Verch Thorsten, Pan Zhen-Kun, Paterson Yvonne
Department of Microbiology, University of Pennsylvania, Philadelphia 19104, USA.
Infect Immun. 2004 Nov;72(11):6418-25. doi: 10.1128/IAI.72.11.6418-6425.2004.
Plasmids represent a powerful tool to rapidly introduce genes into bacteria and help them reach high expression levels. In vaccine development, with live vaccine vectors, this allows greater flexibility and the ability to induce larger antigen amounts through multiple gene copies. However, plasmid retention often requires antibiotic resistance markers, the presence of which has been discouraged in clinical applications by the Food and Drug Administration. Therefore, we developed a Listeria monocytogenes-Escherichia coli shuttle plasmid that is retained by complementation of D-alanine racemase-deficient mutant strains both in vitro and in vivo. Our technology potentially allows the production of antibiotic resistance marker-free DNA vaccines as well as bacterial vaccine vectors devoid of engineered antibiotic resistances. As a proof of concept, we applied the D-alanine racemase complementation system to our Listeria cancer vaccine platform. With a transplantable tumor model, we compared the efficacy of the new Listeria vector to that of an established vector containing a conventional plasmid carrying a tumor-specific antigen. Both vaccine vector systems resulted in long-term regression of established tumors, with no significant difference between them. Thus, the Listeria vaccine vector presented here potentially complies with Food and Drug Administration regulations and could be developed further for clinical use.
质粒是一种强大的工具，可快速将基因导入细菌并帮助它们达到高表达水平。在疫苗开发中，对于活疫苗载体而言，这提供了更大的灵活性，并能够通过多个基因拷贝诱导产生更多的抗原。然而，质粒的保留通常需要抗生素抗性标记，而美国食品药品监督管理局在临床应用中不鼓励使用这类标记。因此，我们开发了一种单核细胞增生李斯特菌-大肠杆菌穿梭质粒，该质粒可通过在体外和体内对D-丙氨酸消旋酶缺陷突变菌株进行互补来实现保留。我们的技术有可能生产无抗生素抗性标记的DNA疫苗以及不含工程化抗生素抗性的细菌疫苗载体。作为概念验证，我们将D-丙氨酸消旋酶互补系统应用于我们的李斯特菌癌症疫苗平台。利用可移植肿瘤模型，我们比较了新型李斯特菌载体与含有携带肿瘤特异性抗原的传统质粒的既定载体的疗效。两种疫苗载体系统均导致既定肿瘤的长期消退，二者之间无显著差异。因此，本文介绍的李斯特菌疫苗载体可能符合美国食品药品监督管理局的规定，并可进一步开发用于临床。
