乙肝病毒核心蛋白病毒样颗粒表面抗原密度对抗体应答水平的影响
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摘要
【目的】为了探究乙肝病毒核心蛋白(HepatitisBviruscoreprotein,HBc)病毒样颗粒(Virus-like particles,VLPs)表面抗原密度对免疫后抗体应答水平的影响,制备了不同抗原密度的HBc VLPs疫苗,并检测了其在小鼠体内的抗体应答水平。【方法】首先制备了N端带有3个甘氨酸的人巨细胞病毒重组抗原域AD-4作为模式抗原,接着通过Sortase A的介导将AD-4连接到HBc VLPs表面上。将系列浓度梯度AD-4抗原在SortaseA介导下分别与相同浓度的HBcVLPs发生反应,制备不同抗原密度的HBc-AD-4 VLPs。将其分别免疫6–8周龄BALB/c小鼠3次,每次免疫间隔2 w,间接ELISA法检测被免疫小鼠血清的抗体应答水平。【结果】结果表明,当HBc VLPs表面抗原密度为44.4%时,即HBc反应浓度∶AD-4反应浓度为1:0.5时,不足以引起高滴度的抗体产生;当HBc VLPs表面抗原密度为64.2%时,即HBc反应浓度∶AD-4反应浓度为1:1时,HBc-AD-4 VLPs诱导的AD-4特异性抗体滴度与100%抗原密度的HBc-AD-4VLPs所引起的抗体滴度相当;当HBcVLPs表面抗原密度大于64.2%时,引起的抗体应答水平不因抗原密度增加而进一步增强。【结论】发现了HBcVLPs表面抗原密度与免疫后抗体滴度呈正相关,然而免疫64.2%抗原密度的HBc VLPs所产生的抗体滴度可达峰值,抗原密度进一步增加,抗体应答水平不会进一步加强。
[Objective] To better understand how antigen density on the surface of hepatitis B virus core protein(HBc) virus-like particles(VLPs) affects their antibody response, we generated HBc VLPs with different antigen densities, and tested their antibody response in mice. [Methods] First, we prepared the recombinant antigen domain 4(AD-4) of human cytomegalovirus(HCMV) as a model antigen, which contains three glycine molecules at its N-terminus, for Sortase A-mediated ligation onto the HBc VLPs. Displaying different densities of antigens onto the surface of VLPs was achieved by using a serial diluted recombinant AD-4 in ligation reactions. After that, HBc-AD-4 VLPs with different antigen densities were applied to 6–8 weeks old BALB/c mice. Each group was inoculated three times at 2-week intervals and the AD-4-specific IgG was detected by indirect ELISA. [Results] When the reaction concentration ratio between HBc and AD-4 is 1:0.5, which HBc surface antigen density is 44.4%, VLPs couldn't induce a high antibody titer. When the reaction concentration ratio between HBc and AD-4 is 1:1, which HBc surface antigen density is 64.2%, VLPs could induce similar highest humoral immune response compared to 100% antigen density HBc VLPs. When HBc surface antigen density is greater than 64.2%, no further enhancement of antibody response was observed by further increasing the antigen density. [Conclusion] In conclusion, we found that antigen density on HBc VLPs is positively correlated with the antibody response. However, it reaches a peak at 64% antigen density, and no further enhancement of antibody response was observed by further increasing the antigen density.
[Objective] To better understand how antigen density on the surface of hepatitis B virus core protein(HBc) virus-like particles(VLPs) affects their antibody response, we generated HBc VLPs with different antigen densities, and tested their antibody response in mice. [Methods] First, we prepared the recombinant antigen domain 4(AD-4) of human cytomegalovirus(HCMV) as a model antigen, which contains three glycine molecules at its N-terminus, for Sortase A-mediated ligation onto the HBc VLPs. Displaying different densities of antigens onto the surface of VLPs was achieved by using a serial diluted recombinant AD-4 in ligation reactions. After that, HBc-AD-4 VLPs with different antigen densities were applied to 6–8 weeks old BALB/c mice. Each group was inoculated three times at 2-week intervals and the AD-4-specific IgG was detected by indirect ELISA. [Results] When the reaction concentration ratio between HBc and AD-4 is 1:0.5, which HBc surface antigen density is 44.4%, VLPs couldn't induce a high antibody titer. When the reaction concentration ratio between HBc and AD-4 is 1:1, which HBc surface antigen density is 64.2%, VLPs could induce similar highest humoral immune response compared to 100% antigen density HBc VLPs. When HBc surface antigen density is greater than 64.2%, no further enhancement of antibody response was observed by further increasing the antigen density. [Conclusion] In conclusion, we found that antigen density on HBc VLPs is positively correlated with the antibody response. However, it reaches a peak at 64% antigen density, and no further enhancement of antibody response was observed by further increasing the antigen density.
引文
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[19]Jegerlehner A,Storni T,Lipowsky G,Schmid M,Pumpens P,Bachmann MF.Regulation of IgG antibody responses by epitope density and CD21-mediated costimulation.European Journal of Immunology,2002,32(11):3305-3314.
[2]Kumar S,Anselmo AC,Banerjee A,Zakrewsky M,Mitragotri S.Shape and size-dependent immune response to antigen-carrying nanoparticles.Journal of Controlled Release,2015,220:141-148.
[3]Benson RA,MacLeod MK,Hale BG,Patakas A,Garside P,Brewer JM.Antigen presentation kinetics control Tcell/dendritic cell interactions and follicular helper T cell generation in vivo.eLife,2015,4:e06994.
[4]Mahmood K,Bright RA,Mytle N,Carter DM,Crevar CJ,Achenbach JE,Heaton PM,Tumpey TM,Ross TM.H5N1VLP vaccine induced protection in ferrets against lethal challenge with highly pathogenic H5N1 influenza viruses.Vaccine,2008,26(42):5393-5399.
[5]Wynne SA,Crowther RA,Leslie AG.The crystal structure of the human hepatitis B virus capsid.Molecular Cell,1999,3(6):771-780.
[6]Zhu P,Liu J,Bess J Jr,Chertova E,Lifson JD,GriséH,Ofek GA,Taylor KA,Roux KH.Distribution and three-dimensional structure of AIDS virus envelope spikes.Nature,2006,441(7095):847-852.
[7]Compans RW,Klenk HD,Caliguiri LA,Choppin PW.Influenza virus proteins.I.Analysis of polypeptides of the virion and identification of spike glycoproteins.Virology,1970,42(4):880-889.
[8]Brewer MG,DiPiazza A,Acklin J,Feng CY,Sant AJ,Dewhurst S.Nanoparticles decorated with viral antigens are more immunogenic at low surface density.Vaccine,2017,35(5):774-781.
[9]Vlasak J,Hoang VM,Christanti S,Peluso R,Li FS,Culp TD.Use of flow cytometry for characterization of human cytomegalovirus vaccine particles.Vaccine,2016,34(20):2321-2328.
[10]Plotkin S.The history of vaccination against cytomegalovirus.Medical Microbiology and Immunology,2015,204(3):247-254.
[11]Dintzis RZ,Middleton MH,Dintzis HM.Studies on the immunogenicity and tolerogenicity of T-independent antigens.The Journal of Immunology,1983,131(5):2196-2203.
[12]Moon JJ,Suh H,Li AV,Ockenhouse CF,Yadava A,Irvine DJ.Enhancing humoral responses to a malaria antigen with nanoparticle vaccines that expand Tfh cells and promote germinal center induction.Proceedings of the National Academy of Sciences of the United States of America,2012,109(4):1080-1085.
[13]Tang SB,Xuan BQ,Ye XH,Huang Z,Qian ZK.A modular vaccine development platform based on sortase-mediated site-specific tagging of antigens onto virus-like particles.Scientific Reports,2016,6:2541.
[14]Walker A,Skamel C,Nassal M.SplitCore:an exceptionally versatile viral nanoparticle for native whole protein display regardless of 3D structure.Scientific Reports,2011,1:5.
[15]Popp MWL,Ploegh HL.Making and breaking peptide bonds:Protein engineering using sortase.Angewandte Chemie-International Edition,2011,50(22):5024-5032.
[16]Galloway AL,Murphy A,de Simone JM,Di J,Herrmann JP,Hunter ME,Kindig JP,Malinoski FJ,Rumley MA,Stoltz DM,Templeman TS,Hubby B.Development of a nanoparticle-based influenza vaccine using the PRINT?technology.Nanomedicine,2013,9(4):523-531.
[17]Paus D,Phan TG,Chan TD,Gardam S,Basten A,Brink R.Antigen recognition strength regulates the choice between extrafollicular plasma cell and germinal center B cell diff erentiation.Journal of Experimental Medicine,2006,203(4):1081-1091.
[18]Crotty S.Follicular helper CD4 T cells(TFH).Annual Review Immunology,2011,29(1):621-663.
[19]Jegerlehner A,Storni T,Lipowsky G,Schmid M,Pumpens P,Bachmann MF.Regulation of IgG antibody responses by epitope density and CD21-mediated costimulation.European Journal of Immunology,2002,32(11):3305-3314.
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