中国药理学与毒理学杂志2025，Vol.39Issue(10)：770-779,10.DOI:10.3867/j.issn.1000-3002.2025.08185
Lamp2b修饰的工程化外泌体SARS-CoV2疫苗增强呼吸道黏膜免疫
Lamp2b modification enhances respiratory mucosal immunity of engineered exosome SARS-CoV-2 vaccine
摘要
Abstract
OBJECTIVE To investigate the impact of lysosomal associated membrane protein 2b(Lamp2b)modification on the mucosal immune efficacy of engineered exosome-based vaccines.METHODS In vitro experiments:The murine dendritic cell line DC2.4 was transfected with a plasmid encoding the Lamp2b-RBD fusion protein.Real-time quantitative PCR and Western blotting were employed to assess Lamp2b-RBD expressions,flow cytometry was used to evaluate the proportion of Lamp2b-RBD-positive cells,and immunofluorescence staining was performed to determine their membrane localization.Exosomes were isolated via ultracentrifugation,and their morphology and particle size distribution were examined using transmission electron microscopy and nanoparticle tracking analysis.Western blotting was applied to confirm exosomal marker proteins[cluster of differentiation 9(CD9),CD63,ALG-2-interacting protein X(Alix),and Golgi marker GM130]and Lamp2b-RBD expression.In vivo experiments:① Female BALB/c mice were divided into the Lamp2b-RBD-Exo group and the lipid nanoparticle(LNP)group,and administered intratracheally for mucosal immunization.Pulmonary reten-tion was assessed by immunofluorescence staining.② Female BALB/c mice were divided into three groups:placebo group(PBS group),Lamp2b-RBD-Exo intratracheal administration group,and Lamp2b-RBD-Exo intramuscular injection group(im).Immunizations were performed on days 0 and 14,and on days 7 and 21.The titers of RBD-specific immunoglobulin G(IgG)in serum and RBD-specific IgA and IgG antibodies in bronchoalveolar lavage fluid were determined by enzyme-linked immunosor-bent assay(ELISA).RESULTS In vitro experiments:Lamp2b-RBD-positive cells accounted for 71.16％.Lamp2b-RBD mRNA levels were upregulated 1 979-fold compared with controls,with Lamp2b-RBD proteins localized on the cell membrane.Purified engineered exosomes displayed regular morphology,expressed CD9,CD63,and Alix but not GM130,had an average diameter of approximately 124 nm,and carried 3 009 pg of RBD protein per 1×109 exosomes.In vivo experiments:At 4 h after administra-tion,fluorescence signals were observed in the lung tissues of both the Lamp2b-RBD-Exo and LNPs groups.At 24 h,the fluorescence signal in the LNPs group shifted to the liver,while in the Lamp2b-RBD-Exo group,the fluorescence expanded from the trachea to the bronchioles and lung tissue,showing significantly better distribution and retention capacity than the LNPs group.Seven days after immuniza-tion,both the Lamp2b-RBD-Exo and Lamp2b-RBD-Exo(im)groups induced RBD-specific IgG antibody titers.At 21 days after immunization,Lamp2b-RBD-Exo elicited a higher level of RBD-specific immune response,with serum IgG titers reaching 1∶8 100 and bronchoalveolar lavage fluid(BALF)IgA titers reaching 1∶300.No RBD-specific IgA antibody titers were detected in the BALF of the Lamp2b-RBD-Exo(im)group.CONCLUSION Lamp2b-RBD modification enables efficient RBD protein loading and enhances pulmonary retention of engineered exosomes,thereby inducing potent antigen-specific mucosal immune responses.关键词
外泌体/疫苗/黏膜免疫/新型冠状病毒/工程化/免疫应答诱导Key words
exosomes/vaccines/mucosal immunity/COVID-19/engineering/immune response induction分类
药学引用本文复制引用
孟繁,任梦杨,邢昊楠,高秀丽,郑爱萍..Lamp2b修饰的工程化外泌体SARS-CoV2疫苗增强呼吸道黏膜免疫[J].中国药理学与毒理学杂志,2025,39(10):770-779,10.基金项目
国家重点研发计划(2023YFC2605000) （2023YFC2605000）
国家自然科学基金(32371440) （32371440）
国家自然科学基金(82104105) （82104105）
国家自然科学基金(32101157) （32101157）
中国博士后基金(2021M693966) National Key Research and Development Program of China(2023YFC2605000) （2021M693966）
General Program of National Natural Science Foundation of China(32371440) （32371440）
National Natural Science Foundation of China(82104105) （82104105）
National Natural Science Foundation of China(32101157) （32101157）
China Postdoctoral Science Foundation(2021M693966) （2021M693966）
