PLGA-HPV16mE7纳米疫苗抗HPV16免疫反应的研究
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摘要
目的:探讨生物可降解材料聚乳酸-羟基乙酸共聚物(PLGA)为载体负载突变后的高危型人乳头瘤病毒16E7(HPV16E7)蛋白的纳米疫苗诱抗肿瘤的免疫反应。方法:表达并纯化定点突变的HPV16 E7蛋白(HPV16mE7)。表征用复乳溶剂挥发法制备的HPV16mE7/PLGA纳米粒。HPV16mE7/PLGA纳米粒皮下免疫C57BL/6(H-2b)小鼠2次,用ELISA检测血清中的抗HPV16E7抗体水平,用Elispot和用乳酸脱氢酶(LDH)释放实验分别检测产生IFN-γ和CTL反应。结果:Western Blot分析证明HPV16mE7蛋白具有和野生型HPV16E7诱导产生的抗体结合的活性。HPV16mE7/PLGA纳米粒粒径大小为(335.6±6.32)nm,Zeta电位为(-6.9±0.41)mV,包封率为62.4%,体外释放呈双相动力学特征,于第9天累积释放达58.2%,纳米悬液在4℃至少稳定1个月。HPV16mE7/PLGA纳米粒免疫的小鼠血清抗E7抗体水平、CTL和产生IFN-γ细胞显著高于HPV16mE7免疫组的小鼠。结论:HPV16mE7/PLGA纳米疫苗具有较单纯的HPV16mE7诱导小鼠产生更强的体液和细胞免疫反应。HPV16mE7/PLGA纳米疫苗可作为HPV16感染相关疾病候选疫苗。
Objective The purpose is to study the anti-tumor immune response with HPV16E7 nano-vaccine based on biodegradable material lactic-co-glycolic acid(PLGA) as carrier. Methods Directly mutated HPV16E7 protein was expressed and purified.HPV16mE7/PLGA nanoparticles(NPs) were prepared by an emulsion/solvent evaporation. method After C57BL/6(H-2b)mice were immunized twice by HPV16mE7/PLGA NPs and HPV16mE7 protein via subcutaneous injection, anti-HPV16E7 antibody levels in serum were measured using ELISA, IFN-γ-producing cells and CTL response were detected using Elispot assay and the release oflactate dehydrogenase(LDH) respectively. Results Western Blot analysis demonstrated that directly mutated HPV16mE7 could bind wild-type derived-anti-HPV16E7 antibody. The size, zeta potentials and entrapped efficiency of HPV16mE7/PLGANPs was 335.6 ± 6.32 nm,-6.9 ± 0.41 mV and 62.4 %, and the release of HPV16mE7 from these NPs presented bi-phase kinetics distribution and accumulative release reached up to 58.2 %on day 9. Nanoparticle suspension could keep stable for at least one month at 4 ℃.The anti-HPV16E7 antibody levels, LDH release and IFN-γ-producing cells derived from mice immunized by HPV16mE7/PLGA NPs was significantly higher than that of mice immunized by HPV16mE7. Conclusions HPV16mE7/PLGA nano-vaccine could induce stronger humoral and cell immune response compared with HPV16mE7 alone. HPV16mE7/PLGA nanovaccine could serve as candidate vaccine for HPV16 infection-associated diseases.
Objective The purpose is to study the anti-tumor immune response with HPV16E7 nano-vaccine based on biodegradable material lactic-co-glycolic acid(PLGA) as carrier. Methods Directly mutated HPV16E7 protein was expressed and purified.HPV16mE7/PLGA nanoparticles(NPs) were prepared by an emulsion/solvent evaporation. method After C57BL/6(H-2b)mice were immunized twice by HPV16mE7/PLGA NPs and HPV16mE7 protein via subcutaneous injection, anti-HPV16E7 antibody levels in serum were measured using ELISA, IFN-γ-producing cells and CTL response were detected using Elispot assay and the release oflactate dehydrogenase(LDH) respectively. Results Western Blot analysis demonstrated that directly mutated HPV16mE7 could bind wild-type derived-anti-HPV16E7 antibody. The size, zeta potentials and entrapped efficiency of HPV16mE7/PLGANPs was 335.6 ± 6.32 nm,-6.9 ± 0.41 mV and 62.4 %, and the release of HPV16mE7 from these NPs presented bi-phase kinetics distribution and accumulative release reached up to 58.2 %on day 9. Nanoparticle suspension could keep stable for at least one month at 4 ℃.The anti-HPV16E7 antibody levels, LDH release and IFN-γ-producing cells derived from mice immunized by HPV16mE7/PLGA NPs was significantly higher than that of mice immunized by HPV16mE7. Conclusions HPV16mE7/PLGA nano-vaccine could induce stronger humoral and cell immune response compared with HPV16mE7 alone. HPV16mE7/PLGA nanovaccine could serve as candidate vaccine for HPV16 infection-associated diseases.
引文
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[3]Okada H,Doken Y,Ogawa Y,et al.Preparation of threemonth depot injectable microspheres of leuprorelin acetate using biodegradable polymers[J].Pharmaceutical Research,1994,11(8):1143-1147.
[4]Yamaguchi Y,Takenaga M,Kitagawa A,et al.Insulinloaded biodegradable PLGA microcapsules:initial burst release controlled by hydrophilic additives[J].Journal of Controlled Release,2002,81(3):235-249.
[5]Yang C,Jiang L,Bu S,et al.Intravitreal administration of dexamethasone-loaded PLGA-TPGS nanoparticles for the treatment of posterior segment diseases[J].Journal of Biomedical Nanotechnology,2013,9(9):1617-1623.
[6]Zheng Y,Zhang YJ,Ma YD,et al.Enhancement of immunotherapeutic effects of HPV16E7 on cervical cancer by fusion with CTLA4 extracellular region[J].Journal of Microbiology,2008,46(6):410-418.
[7]Khallouf H,Grabowska AK,Riemer AB.Therapeutic Vaccine Strategies against Human Papillomavirus.Vaccines[J].Vaccines,2014,2(2):422-462.
[8]Chen YL,Chang MC,Chen CA,et al.Depletion of regulatory T lymphocytes reverses the imbalance between pro-and antitumor immunities via enhancing antigen-specific T cell immune responses[J].Plos One,2012,7(10):e47190.
[9]Ding Z,Ou R,Ni B,et al.Cytolytic activity of the human papillomavirus type 16 E711-20 epitope-specific cytotoxic T lymphocyte is enhanced by heat shock protein 110 in HLA-A*0201 transgenic mice[J].Clin Vaccine Immune,2013,20(7):1027-1033.
[10]Vishnoi K,Mahata S,Tyagi A,et al.Cross-talk between Human Papillomavirus Oncoproteins and Hedgehog Signaling Synergistically Promotes Stemness in Cervical Cancer Cells[J].Sci Rep,2016,28(6):34377.
[11]Senba M,Mori N.Mechanisms of virus immune evasion lead to development from chronic inflammation to cancer formation associated with human papillomavirus infection[J].Oncol Rev,2012,6(2):e17.
[12]Yang A,Jeang J,Cheng K,et al.Current state in the development of candidate therapeutic HPV vaccines[J].Expert Rev Vaccines,2016,15(8):989-1007.
[13]Skeate JG,Woodham AW,Einstein MH,et al.Current therapeutic vaccination and immunotherapy strategies for HPV-related diseases[J].Hum Vaccine Immunother,2016,12(6):1418-1429.
[14]Watkins R,Wu L,Zhang C,et al.Natural product-based nanomedicine:recent advances and issues[J].Int JNanomedicine,2015,28(10):6055-6074.
[15]Zhang J,Chen Y,Li X,et al.The influence of different long-circulating materials on the pharmacokinetics of liposomal vincristine sulfate[J].Int J Nanomedicine,2016,11(8):4187-4197.
[16]Fabiano A,Chetoni P,Zambito Y.Mucoadhesive nanosized supramolecular assemblies for improved pre-corneal drug residence time[J].Drug Dev Ind Pharm,2015,41(12):2069-2076.
[17]Namdee K,Carrasco-Teja M,Fish MB,et al.Effect of variation in hemorheology between human and animal blood on the binding efficacy of vascular-targeted carriers[J].Sci Rep,2015,11(5):11631.
[18]Jahan ST,Haddadi A.Investigation and optimization of formulation parameters on preparation of targeted anti-CD205tailored PLGA nanoparticles[J].Int J Nanomedicine,2015,10(10):7371-7384
[19]Avgoustakis K,Beletsi A,Panagi Z,et al.PLGA-mPEGnanoparticles of cisplatin:in vitro nanoparticle degradation,in vitro drug release and in vivo drug residence in blood properties[J].Journal of Controlled Release,2002,79(1-3):123-135.
[20]Prabha S,Labhasetwar V.Critical determinants in PLGA/PLA nanoparticle-mediated gene expression[J].Pharmaceutical Research,2004,21(2):354-364.
[21]Vargas A,Lange N,Arvinte T,et al.Toward the understanding of the photodynamic activity of m-THPPencapsulated in PLGA nanoparticles:correlation between nanoparticle properties and in vivo activity[J].Journal of Drug Targeting,2009,17(8):599-609.
[22]Yang R,Shim WS,Cui FD,et al.Enhanced electrostatic interaction between chitosan-modified PLGA nanoparticle and tumor[J].International Journal of Pharmaceutics,2009,371(1-2):142-147.
[23]Florindo HF,Pandit S,Gon?alves LM,et al.Antibody and cytokine-associated immune responses to S.equi antigens entrapped in PLA nanospheres[J].Biomaterials,2009,30(28):5161-5169.