结核分枝杆菌Ag85B-mRNA疫苗的体外合成及其免疫原性研究
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摘要
目的体外转录结核分枝杆菌Ag85B-mRNA并评价其免疫原性。方法结合密码子偏好性、GC含量和mRNA二级结构的自由能,优化Ag85B编码区序列,并在其两端插入β球蛋白3’和5’UTR序列。合成目的序列,进行体外转录。经琼脂糖凝胶电泳鉴定正确,体外转染细胞验证其表达后,联合鱼精蛋白免疫Balb/c小鼠,检测特异性细胞免疫应答。结果 Ag85B优化序列二级结构分析显示,其具有更高的稳定性。成功体外合成稳定的Ag85B-mRNA。Western blot检测Ag85B-mRNA转染293T细胞24、48 h,均有清晰特异性的目的蛋白条带。小鼠免疫试验结果显示,Ag85B-mRNA可诱导针对结核分枝杆菌Ag85B分泌高水平IFN-γ的Th1型免疫应答。结论成功体外合成稳定的Ag85B-mRNA疫苗,具有较好的免疫原性,为新型结核疫苗的研制提供新思路。
This study was performed to transcript Mycobacterium tuberculosis Ag85 B-mRNA and evaluate its immunogenicity in vitro. The Ag85 B coding sequence was optimized by combining codon preference, GC content and free energy of mRNA secondary structure, while the β globin 3'and 5'UTR sequences were inserted at both ends.The sequence of interest is synthesized and subjected to in vitro transcription, then identified by agarose gel electrophoresis. After cell transfection and expression confirmation in vitro, the Ag85 B-mRNA was used to immunize Balb/c mice with protamine for evaluating specific cellular immune responses. Secondary structure analysis showed that optimized sequence of Ag85 B has higher stability. The stable Ag85 B-mRNA was constructed in vitro. Western blot showed after Ag85 B-mRNA transfection into 293 T cells for 24 and 48 hours, there were clear and specific target protein bands. The results of mouse immunoassay showed that Ag85 B-mRNA could induce high level of IFN-γ against mycobacterial Ag85 B which was responsible for Th1-type immune response. In conclusion,the synthesized stable Ag85 B-mRNA vaccine in vitro has good immunogenicity, which lays a foundation for the improvement of mRNA vaccine research and provides new ideas for the development of tuberculosis vaccine.
This study was performed to transcript Mycobacterium tuberculosis Ag85 B-mRNA and evaluate its immunogenicity in vitro. The Ag85 B coding sequence was optimized by combining codon preference, GC content and free energy of mRNA secondary structure, while the β globin 3'and 5'UTR sequences were inserted at both ends.The sequence of interest is synthesized and subjected to in vitro transcription, then identified by agarose gel electrophoresis. After cell transfection and expression confirmation in vitro, the Ag85 B-mRNA was used to immunize Balb/c mice with protamine for evaluating specific cellular immune responses. Secondary structure analysis showed that optimized sequence of Ag85 B has higher stability. The stable Ag85 B-mRNA was constructed in vitro. Western blot showed after Ag85 B-mRNA transfection into 293 T cells for 24 and 48 hours, there were clear and specific target protein bands. The results of mouse immunoassay showed that Ag85 B-mRNA could induce high level of IFN-γ against mycobacterial Ag85 B which was responsible for Th1-type immune response. In conclusion,the synthesized stable Ag85 B-mRNA vaccine in vitro has good immunogenicity, which lays a foundation for the improvement of mRNA vaccine research and provides new ideas for the development of tuberculosis vaccine.
引文
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[3]Kaufmann SH,Weiner J,von Reyn CF.Novel approaches to tuberculosis vaccine development[J].Int J Infect Dis,2017,56:263-267.
[4]Pardi N,Hogan MJ,Porter FW,et al.mRNA vaccines-a new era in vaccinology[J].Nat Rev Drug Dis,2018,17(4):261-279.
[5]Nieuwenhuizen NE,Kaufmann SH.Next-generation vaccines based on Bacille Calmette-Guerin[J].Front Immunol,2018,9:121.
[6]Pollard C,De Koker S,Saelens X,et al.Challenges and advances towards the rational design of mRNA vaccines[J].Trends Mol Med,2013,19(12):705-713.
[7]Sahin U,Kariko K,Tureci O.mRNA-based therapeuticsdeveloping a new class of drugs[J].Nat Rev Drug Dis,2014,13(10):759-780.
[8]Ulmer JB,Geall AJ.Recent innovations in mRNAvaccines[J].Curr Opin Immunol,2016,41:18-22.
[9]De Beuckelaer A,Grooten J,De Koker S.Type Iinterferons modulate CD8(+)T cell immunity to mRNAvaccines[J].Trends Mol Med,2017,23(3):216-226.
[10]Thess A,Grund S,Mui BL,et al.Sequence-engineered mRNA without chemical nucleoside modifications enables an effective protein therapy in large animals[J].Mol Ther,2015,23(9):1456-1464.
[11]Holtkamp S,Kreiter S,Selmi A,et al.Modification of antigen-encoding RNA increases stability,translational efficacy,and T-cell stimulatory capacity of dendritic cells[J].Blood,2006,108(13):4009-4017.
[12]Elango N,Elango S,Shivshankar P,et al.Optimized transfection of mRNA transcribed from a d(A/T)100 tailcontaining vector[J].Biochem Bioph Res Communications,2005,330(3):958-966.
[13]Kauffman KJ,Mir FF,Jhunjhunwala S,et al.Efficacy and immunogenicity of unmodified and pseudouridinemodified mRNA delivered systemically with lipid nanoparticles in vivo[J].Biomaterials,2016,109:78-87.
[14]Isaacs A,Cox RA,Rotem Z.Foreign nucleic acids as the stimulus to make interferon[J].Lancet,1963,2(7299):113-116.
[15]Kariko K,Muramatsu H,Ludwig J,et al.Generating the optimal mRNA for therapy:HPLC purification eliminates immune activation and improves translation of nucleosidemodified,protein-encoding mRNA[J].Nucleic Acids Res,2011,39(21):e142.
[16]Petsch B,Schnee M,Vogel AB,et al.Protective efficacy of in vitro synthesized,specific mRNA vaccines against influenza A virus infection[J].Nat Biotechnol,2012,30(12):1210-1216.
[17]VanBlargan LA,Himansu S,Foreman BM,et al.An m RNAvaccine protects mice against multiple tick-transmitted flavivirus infections[J].Cell Rep,2018,25(12):3382-3392e3383.
[18]Chahal JS,Khan OF,Cooper CL,et al.Dendrimer-RNAnanoparticles generate protective immunity against lethal Ebola,H1N1 influenza,and Toxoplasma gondii challenges with a single dose[J].Proc Nat Acad Sci U S A,2016,113(29):E4133-4142.
[19]Pardi N,Hogan MJ,Pelc RS,et al.Zika virus protection by a single low-dose nucleoside-modified mRNAvaccination[J].Nature,2017,543(7644):248-251.
[20]Maruggi G,Chiarot E,Giovani C,et al.Immunogenicity and protective efficacy induced by self-amplifying mRNAvaccines encoding bacterial antigens[J].Vaccine,2017,35(2):361-368.
[21]Alberer M,Gnad Vogt U,Hong HS,et al.Safety and immunogenicity of a mRNA rabies vaccine in healthy adults:an open-label,non-randomised,prospective,firstin-human phase 1 clinical trial[J].Lancet,2017,390(10101):1511-1520.
[22]Boczkowski D,Lee J,Pruitt S,et al.Dendritic cells engineered to secrete anti-GITR antibodies are effective adjuvants to dendritic cell-based immunotherapy[J].Cancer Gene Ther,2009,16(12):900-911.
[23]Pruitt SK,Boczkowski D,de Rosa N,et al.Enhancement of anti-tumor immunity through local modulation of CTLA-4and GITR by dendritic cells[J].Eur J Immunol,2011,41(12):3553-3563.
[24]Pardi N,Secreto AJ,Shan X,et al.Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1challenge[J].Nat Commun,2017,8:14630.