轮状病毒VP4~*高聚体的制备及其免疫保护性评价
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摘要
在前期工作中发现,截短的轮状病毒VP4~*蛋白(aa26–476)在大肠杆菌中能够以可溶形式表达,且在小鼠模型中具有较高的免疫原性和免疫保护性。本研究通过颗粒化进一步提高VP4~*蛋白的免疫保护性。通过37℃水浴加热处理24h使VP4~*蛋白多聚化,通过高效液相色谱、透射电镜、分析超离等分析VP4~*蛋白颗粒化程度,通过酶联免疫吸附试验分析颗粒化对VP4~*蛋白与中和抗体反应性的影响;通过差示量热法分析VP4~*高聚体的热稳定性;最后,通过小鼠母传抗体模型研究颗粒化对VP4~*免疫原性和免疫保护性的影响。结果表明,VP4~*蛋白高聚体结构均一,并且相比三聚体,具有更高热稳定性和中和抗体结合活性;在内毒素<20 EU/mg的条件下,与铝佐剂混合,刺激小鼠产生更高滴度的中和抗体;对轮状病毒导致的腹泻具有更高的免疫保护性。综上所述,VP4~*高聚体的研究为轮状病毒基因工程亚单位疫苗的研制提供了更广阔的思路。
In previous studies, we found that truncated rotavirus VP4~*(aa 26–476) could be expressed in soluble form in Escherichia coli and confer high protection against rotavirus in the mouse mode. In this study, we further improved the immunogenicity of VP4~* by polymerization. The purified VP4~* was polymerized through incubation at 37 ℃ for 24 h, and then the homogeneity of the particles was analyzed by HPLC, TEM and AUC, while the thermal stability and antigenicity was analyzed by DSC and ELISA, respectively. Finally, the immunogenicity and protective efficacy of the polymers analyzed by a mouse maternal antibody model. The results showed that VP4~* aggregated into homogeneous polymers, with high thermostability and neutralizing antibody binding activity. In addition, VP4~* polymers(endotoxin <20 EU/dose) stimulated higher neutralizing antibodies and confer higher protection against rotavirus-induced diarrhoea compared with the VP4~* trimers when immunized with aluminium adjuvant. In summary, the study in VP4~* polymers provides a new strategy for the development of recombinant rotavirus vaccines.
In previous studies, we found that truncated rotavirus VP4~*(aa 26–476) could be expressed in soluble form in Escherichia coli and confer high protection against rotavirus in the mouse mode. In this study, we further improved the immunogenicity of VP4~* by polymerization. The purified VP4~* was polymerized through incubation at 37 ℃ for 24 h, and then the homogeneity of the particles was analyzed by HPLC, TEM and AUC, while the thermal stability and antigenicity was analyzed by DSC and ELISA, respectively. Finally, the immunogenicity and protective efficacy of the polymers analyzed by a mouse maternal antibody model. The results showed that VP4~* aggregated into homogeneous polymers, with high thermostability and neutralizing antibody binding activity. In addition, VP4~* polymers(endotoxin <20 EU/dose) stimulated higher neutralizing antibodies and confer higher protection against rotavirus-induced diarrhoea compared with the VP4~* trimers when immunized with aluminium adjuvant. In summary, the study in VP4~* polymers provides a new strategy for the development of recombinant rotavirus vaccines.
引文
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[11]Becker-Dreps S,Vilchez S,Velasquez D,et al.Rotavirus-specific IgG antibodies from mothers'serum may inhibit infant immune responses to the pentavalent rotavirus vaccine.Pediatr Infect Dis J,2015,34(1):115-116.
[12]Chilengi R,Simuyandi M,Beach L,et al.Association of maternal immunity with rotavirus vaccine immunogenicity in zambian infants.PLoS ONE,2016,11(3):e0150100.
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[18]Liu X,Sun MS.Procaryotic expression of rotavirus VP4gene,protein purification and animal immunization.Progr Vet Med,2006,27(12):88-90(in Chinese).刘馨,孙茂盛.轮状病毒VP4基因的原核表达、蛋白纯化及动物免疫试验.动物医学进展,2006,27(12):88-90.
[19]Lee J,Babiuk LA,Harland R,et al.Immunological response to recombinant VP8*subunit protein of bovine rotavirus in pregnant cattle.J Gen Virol,1995,76(10):2477-2483.
[20]Favacho ARM,Kurtenbach E,Sardi SI,et al.Cloning,expression,and purification of recombinant bovine rotavirus hemagglutinin,VP8*,in Escherichia coli.Protein Expr Purif,2006,46(2):196-203.
[21]Wen XB,Cao DJ,Jones RW,et al.Construction and characterization of human rotavirus recombinant VP8*subunit parenteral vaccine candidates.Vaccine,2012,30(43):6121-6126.
[22]Wen XB,Wen K,Cao DJ,et al.Inclusion of a universal tetanus toxoid CD4+T cell epitope P2 significantly enhanced the immunogenicity of recombinant rotavirusΔVP8*subunit parenteral vaccines.Vaccine,2014,32(35):4420-4427.
[23]Li YJ,Xue MG,Yu LQ,et al.Expression and characterization of a novel truncated rotavirus VP4 for the development of a recombinant rotavirus vaccine.Vaccine,2018,36(16):2086-2092.
[24]Levin D,Constant S,Pasqualini T,et al.Role of dendritic cells in the priming of CD4+T lymphocytes to peptide antigen in vivo.J Immunol,1993,151(12):6742-6750.
[25]Banchereau J,Steinman RM.Dendritic cells and the control of immunity.Nature,1998,392(6673):245-252.
[26]Tuyaerts S,Van Meirvenne S,Bonehill A,et al.Expression of human GITRL on myeloid dendritic cells enhances their immunostimulatory function but does not abrogate the suppressive effect of CD4+CD25+regulatory T cells.J Leukoc Biol,2007,82(1):93-105.
[27]Li SW,Tang XH,Seetharaman J,et al.Dimerization of hepatitis E virus capsid protein E2s domain is essential for virus-host interaction.PLoS Pathog,2009,5(8):e1000537.
[28]Xue MG,Yu LQ,Che YJ,et al.Characterization and protective efficacy in an animal model of a novel truncated rotavirus VP8 subunit parenteral vaccine candidate.Vaccine,2015,33(22):2606-2613.
[29]Xue MG,Yu LQ,Jia LZ,et al.Immunogenicity and protective efficacy of rotavirus VP8*fused to cholera toxin B subunit in a mouse model.Hum Vaccin Immunother,2016,12(11):2959-2968.
[30]Li TD,Lin HJ,Yu LQ,et al.Development of an enzyme-linked immunospot assay for determination of rotavirus infectivity.J Virol Methods,2014,209:7-14.
[31]Yuan LJ,Geyer A,Hodgins DC,et al.Intranasal administration of 2/6-rotavirus-like particles with mutant Escherichia coli heat-labile toxin(LT-R192G)induces antibody-secreting cell responses but not protective immunity in gnotobiotic pigs.J Virol,2000,74(19):8843-8853.
[32]Li TD,Lin HJ,Zhang Y,et al.Improved characteristics and protective efficacy in an animal model of E.coli-derived recombinant double-layered rotavirus virus-like particles.Vaccine,2014,32(17):1921-1931.
[2]Parashar UD,Glass RI.Rotavirus vaccines-early success,remaining questions.N Engl J Med,2009,360(11):1063-1065.
[3]Harris VC,Armah G,Fuentes S,et al.Significant correlation between the infant gut microbiome and rotavirus vaccine response in rural ghana.J Infect Dis,2017,215(1):34-41.
[4]Cunliffe NA,Witte D,Ngwira BM,et al.Efficacy of human rotavirus vaccine against severe gastroenteritis in Malawian children in the first two years of life:a randomized,double-blind,placebo controlled trial.Vaccine,2012,30(S1):A36-A43.
[5]Armah GE,Sow SO,Breiman RF,et al.Efficacy of pentavalent rotavirus vaccine against severe rotavirus gastroenteritis in infants in developing countries in sub-Saharan Africa:a randomised,double-blind,placebocontrolled trial.Lancet,2010,376(9741):606-614.
[6]Payne DC,Edwards KM,Bowen MD,et al.Sibling transmission of vaccine-derived rotavirus(RotaTeq)associated with rotavirus gastroenteritis.Pediatrics,2010,125(2):e438-e441.
[7]Yen C,Jakob K,Esona MD,et al.Detection of fecal shedding of rotavirus vaccine in infants following their first dose of pentavalent rotavirus vaccine.Vaccine,2011,29(24):4151-4155.
[8]Moon SS,Wang YH,Shane AL,et al.Inhibitory effect of breast milk on infectivity of live oral rotavirus vaccines.Pediatr Infect Dis J,2010,29(10):919-923.
[9]Moon SS,Tate JE,Ray P,et al.Differential profiles and inhibitory effect on rotavirus vaccines of nonantibody components in breast milk from mothers in developing and developed countries.Pediatr Infect Dis J,2013,32(8):863-870.
[10]Appaiahgari MB,Glass R,Singh S,et al.Transplacental rotavirus IgG interferes with immune response to live oral rotavirus vaccine ORV-116E in Indian infants.Vaccine,2014,32(6):651-656.
[11]Becker-Dreps S,Vilchez S,Velasquez D,et al.Rotavirus-specific IgG antibodies from mothers'serum may inhibit infant immune responses to the pentavalent rotavirus vaccine.Pediatr Infect Dis J,2015,34(1):115-116.
[12]Chilengi R,Simuyandi M,Beach L,et al.Association of maternal immunity with rotavirus vaccine immunogenicity in zambian infants.PLoS ONE,2016,11(3):e0150100.
[13]Estes MK,Graham DY,Mason BB.Proteolytic enhancement of rotavirus infectivity:molecular mechanisms.J Virol,1981,39(3):879-888.
[14]Settembre EC,Chen JZ,Dormitzer PR,et al.Atomic model of an infectious rotavirus particle.EMBO J,2011,30(2):408-416.
[15]Abdelhakim AH,Salgado EN,Fu XF,et al.Structural correlates of rotavirus cell entry.PLoS Pathog,2014,10(9):e1004355.
[16]Mackow ER,Vo PT,Broome R,et al.Immunization with baculovirus-expressed VP4 protein passively protects against simian and murine rotavirus challenge.JVirol,1990,64(4):1698-1703.
[17]Trask SD,McDonald SM,Patton JT.Structural insights into the coupling of virion assembly and rotavirus replication.Nat Rev Microbiol,2012,10(3):165-177.
[18]Liu X,Sun MS.Procaryotic expression of rotavirus VP4gene,protein purification and animal immunization.Progr Vet Med,2006,27(12):88-90(in Chinese).刘馨,孙茂盛.轮状病毒VP4基因的原核表达、蛋白纯化及动物免疫试验.动物医学进展,2006,27(12):88-90.
[19]Lee J,Babiuk LA,Harland R,et al.Immunological response to recombinant VP8*subunit protein of bovine rotavirus in pregnant cattle.J Gen Virol,1995,76(10):2477-2483.
[20]Favacho ARM,Kurtenbach E,Sardi SI,et al.Cloning,expression,and purification of recombinant bovine rotavirus hemagglutinin,VP8*,in Escherichia coli.Protein Expr Purif,2006,46(2):196-203.
[21]Wen XB,Cao DJ,Jones RW,et al.Construction and characterization of human rotavirus recombinant VP8*subunit parenteral vaccine candidates.Vaccine,2012,30(43):6121-6126.
[22]Wen XB,Wen K,Cao DJ,et al.Inclusion of a universal tetanus toxoid CD4+T cell epitope P2 significantly enhanced the immunogenicity of recombinant rotavirusΔVP8*subunit parenteral vaccines.Vaccine,2014,32(35):4420-4427.
[23]Li YJ,Xue MG,Yu LQ,et al.Expression and characterization of a novel truncated rotavirus VP4 for the development of a recombinant rotavirus vaccine.Vaccine,2018,36(16):2086-2092.
[24]Levin D,Constant S,Pasqualini T,et al.Role of dendritic cells in the priming of CD4+T lymphocytes to peptide antigen in vivo.J Immunol,1993,151(12):6742-6750.
[25]Banchereau J,Steinman RM.Dendritic cells and the control of immunity.Nature,1998,392(6673):245-252.
[26]Tuyaerts S,Van Meirvenne S,Bonehill A,et al.Expression of human GITRL on myeloid dendritic cells enhances their immunostimulatory function but does not abrogate the suppressive effect of CD4+CD25+regulatory T cells.J Leukoc Biol,2007,82(1):93-105.
[27]Li SW,Tang XH,Seetharaman J,et al.Dimerization of hepatitis E virus capsid protein E2s domain is essential for virus-host interaction.PLoS Pathog,2009,5(8):e1000537.
[28]Xue MG,Yu LQ,Che YJ,et al.Characterization and protective efficacy in an animal model of a novel truncated rotavirus VP8 subunit parenteral vaccine candidate.Vaccine,2015,33(22):2606-2613.
[29]Xue MG,Yu LQ,Jia LZ,et al.Immunogenicity and protective efficacy of rotavirus VP8*fused to cholera toxin B subunit in a mouse model.Hum Vaccin Immunother,2016,12(11):2959-2968.
[30]Li TD,Lin HJ,Yu LQ,et al.Development of an enzyme-linked immunospot assay for determination of rotavirus infectivity.J Virol Methods,2014,209:7-14.
[31]Yuan LJ,Geyer A,Hodgins DC,et al.Intranasal administration of 2/6-rotavirus-like particles with mutant Escherichia coli heat-labile toxin(LT-R192G)induces antibody-secreting cell responses but not protective immunity in gnotobiotic pigs.J Virol,2000,74(19):8843-8853.
[32]Li TD,Lin HJ,Zhang Y,et al.Improved characteristics and protective efficacy in an animal model of E.coli-derived recombinant double-layered rotavirus virus-like particles.Vaccine,2014,32(17):1921-1931.