小分子化合物与铝佐剂联用对铜绿假单胞菌疫苗候选抗原PA0833保护效果的影响及机制初步研究  

Effects of small molecule compounds combined with aluminum adjuvant on the protective efficacy of candidate antigen PA0833 from Pseudomonas aeruginosa and the underlying mechanisms

作　　者：孙恬峻 张晓丽 夏真萍 赵卓 章金勇 王乂[1] Sun Tianjun;Zhang Xiaoli;Xia Zhenping;Zhao Zhuo;Zhang Jinyong;Wang Yi(Key Laboratory of Marine Drugs,Ministry of Education,School of Medicine and Pharmacy,Ocean University of China,Qingdao 266003,China;Department of Microbiology and Biochemical Pharmacy,College of Pharmacy and Laboratory Medicine,Army Medical University,Chongqing 400038,China;Department of Clinical Hematology,College of Pharmacy and Laboratory Medicine,Army Medical University,Chongqing 400038,China)

机构地区：[1]中国海洋大学医药学院,海洋药物教育部重点实验室,青岛266003 [2]陆军军医大学药学与检验医学系微生物与生化药学教研室,重庆400038 [3]陆军军医大学药学与检验医学系临床血液学教研室,重庆400038

出　　处：《中华微生物学和免疫学杂志》2024年第3期189-197,共9页Chinese Journal of Microbiology and Immunology

基　　金：重庆市自然科学基金面上项目(cstc2021jcyj-msxmX0496);国家自然科学基金面上项目(32170938)。

摘　　要：目的评价二乙基二硫代氨基甲酸钠(sodium diethyldithiocarbamate,DTC)、左旋咪唑(levamisole,LMS)、咪喹莫特(imiquimod,Imi)3个小分子化合物对铜绿假单胞菌疫苗候选抗原PA0833免疫原性和保护效果的影响,并进一步对其机制进行初步研究。方法抗原PA0833分别与上述3种小分子以及铝佐剂配伍制备疫苗,通过肌肉注射的方式于0、14、21 d分别免疫BALB/c小鼠。收集3次免疫后血清,ELISA测定PA0833特异性IgG抗体水平。采用铜绿假单胞菌PAO1感染免疫后的小鼠,通过生存率、体重变化、临床评分、炎症因子检测和肺组织病理学改变等评价疫苗的保护效果。在此基础上,对小鼠连续7 d肌肉注射DTC,取肺脏组织进行转录组测序分析,并通过流式细胞术检测其对抗原提呈细胞活化的影响。结果联用小分子免疫组能够有效提高PA0833特异性IgG抗体效价,降低细菌感染后肺组织中细菌负荷和TNF-α、IL-6、IL-1β炎症因子水平,显著提高细菌感染后的生存率,其中DTC效果最好。转录组测序结果表明,相比于PBS对照组,DTC处理组样本有121个基因表达上调、18个基因下调;差异表达基因主要富集在免疫信号通路,IL-17信号通路活化明显。流式细胞术结果表明DTC能促进脾细胞中Th17细胞分化,且能促进抗原提呈细胞活化。结论本研究中的3种小分子都能够有效地增强抗原的免疫原性,其中DTC效果最优,可用于新型疫苗佐剂的研发。Objective To evaluate the impact of three small compounds,namely sodium diethyldithiocarbamate(DTC),levamisole(LMS)and imiquimod(Imi),on the immunogenicity and protective efficacy of the candidate antigen PA0833 from Pseudomonas aeruginosa(Pa)and analyze the underlying mechanisms.Methods PA0833 was formulated with aluminum adjuvant and the above small compounds,respectively.BALB/c mice were immunized with these vaccines intramuscularly on days 0,14 and 21.Serum samples were collected and the levels of PA0833-specific IgG were measured by ELISA.The protective efficacy of these vaccines was evaluated by assessment of survival rates,body weights,clinical scores,inflammatory factors,and histopathological changes after infecting the immunized mice with Pa PAO1 strains.Besides,the mice were injected with DTC intramuscularly for seven consecutive days to analyze the mechanism of DTC in enhancing immune response using transcriptome sequencing and flow cytometry.Results All these small compounds were capable of effectively enhancing the immunogenicity of PA0833 formulated with aluminum adjuvant,reducing bacterial loads in lung tissues,inhibiting the secretion of TNF-α,IL-6 and IL-1β,and improving mouse survival rates upon Pa infection.DTC was more effective than the other two compounds.Transcriptome sequencing identified 121 up-regulated genes and 18 down-regulated genes in DTC-treated group as compared with PBS control group.These differentially expressed genes were significantly enriched in immune pathways,with a strong activation of the IL-17 pathway.Flow cytometry analysis demonstrated significant activation of dendritic cells and proliferation of Th17 cells in splenocytes in DTC-treated group as compared with PBS control group.Conclusions All three small compounds are able of effectively enhance antigen immunogenicity with DTC being the most effective,indicating that DTC can be used as a novel adjuvant in vaccine development.

关 键 词：佐剂 二乙基二硫代氨基甲酸钠(DTC) 铜绿假单胞菌 疫苗 

分 类 号：R392-33[医药卫生—免疫学]