菊糖作为结核分枝杆菌和人乳头瘤病毒疫苗佐剂的免疫原性研究
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摘要
目的研究菊糖的免疫佐剂效果,探讨菊糖与结核分枝杆菌(Mtb)ESAT-CFP10融合蛋白EC、人乳头瘤病毒16型E6-E7融合蛋白E6E7联合免疫小鼠的免疫应答特征。方法将菊糖(Inulin)分别与E6E7、EC共价结合制备E6E7-I、EC-I疫苗,采用E6E7-I、EC-I免疫小鼠,通过比较小鼠血清中抗原特异性IgG滴度,脾淋巴细胞抗原特异性IFN-γ、TNF-α分泌水平和CD4~+T、CD8~+T亚群分布,评价菊糖的疫苗佐剂功能。结果 (1)第7、14、21 d,菊糖结合的EC-I、E6E7-I组抗原特异性抗体IgG滴度分别高于单独抗原EC组、E6E7组(P<0.05),且未检出抗菊糖抗体。(2)EC-I、E6E7-I能分别诱导出高于EC组、E6E7的脾淋巴细胞IFN-γ、TNF-α分泌水平(P<0.05)。(3)EC-I组、EC组、PBS组的方差分析结果表明,EC-I组CD8~+T、CD4~+T细胞亚群所占百分比高于EC组(P<0.05;P=0.017);E6E7-I组的CD8~+T、CD4~+T细胞亚群所占百分比高于E6E7-I组,分别(P=0.007;0.016)。结论菊糖佐剂共价结合E6E7、EC蛋白,能有效激活机体的抗原特异性体液免疫和细胞免疫,且佐剂自身免疫原性低,提示菊糖作为HPV16型宫颈癌疫苗、TB疫苗佐剂具有良好的佐剂效应。
Objective The aim of this study was to evaluate the immunogenicity and adjuvant effect of Jerusalem artichoke polysaccharide as vaccine adjuvant, and improve the immunogenicity of human papilloma carcinogenic antigen E6E7 fusion protein and tuberculous bacillus antigen EC fusion protein. Methods Inulin was combined with E6E7 and EC respectively to prepare I-E6E7 and I-EC as antigens and immunized mice, respectively. Then the immune response characteristics of mice were investigated. The specific IgG antibody titer, antigen specific IFN-γ, TNF-α expression in the mouse spleen lymphocyte were detected. The rations of CD4~+T cells and CD8~+T cells in spleen lymphocyte upon restimulation with EC or E6E7 in vitro and serum biochemical indexes were detected. Results The research showed that when inulin was used as an adjuvant to modify the antigen protein, at first, it could improve the antibody titer of the antigen protein, and fail to detect the anti-polysaccharide IgG antibody(P<0.05). And secondly, the higher secretion levels of IFN-γ and TNF-α than those of the control group were induced(P<0.05). At last, the proportion of CD4~+T and CD8~+T subgroups of spleen lymphocytes were significantly increased(P<0.05). Conclusion As a vaccine adjuvant, inulin covalent binding antigen activated the cellular and humoral immunity of the body, and its immunogenicity was weak. It may have good efficacy.
Objective The aim of this study was to evaluate the immunogenicity and adjuvant effect of Jerusalem artichoke polysaccharide as vaccine adjuvant, and improve the immunogenicity of human papilloma carcinogenic antigen E6E7 fusion protein and tuberculous bacillus antigen EC fusion protein. Methods Inulin was combined with E6E7 and EC respectively to prepare I-E6E7 and I-EC as antigens and immunized mice, respectively. Then the immune response characteristics of mice were investigated. The specific IgG antibody titer, antigen specific IFN-γ, TNF-α expression in the mouse spleen lymphocyte were detected. The rations of CD4~+T cells and CD8~+T cells in spleen lymphocyte upon restimulation with EC or E6E7 in vitro and serum biochemical indexes were detected. Results The research showed that when inulin was used as an adjuvant to modify the antigen protein, at first, it could improve the antibody titer of the antigen protein, and fail to detect the anti-polysaccharide IgG antibody(P<0.05). And secondly, the higher secretion levels of IFN-γ and TNF-α than those of the control group were induced(P<0.05). At last, the proportion of CD4~+T and CD8~+T subgroups of spleen lymphocytes were significantly increased(P<0.05). Conclusion As a vaccine adjuvant, inulin covalent binding antigen activated the cellular and humoral immunity of the body, and its immunogenicity was weak. It may have good efficacy.
引文
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[13]Hayashi M,Aoshi T,Haseda Y,et al.Advax,a delta inulin microparticle,potentiates in-built adjuvant property of co-administered vaccines[J].Ebiomedicine,2017,15:127-136.
[14]雷建平.关于CD8细胞在结核病免疫治疗效果评价中的意义之我见[J].中国防痨杂志,2002,24(1):65-66.
[15]Honda-Okubo Y,Saade F,Petrovsky N,et al.Advax?,a polysaccharide adjuvant derived from delta inulin,provides improved influenza vaccine protection through broad-based enhancement of adaptive immune responses[J].Vaccine,2012,30(36):5373-5381.
[16]Hua S,Yu WL,Hu CY,et al.Conjugation of the CRM197-inulin conjugate significantly increases the immunogenicity of Mycobacterium tuberculosis CFP10-TB10.4 fusion protein[J].Bioorganic and Medicinal Chemistry,2017,25(21):5968-5974.
[17]Wang LX,Barclay T,Song YM,et al.Investigation of the biodistribution,breakdown and excretion of delta inulin adjuvant[J].Vaccine,2017,35(34):4382-4388.
[18]Azmi F,Fuaad AA,Skwarczynski M,et al.Recent progress in adjuvant discovery for peptide-based subunit vaccines[J].Human Vaccines and Immunotherapeutics,2014,10(3):778-796.
[2]孙晓敏,陈勇.人用疫苗佐剂的安全性[J].微生物学免疫学进展,2018,46(1):1-6.
[3]聂伟,张永祥.多糖的免疫调节作用及其作用机制研究进展[J].中国药理学通报,1999,15(6):484.
[4]倪峰.免疫活性的中药多糖[J].福建中医学院学报,2001,11(1):57-58.
[5]Petrovsky N,Cooper PD.Advax(TM),a novel microcrystalline polysaccharide particle engineered from delta inulin,provides robust adjuvant potency together with tolerability and safety[J].Vaccine,2015,33(44,SI):5920-5926.
[6]Saade F,Honda-Okubo Y,Trec S,et al.A novel hepatitis B vaccine containing Advax(TM),a polysaccharide adjuvant derived from delta inulin,induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing[J].Vaccine,2013,31(15):1999-2007.
[7]Cooper PD.Vaccine adjuvants based on gamma inulin[J].Pharmaceutical Biotechnology,1995,6:559-80.
[8]Cooper PD,Mccomb C,Steele EJ.The adjuvanticity of algammulin,a new vaccine adjuvant[J].Vaccine,1991,9(6):408-415.
[9]Cooper PD,Steele EJ.Algammulin,a new vaccine adjuvant comprising gamma inulin particles containing alum:preparation and in vitro properties[J].Vaccine,1991,9(5):351-357.
[10]Tindle RW.Vaccines for human papillomavirus infection and anogenital disease[M].Austin:R.G Landes Company,1999:91-104.
[11]Yu W,Hu T.Conjugation with an inuin-chitosan adjuvant markedly improves the immunogenicity of mycobacterium tuberculosis CFP10-TB10.4 fusion protein[J].Molecular Pharmaceutics,2016,13(11):3626-3635.
[12]Huang QR,Yu WL,Hu T.Potent Antigen-Adjuvant delivery system by conjugation of mycobacterium tuberculosis Ag85B-HspXfusion protein with Arabinogalactan-Poly(I:C)conjugate[J].Bioconjugate chemistry,2016,27(4):1165-1174.
[13]Hayashi M,Aoshi T,Haseda Y,et al.Advax,a delta inulin microparticle,potentiates in-built adjuvant property of co-administered vaccines[J].Ebiomedicine,2017,15:127-136.
[14]雷建平.关于CD8细胞在结核病免疫治疗效果评价中的意义之我见[J].中国防痨杂志,2002,24(1):65-66.
[15]Honda-Okubo Y,Saade F,Petrovsky N,et al.Advax?,a polysaccharide adjuvant derived from delta inulin,provides improved influenza vaccine protection through broad-based enhancement of adaptive immune responses[J].Vaccine,2012,30(36):5373-5381.
[16]Hua S,Yu WL,Hu CY,et al.Conjugation of the CRM197-inulin conjugate significantly increases the immunogenicity of Mycobacterium tuberculosis CFP10-TB10.4 fusion protein[J].Bioorganic and Medicinal Chemistry,2017,25(21):5968-5974.
[17]Wang LX,Barclay T,Song YM,et al.Investigation of the biodistribution,breakdown and excretion of delta inulin adjuvant[J].Vaccine,2017,35(34):4382-4388.
[18]Azmi F,Fuaad AA,Skwarczynski M,et al.Recent progress in adjuvant discovery for peptide-based subunit vaccines[J].Human Vaccines and Immunotherapeutics,2014,10(3):778-796.