以pH敏感型LAH肽为载体的高效抗HIV DNA 黏膜疫苗及其干粉吸入剂处方研究

DNA vaccine has a huge potential in providing immune protection against various infectious diseases, including AIDS, which remains one of the world’s most significant public health challenges. To achieve effective protection against infectious pathogens, the vaccine must induce effective immune response, especially T cell responses. Low immunogenicity is a problem of DNA vaccine and this could be partly due to the inefficient cellular uptake of plasmid DNA. One of the strategies to improve the efficacy of DNA vaccine is to develop a safe and effective DNA delivery system. Pulmonary vaccination is attractive because this non-invasive immunization method could induce immunity at mucosal sites through which many pathogens enter the body. By formulating DNA vaccines as inhalable powder could also enhance the stability and shelf-life of the vaccines. Previously, our group has demonstrated that pH responsive LAH4-L1 peptide is effective in mediating DNA transfection in lung tissues, and the LAH4-L1/DNA complexes can be successfully formulated into inhalable dry powder by spray drying or spray freeze drying. Moreover, by incorporating nuclear localizing signal (NLS) into the peptides (LAH4-L1-PK), the transfection efficiency could be improved. To further enhance T cell immunity, soluble programmed death-1 (sPD-1) based DNA vaccine which has been shown by our team to target the expressed antigens to dendritic cells (DCs) will be employed. The objective of this study is to develop a highly efficient inhalable dry powder formulation of DNA vaccines, using pH responsive peptides to deliver DNA encoding HIV-1 GAG p24 antigen fused with sPD-1, to induce immune response so that they could offer protection against HIV-1 and possibly other infectious diseases.

研发预防艾滋病的疫苗意义重大。有效的HIV疫苗必须能诱导强效的细胞免疫应答。为了克服细胞不能有效地摄取质粒DNA导致的低免疫原性问题，开发安全有效的DNA传递系统是提高DNA疫苗效果的重要策略。新型肺内DNA免疫不仅能诱导系统免疫应答，还能提供黏膜免疫保护。在前期研究中，本课题组以pH敏感型LAH4-L1肽为载体实现了有效的肺内DNA转染，并通过喷雾干燥和喷雾冷冻干燥技术成功制备出DNA干粉吸入剂，增加了疫苗稳定性。我们对该肽载体进行进一步改造，增加了核定位序列以提高其转染效率。为了增强T细胞免疫应答的能力，本课题组此前还通过设计基于可溶性程序性死亡因子-1（sPD-1）的DNA疫苗成功实现了目标抗原的树突状细胞靶向性。本项目在此基础上，以pH敏感型肽为载体，传递sPD-1、HIV-1 GAG p24抗原融合质粒，并优化其干粉吸入剂处方组成，为发展新型高效抗HIV DNA疫苗提供实验依据。

获得性免疫综合征（艾滋病）是全球性健康难题，研究开发安全有效的预防艾滋病的疫苗意义重大。有效的HIV疫苗必须能诱导强效的细胞免疫应答。但是，一般注射疫苗无法激活病原体入侵位点的黏膜免疫应答。肺内核酸表达免疫可以有效地对抗通过黏膜表面入侵人体的病原体，从而预防感染。因此，本课题研发DNA与mRNA干粉疫苗，通过肺部给药的方式转染呼吸道黏膜并诱导免疫应答，以预防人类免疫缺陷病毒。为了提高DNA进入细胞核的能力，本项目在pH敏感型LAH肽载体的基础上，设计了加核定位序列修饰(NLS)的肽基因载体，PK1和PK2。本项目同样使用多肽传递系统，聚乙二醇修饰的KL4多肽以提高mRNA的传递效率。目前，本课题已成功使用喷雾冷冻干燥及喷雾干燥法制备出DNA和mRNA干粉，并进行处方优化以提高干粉制剂的可吸入百分比。其中所研发的mRNA干粉制剂已经证明在小鼠体内安全并且有理想的转染效率。本研究发展的肺部核酸传递系统及制剂不仅有望成为安全有效的预防艾滋病疫苗，也可为其他传染性疾病的核酸疫苗研究提供帮助。