Simon Benjamin E, Ybarra Noel, Bonneval Aimée O, Barry Ronald A
Department of Veterans Affairs Medical Center, Portland, Oregan, USA.
Biotechnol Prog. 2006 Sep-Oct;22(5):1394-9. doi: 10.1021/bp060177i.
Optimal DNA vaccine efficacy requires circumventing several obstacles, including low immunogenicity, a need for adjuvant, and the costs of purifying injection grade plasmid DNA. Bacterial delivery of plasmid DNA may provide an efficient and low-cost alternative to plasmid purification and injection. Also, the bacterial vector may exhibit potential as an immune adjuvant in vivo. Thus, we elected to examine the use of cell-wall-deficient Listeria monocytogenes as a DNA delivery vehicle in vitro. First, the D-alanine-deficient (Deltadal-dat) L. monocytogenes strain DP-L3506, which undergoes autolysis inside eukaryotic host cells in the absence of D-alanine, was transformed with a plasmid encoding green fluorescent protein (GFP) under control of the CMV promoter (pAM-EGFP). Then COS-7 and MC57G cell lines were infected with the transformed DP-L3506 at various multiplicities of infection (MOI) in the presence or absence of D-alanine. Subsequent GFP expression was observed in both cell lines by 24 h post-infection with DP-L3506(pAM-EGFP). Notably, no GFP positive cells were observed when D-alanine was omitted. Although transfection efficiency initially increased as a result of D-alanine supplementation, high concentration or long-term supplementation led to sustained bacterial growth that killed the infected host cells, resulting in fewer GFP-expressing cells. Thus, efficient DNA delivery by transformed bacteria must balance bacterial invasion and survival with target cell health and survival.
最佳的DNA疫苗效力需要克服几个障碍，包括低免疫原性、对佐剂的需求以及纯化注射级质粒DNA的成本。通过细菌递送质粒DNA可能为质粒纯化和注射提供一种高效且低成本的替代方法。此外，细菌载体在体内可能具有作为免疫佐剂的潜力。因此，我们选择研究细胞壁缺陷型单核细胞增生李斯特菌作为体外DNA递送载体的用途。首先，用在巨细胞病毒（CMV）启动子（pAM-EGFP）控制下编码绿色荧光蛋白（GFP）的质粒转化缺乏D-丙氨酸（Deltadal-dat）的单核细胞增生李斯特菌菌株DP-L3506，该菌株在没有D-丙氨酸的情况下会在真核宿主细胞内自溶。然后，在有或没有D-丙氨酸存在的情况下，以不同的感染复数（MOI）用转化后的DP-L3506感染COS-7和MC57G细胞系。在用DP-L3506（pAM-EGFP）感染后24小时，在两种细胞系中均观察到了随后的GFP表达。值得注意的是，当省略D-丙氨酸时，未观察到GFP阳性细胞。尽管由于补充D-丙氨酸，转染效率最初有所提高，但高浓度或长期补充会导致细菌持续生长，从而杀死被感染的宿主细胞，导致表达GFP的细胞减少。因此，通过转化细菌进行有效的DNA递送必须在细菌入侵和存活与靶细胞健康和存活之间取得平衡。
