5型肺炎链球菌荚膜多糖与CRM197蛋白结合疫苗的制备及免疫原性的研究
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摘要
目的:研制5型肺炎链球菌荚膜多糖(PN5PS)与白喉无毒突变体(CRM197)结合疫苗。方法:采用还原胺化法制备结合物,即以肺炎5型多糖的邻位羟基作为活化位点,用高碘酸钠进行氧化,形成的醛基与CRM197蛋白上的氨基生成席夫碱反应,在氰基硼氢化钠的作用下形成共价结合物,并将制备的结合物与两种不同铝佐剂吸附,用间接ELISA法检测大鼠血清中针对肺炎5型多糖的抗体效价,比较结合疫苗与不同佐剂吸附前后的免疫原性。结果:当多糖活化度为7. 3时,总糖及蛋白的回收率较高,游离糖含量较低,糖蛋白结合比在1. 2~2.7时结合疫苗具有较强的免疫原性,结合比为1. 9时结合疫苗的免疫原性最强;结合物吸附磷酸铝佐剂后所产生的抗体水平略高于氢氧化铝。结论:通过还原胺化法制备的PN5PSCRM197结合疫苗具有较好的免疫原性。
Objective: To develop the method of combination of pneumococcal type 5 capsular polysaccharide(PN5 PS) with diphtheria non-toxic mutant(CRM197) conjugate. Methods: The PN5 PS was conjugated to protein with reductive amination method. The vicinal hydroxyl group of pneumonia type 5 polysaccharide was used as the activation site. It was oxidized by sodium periodate,and the aldehyde produced Schiff base reaction with amino of CRM197 protein to form a covalent conjugate under the action of sodium cyanoborohydride. Conjugates were adsorbed to different aluminum adjuvants and immunogenicity was evaluated in rat model.Antibody titers against the pneumococcal type 5 capsular polysaccharide in rat sera were determined by indirect ELISA. Results: Under the condition that degree of oxidation(DO) was 7. 3,the recovery of conjugate was optimal,and the content of free Ps was low. The immunogenicity of the conjugate vaccine was good when the proportion of polysaccharide and protein in the product is 1. 2-2. 7 and the best when the proportion of polysaccharide and protein in the product was 1. 9. Aluminum phosphate induced higher antibody titer compared with the aluminum hydroxide. Conclusion: The immunogenicity of the PN5 PS protein conjugate vaccine prepared with reductive amination method is good.
Objective: To develop the method of combination of pneumococcal type 5 capsular polysaccharide(PN5 PS) with diphtheria non-toxic mutant(CRM197) conjugate. Methods: The PN5 PS was conjugated to protein with reductive amination method. The vicinal hydroxyl group of pneumonia type 5 polysaccharide was used as the activation site. It was oxidized by sodium periodate,and the aldehyde produced Schiff base reaction with amino of CRM197 protein to form a covalent conjugate under the action of sodium cyanoborohydride. Conjugates were adsorbed to different aluminum adjuvants and immunogenicity was evaluated in rat model.Antibody titers against the pneumococcal type 5 capsular polysaccharide in rat sera were determined by indirect ELISA. Results: Under the condition that degree of oxidation(DO) was 7. 3,the recovery of conjugate was optimal,and the content of free Ps was low. The immunogenicity of the conjugate vaccine was good when the proportion of polysaccharide and protein in the product is 1. 2-2. 7 and the best when the proportion of polysaccharide and protein in the product was 1. 9. Aluminum phosphate induced higher antibody titer compared with the aluminum hydroxide. Conclusion: The immunogenicity of the PN5 PS protein conjugate vaccine prepared with reductive amination method is good.
引文
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[2]Avery OT,Dubos R.The protective action of a specific enzyme against type III pneumococcus infection in mice[J].J Exp Med,1931,54(1):73-89.
[3]Novotny LA,Partida-Sánchez S,Munson RS,et al.Differential uptake and processing of a Haemophilus influenzae P5-derived immunogen by chinchilla dendritic cells[J].Infection Immunity,2008,76(3):967-977.
[4]Toropainen M,Raitolehto A,Henckaerts I,et al.Pneumococcal Haemophilus influenzae protein D conjugate vaccine induces antibodies that inhibit glycerophosphodiester phosphordiesterase activity of protein D[J].Infection Immunity,2008,76(10):4546-4553.
[5]Backhaus E,Berg S,Andersson R,et al.Epidemiology of invasive pneumococcal infections:manifestations,incidence and case fatality rate correlated to age,gender and risk factors[J].BMC Infect Dis,2016,16(1):367-379.
[6]Herrero FS,Perez TL,Olivas JB,et al.Bacteremic Streptococcus pneumoniae in community-acquired pneumonia:an update[J].Current Respiratory Med Rev,2010,6(3):188-193.
[7]Beatiy JA,Majumdar SR,Tyrrell GJ,et al.Prognostic factors associated with mortality and major in-hospital complications in patients with bacteremic pneumococcal pneumonia:Population-based study[J].Medicine(Baltimore),2016,95(46):5179-5184.
[8]陆林,刘晓强.23价肺炎球菌多糖疫苗研究进展[J].中国新药杂志,2012,21(10):1099-1102.Lu L,Liu XQ.Advance in 23-valent pneumococcal polysaccharide vaccine[J].Chin J New Drugs,2012,21(10):1099-1102.
[9]Shen SJ.Serotypes and epidemiological study of pneumococcal infection in 18 provines(cities)in China[J].Zhonghua Liuxing Bing Xue Za Zhi,1989,10(3):133-137.
[10]朱学喆.肺炎链球菌疫苗研制进展[J].微生物免疫学进展,2013,41(1):58-64.Zhu XZ.Research progress on pneumococcal vaccine[J].Progress Microbiol Immunol,2013,41(1):58-64.
[11]陈晓航,王剑虹,任克明,等.5型肺炎链球菌荚膜多糖结合疫苗的不同糖蛋白比与免疫原性的关系[J].微生物学免疫学进展,2006,34(4):19-22.Chen XH,Wang JH,Ren KM,et al.Comparison on the immunogenicity in the different proportion of penumococcal polysaccharides and protein for type 5 of Streptococcus pneumonia[J].Progress Microbiol Immunol,2006,34(4):19-22.
[12]黄镇,陈玉秋,李薇,等.CDAP活化多糖制备5型肺炎链球菌荚膜多糖-破伤风类毒素结合疫苗[J].中国生物制品学杂志,2010,23(5):493-496.Huang Z,Chen YQ,Li W,et al.Preparation of type 5 Streptococcus pneumoniae capsular polysaccharide-TT conjugate vaccine by activation of polysaccharide with CDAP[J].Chin J Biologics,2010,23(5):493-496.
[13]Romaniuk SI,Kolybo DV,Komisarenko SV.Recombinant diphtheria toxinderivatives:perspectives of application[J].Russian J Bioorganic Chem,2012,38(6):565-577.
[14]陈玉秋,何建东,范荣坤,等.22F肺炎球菌荚膜多糖的活化及其多糖蛋白结合物的免疫原性[J].中国生物制品学杂志,2015,28(5):479-487.Chen YQ,He JD,Fang RK,et al.Activation of type 22F Streptococcus pneumoniae capsular polysaccharide and immunogenicity of polysaccharide-protein conjugate[J].Chin J Biol,2015,28(5):479-487.
[15]尹丹丹,周觉非,刘威,等.5型肺炎链球菌荚膜多糖与破伤风类毒素的结合及其影响因素探讨[J].生物医药前沿,2013,22(16):1895-1900.Yin DD,Zhou JF,Liu W,et al.Preparation of Streptococcus pneumonia type 5 capsular polysaccharide-TT conjugate and the key synthetic parameters[J].Chin J New Drugs,2013,22(16):1895-1900.