毒素(TT)与rCTB为蛋白载体的黏膜投递型疫茸的免疫效果进行初步探讨.方法 首先通过基因工程手段获得具有五聚体结构的rCTB.再将rCTB五聚体蛋白利用化学方法(ADH方法)与A群脑膜炎球菌多糖(GAMP)耦联,获得多糖蛋白结合物GAMP-rCTB,并将其与TT为蛋白载体的A群流脑多糖蛋白结合物(GAMP-TT)以滴鼻和注射途径免疫BALB/c小鼠,并对其进行免疫学评价.结果 以rCTB和TT为载体的A群流脑多糖蛋白结合物,通过黏膜投递途径均可在血清中产生相对较高的多糖特异性IgG抗体,在肺部盥洗液和小肠黏膜也产生了相应的特异性IgA抗体.结论 rCTB和TT均可作为黏膜投递型多糖结合疫苗的候选蛋白载体.以rCTB为载体的多糖蛋白结合物,黏膜途径可能在免疫功能方面优于注射途径.     

肺炎球菌荚膜多糖和溶血素结合疫苗的制备及其免疫原性的研究

目的 研制肺炎球菌荚膜多糖（PS）与肺炎球菌溶血素（蛋白）偶联结合疫苗.方法 用PCR扩增肺炎球菌溶血素（Pn）基因,将其克隆入表达载体pET-28a质粒中,然后转化大肠杆菌JM109（DE3）宿主细胞,并以IPTG诱导进行蛋白质表达,对所得蛋白质用固相Ni-NTA树脂作纯化,再用SDS-PAGE鉴定表达的纯化蛋白（rPn）.以福尔马林脱毒rPn,去除其溶血活性,然后用氨基还原法将脱毒的rPn与PS（19F血清型）偶联结合,加入铝佐剂制备成疫苗.以该疫苗腹腔注射接种小鼠,用ELISA法检测小鼠血清中抗PS及抗Pn的体水平.结果 成功扩增和克隆了Pn基因,经诱导表达得到rPn蛋白,纯化后纯度达90%以上.PS与Pn结合物经凝胶层析柱分离,其洗脱峰较PS与Pn二峰明显前移.用该结合疫苗免疫的小鼠血清中抗PS（19F）抗体及抗Pn抗体的效价均明显高于阴性对照组（P＜0.01）.结论 用基因工程技术制备的肺炎球菌溶血素作为蛋白载体,脱毒后与肺炎球菌多糖偶联而成的结合疫苗能在小鼠体内诱导出明显的免疫应答反应.     

rCTB和TT为蛋白载体的A群流脑多糖黏膜免疫初步研究

目的 对重组霍乱毒素B亚单位(rCTB)作为多糖蛋白结合疫苗候选载体的可行性进行分析,并对以破伤风类毒素(TT)与rCTB为蛋白载体的黏膜投递型疫茸的免疫效果进行初步探讨.方法 首先通过基因工程手段获得具有五聚体结构的rCTB.再将rCTB五聚体蛋白利用化学方法(ADH方法)与A群脑膜炎球菌多糖(GAMP)耦联,获得多糖蛋白结合物GAMP-rCTB,并将其与TT为蛋白载体的A群流脑多糖蛋白结合物(GAMP-TT)以滴鼻和注射途径免疫BALB/c小鼠,并对其进行免疫学评价.结果 以rCTB和TT为载体的A群流脑多糖蛋白结合物,通过黏膜投递途径均可在血清中产生相对较高的多糖特异性IgG抗体,在肺部盥洗液和小肠黏膜也产生了相应的特异性IgA抗体.结论 rCTB和TT均可作为黏膜投递型多糖结合疫苗的候选蛋白载体.以rCTB为载体的多糖蛋白结合物,黏膜途径可能在免疫功能方面优于注射途径.     

Synthesis and immunological properties of conjugates composed of group B streptococcus type III capsular polysaccharide covalently bound to tetanus toxoid

A synthetic scheme for covalently binding group B streptococcus type III to tetanus toxoid (TT), using adipic acid dihydrazide as a spacer, is described. Type III alone or as a conjugate with TT was injected subcutaneously into laboratory mice, and the type-specific and TT antibody responses elicited by these immunogens were assayed. Type III-TT elicited significantly higher levels of type-specific antibodies after each immunization than did the type III alone. These levels were related to the dosage of the conjugate, enhanced by Freund adjuvant, and exhibited booster responses. Type III alone elicited only immunoglobulin M (IgM) antibodies in Swiss albino mice and mostly IgM and low levels of IgG antibodies of the IgG3 subclass in BALB/c mice. Type III-TT conjugates, in contrast, elicited mostly IgG antibodies in both strains of mice. IgA type III antibodies were not detected. The first two immunizations with the conjugates elicited type III antibodies in the IgG1 and in the IgG3 subclasses. Low levels of IgG2a type III antibodies were detected after a third injection of type III-TT. Conjugate-induced antibodies facilitated opsonization of group B streptococcus type III organisms and did not react with the structurally related pneumococcus type 14. TT alone or as a component of type III-TT induced mostly antibodies of the IgG class: IgG1 levels were the highest of the four subclasses. No IgA TT antibodies were detected. The conjugation procedure, therefore, enhanced the immunogenicity of and conferred T-cell dependent properties to the type III while preserving the immunogenicity of the TT component. The T-cell dependent properties of the conjugates were responsible for stimulating IgG type III antibodies which could be boosted. Evaluation of type III-TT conjugates in antibody-negative women of child-bearing age is planned.     

Capsular polysaccharide-protein conjugate vaccines of carbotype 1 Vibrio vulnificus: construction, immunogenicity, and protective efficacy in a murine model.

Vibrio vulnificus causes septicemia and wound infections in immunocompromised humans. The capsular polysaccharide of Vibrio vulnificus (VvPS) is critical for virulence. We synthesized conjugate vaccines of carbotype 1 VvPS under conditions and in formulations suitable for human use. Purified VvPS was conjugated to tetanus toxoid (TT) or to inactivated V. vulnificus cytolysin or elastase by two different schemes. All conjugates elicited elevated anticapsular immunoglobulin G (IgG) and IgM and antiprotein IgG responses in mice compared with saline placebo. The conjugates prepared through caboxyl activation of VvPS (VvPS-TTa, VvPS-cytolysin, and VvPS-elastase) were more immunogenic than the one prepared through hydroxyl activation (VvPS-TTb). The protective efficacy of conjugated and unconjugated formulations of VvPS and that of protein carriers were evaluated in a mouse septicemia model. Eighty percent of mice actively immunized with VvPS-TTa vaccine survived challenge with carbotype 1 V. vulnificus, while VvPS-cytolysin and VvPS-elastase conjugates conferred 44 and 40% protection, respectively. Control mice immunized with VvPS, cytolysin, or elastase alone, or saline only, showed 70 to 100% mortality. VvPS-TTa vaccine is nontoxic, immunogenic, and protective in mice.     

Further studies on the immunogenicity of Haemophilus influenzae type b and pneumococcal type 6A polysaccharide-protein conjugates

Conjugates were prepared by carbodiimide-mediated coupling of adipic acid hydrazide derivatives of Haemophilus influenzae type b (Hib), Escherichia coli K100, and pneumococcal 6A (Pn6A) polysaccharides with tetanus toxoid (TT), as an example of a “useful” carrier, and horseshoe crab hemocyanin (HCH), as an example of a “nonsense” carrier. These conjugates were injected into NIH mice, and their serum antibody responses to the polysaccharides and proteins were characterized. As originally reported, Hib conjugates increased the immunogenicity of the capsular polysaccharide and elicited greater than the estimated protective levels of anti-Hib antibodies in most recipients after one injection and in all after the third injection (Schneerson et al., J. Exp. Med. 152:361-376, 1980). Both Hib conjugates induced similar anti-Hib responses. The K100-HCH conjugate was more immunogenic than the K100-TT conjugate and elicited anti-Hib responses similar to the Hib conjugates after the third injection. Simultaneous injection of the K100 and the Hib conjugates did not enhance the anti-Hib response. The Pn6A-TT conjugate induced low levels of anti-Hib antibodies; when injected simultaneously with the Hib conjugates, the anti-Hib response was enhanced, as all mice responded after the first injection and with higher levels of anti-Hib than observed with the Hib conjugates alone (P < 0.05). The Pn6A conjugates were not as immunogenic as the Hib conjugates. Pn6A-TT was more effective than was Pn6A-HCH; it elicited anti-Pn6A (>100 ng of antibody nitrogen per ml) in 6 of 10 mice after the third injection. The addition of the Hib-HCH conjugate to the Pn6A-TT conjugate increased the anti-Pn6A response with a higher geometric mean antibody titer, and 9 of 10 mice responded after the third injection. A preparation of diphtheria toxoid, TT, and pertussis vaccine increased the anti-Hib antibody levels after the first injection only in mice receiving Hib-TT, but not in mice receiving Hib-HCH, suggesting that additional carrier protein (TT) enhanced the anti-polysaccharide response. Simultaneous injection of Hib and Pn6A conjugates with the same or different carriers resulted in an enhanced serum antibody response to each polysaccharide. The anti-tetanus toxin response reached protective levels (>0.01 U/ml) in most mice after the first injection and in all mice after the second and third injections of TT conjugates. A progressive increase in the anti-HCH response with each additional injection was noted in animals receiving HCH conjugates. Animals receiving the diphtheria toxoid-TT-pertussis vaccine preparation responded with a greater increase in anti-carrier antibody than those receiving the conjugates alone. This method of synthesis provided conjugates capable of inducing protective levels of antibodies to both the polysaccharides and carrier proteins.     

rCTB和TT为蛋白载体的A群流脑多糖黏膜免疫初步研究

目的 对重组霍乱毒素B亚单位(rCTB)作为多糖蛋白结合疫苗候选载体的可行性进行分析,并对以破伤风类毒素(TT)与rCTB为蛋白载体的黏膜投递型疫茸的免疫效果进行初步探讨.方法 首先通过基因工程手段获得具有五聚体结构的rCTB.再将rCTB五聚体蛋白利用化学方法(ADH方法)与A群脑膜炎球菌多糖(GAMP)耦联,获得多糖蛋白结合物GAMP-rCTB,并将其与TT为蛋白载体的A群流脑多糖蛋白结合物(GAMP-TT)以滴鼻和注射途径免疫BALB/c小鼠,并对其进行免疫学评价.结果 以rCTB和TT为载体的A群流脑多糖蛋白结合物,通过黏膜投递途径均可在血清中产生相对较高的多糖特异性IgG抗体,在肺部盥洗液和小肠黏膜也产生了相应的特异性IgA抗体.结论 rCTB和TT均可作为黏膜投递型多糖结合疫苗的候选蛋白载体.以rCTB为载体的多糖蛋白结合物,黏膜途径可能在免疫功能方面优于注射途径.     

rCTB和TT为蛋白载体的A群流脑多糖黏膜免疫初步研究

目的 对重组霍乱毒素B亚单位(rCTB)作为多糖蛋白结合疫苗候选载体的可行性进行分析,并对以破伤风类毒素(TT)与rCTB为蛋白载体的黏膜投递型疫茸的免疫效果进行初步探讨.方法 首先通过基因工程手段获得具有五聚体结构的rCTB.再将rCTB五聚体蛋白利用化学方法(ADH方法)与A群脑膜炎球菌多糖(GAMP)耦联,获得多糖蛋白结合物GAMP-rCTB,并将其与TT为蛋白载体的A群流脑多糖蛋白结合物(GAMP-TT)以滴鼻和注射途径免疫BALB/c小鼠,并对其进行免疫学评价.结果 以rCTB和TT为载体的A群流脑多糖蛋白结合物,通过黏膜投递途径均可在血清中产生相对较高的多糖特异性IgG抗体,在肺部盥洗液和小肠黏膜也产生了相应的特异性IgA抗体.结论 rCTB和TT均可作为黏膜投递型多糖结合疫苗的候选蛋白载体.以rCTB为载体的多糖蛋白结合物,黏膜途径可能在免疫功能方面优于注射途径.     

rCTB和TT为蛋白载体的A群流脑多糖黏膜免疫初步研究

目的 对重组霍乱毒素B亚单位(rCTB)作为多糖蛋白结合疫苗候选载体的可行性进行分析,并对以破伤风类毒素(TT)与rCTB为蛋白载体的黏膜投递型疫茸的免疫效果进行初步探讨.方法 首先通过基因工程手段获得具有五聚体结构的rCTB.再将rCTB五聚体蛋白利用化学方法(ADH方法)与A群脑膜炎球菌多糖(GAMP)耦联,获得多糖蛋白结合物GAMP-rCTB,并将其与TT为蛋白载体的A群流脑多糖蛋白结合物(GAMP-TT)以滴鼻和注射途径免疫BALB/c小鼠,并对其进行免疫学评价.结果 以rCTB和TT为载体的A群流脑多糖蛋白结合物,通过黏膜投递途径均可在血清中产生相对较高的多糖特异性IgG抗体,在肺部盥洗液和小肠黏膜也产生了相应的特异性IgA抗体.结论 rCTB和TT均可作为黏膜投递型多糖结合疫苗的候选蛋白载体.以rCTB为载体的多糖蛋白结合物,黏膜途径可能在免疫功能方面优于注射途径.