载铜绿假单胞菌OprF和PcrV基因联合DNA疫苗的水凝胶缓释系统的构建及免疫效力评价

Construction and immune efficacy evaluation of a hydrogel sustained-release system containing a combined DNA vaccine of Pseudomonas aeruginosa OprF and PcrV genes

目的构建载铜绿假单胞菌(PA)外膜蛋白F(OprF)和V抗原(PcrV)基因联合DNA疫苗的PAEH/PEG DA水凝胶缓释系统,并评价该系统的体内免疫效力。方法采用简单混合法制备载PA联合DNA疫苗的PAEH/PEG DA水凝胶,测定其凝胶时间,并评价其体外细胞毒性(DC 2.4)、体外降解性能及累积释放率。将9只小鼠随机分为30 min组、2 d组、7 d组,每组3只,通过注射部位凝胶体积变化及皮肤组织病理切片分别评价水凝胶的体内降解性能和安全性;将18只小鼠随机分为6组(n=3):对照组(PBS)、水凝胶组(Gel)、En/pVAX1-OprF组(O)、En/pVAX1-PcrV组(P)、En/(pVAX1-OprF+pVAX1-PcrV)组(OP)、水凝胶+En/(pVAX1-OprF+pVAX1-PcrV)组(GOP),每只小鼠均免疫3次,每次间隔10 d,末次免疫2周后处死小鼠,检测血清特异性IgG抗体、脾淋巴细胞刺激指数(SI)及细胞上清液中γ干扰素(IFN-γ)水平。结果该水凝胶缓释系统约30 min即可形成凝胶态,体外对DC 2.4细胞无明显毒性,体外36 h时可释放约85%的质粒DNA。该水凝胶体内外降解时间基本一致,7 d内即可完全降解,且具有良好的体内降解性能和安全性;体内免疫实验结果显示,与PBS组比较,Gel组特异性IgG抗体水平、脾淋巴细胞SI及IFN-γ水平无明显变化(P>0.05);与相应的DNA疫苗组(O组或P组)和PBS组比较,OP组和GOP组特异性IgG抗体水平、脾淋巴细胞SI和IFN-γ水平均明显升高(P<0.05,P<0.01);而与OP组比较,GOP组特异性IgG抗体水平、脾淋巴细胞SI和IFN-γ水平均明显升高(P<0.05,P<0.01)。结论载PA OprF和PcrV基因联合DNA疫苗的PAEH/PEG DA水凝胶缓释系统能够缓慢释放联合DNA疫苗,进一步增强联合DNA疫苗的免疫效力,是研发PA疫苗有前景的策略之一。

Objective To construct a PAEH/PEG DA hydrogel sustained-release system containing combined Pseudomonas aeruginosa(PA)outer membrane protein F(OprF)and Pseudomonas V antigen(PcrV)gene DNA vaccine,and evaluate the in vivo immune efficacy of the system.Methods The PAEH/PEG DA hydrogel containing combined PA DNA vaccine was prepared with a simple mixing method.The gelation time was tested,and the cytotoxicity(DC 2.4),degradation and cumulative release rate in vitro of the hydrogel were evaluated.Nine mice were randomly divided into 3 groups(3 each):30 min group,2 d group and 7 d group,the in vivo degradability and security of the hydrogel were evaluated by gelation volume changes at the injection site and histopathological sections of the skin,respectively.Eighteen mice were randomly divided into 6 groups(3 each):control group(PBS),hydrogel group(Gel),En/pVAX1-OprF group(O),En/pVAX1-PcrV group(P),En/(pVAX1-OprF+pVAX1-PcrV)group(OP),and hydrogel+En/(pVAX1-OprF+pVAX1-PcrV)group(GOP).Mice were immunized 3 times with a 10-day interval,then sacrificed 2 weeks after last immunization.The levels of serum specific IgG antibody,splenic lymphocyte stimulation index(SI)and interferon-γ(IFN-γ)in the supernatant of splenic lymphocytes were detected.Results The PAEH/PEG DA hydrogel sustained-release system required only about 30 min to form a gelation state.There was no significant toxicity to DC 2.4 cells in vitro,and approximately 85%of plasmid DNA was released after 36 hours of in vitro release.The degradation time of the hydrogel was nearly the same in vitro and in vivo.It could be almost completely degraded in about 7 days,and had good in vivo biodegradability and biosafety.The results of in vivo immune test showed that,compared with PBS group,no significant changes existed in the levels of specific IgG antibodies,splenic lymphocyte SI and IFN-γin the Gel group(P>0.05).Compared with the corresponding DNA vaccine groups(O group or P group)and PBS group,the specific IgG antibody levels,spleen lymphocyte SI and IFN-γlevel increased obviously in OP group and GOP group(P<0.05,P<0.01).While compared with OP group,the levels of specific IgG antibodies,splenic lymphocyte SI and IFN-γincreased markedly in GOP group(P<0.05,P<0.01).Conclusion The PAEH/PEG DA hydrogel sustained-release systems containing PA OprE and PcrF gene combined DNA vaccines,which can slowly release the combined DNA vaccine and further enhance the immune efficacy of combined DNA vaccine,are one of the promising strategies for the development of PA vaccines.