Mader H J, Szostak M P, Hensel A, Lubitz W, Haslberger A G
Institute of Microbiology and Genetics, University of Vienna, Austria.
Vaccine. 1997 Feb;15(2):195-202. doi: 10.1016/s0264-410x(96)00141-7.
Gram-negative bacterial ghosts produced by controlled expression of the plasmid-encoded lysis gene E offers a promising approach in non-living vaccine technology. Bacterial cell wall complex and hence the antigenic determinants of the living cells are not affected by denaturation due to cell killing. However, the endotoxin content of the Gram-negative cell wall has been discussed as a potential problem for this kind of whole cell or envelope vaccines. Here we show that bacterial ghosts prepared from Escherichia coli O26:B6 and Salmonella typhimurium C5 induce dose-dependent antibody responses against bacterial cells or their corresponding lipopolysaccharides (LPS) in doses 25 ng kg-1 when administered intravenously to rabbits in a standard immunization protocol. No differences between the immune responses of the rabbits were observed when comparing equivalent doses of bacterial ghosts and antibiotic-treated whole cells. The results indicate that the bacterial ghosts exhibit all the antigenic properties of the living cells. No significant fever responses in rabbits have been recorded in doses of < 250 ng kg-1 E. coli O26:B6 ghosts and up to doses of 250 ng kg-1 S. typhimurium C5 ghosts when applying test methods recommended by the US pharmacopoeia. These findings correlate with cell culture experiments where doses 100 ng ml-1 of bacterial ghosts were needed for the release of tumour necrosis factor alpha (TNF alpha) and prostaglandin E2 (PGE2) from RAW mouse macrophage cultures. Free LPS of Salmonella abortus equi commonly used as a LPS-standard, however, stimulated TNF alpha and PGE2 synthesis of RAW cells in doses of 1 ng ml-1. The endotoxic activity of our bacterial preparations analysed by a standard limulus amoebocyte lysate and 2-keto-3-deoxyoctonate assay correlated with the capacity to stimulate the release of PGE2 and TNF alpha in RAW mouse macrophage cultures and the endotoxic responses in rabbits. It can be concluded that these in vitro systems can be used as easy predictive test systems for preparations of bacterial vaccines, particularly for bacterial ghosts.
通过质粒编码的裂解基因E的可控表达产生的革兰氏阴性菌菌影，为非活性疫苗技术提供了一种很有前景的方法。细菌细胞壁复合物以及活细胞的抗原决定簇不会因细胞死亡导致的变性而受到影响。然而，革兰氏阴性细胞壁的内毒素含量已被视为这类全细胞或包膜疫苗的一个潜在问题。在此我们表明，按照标准免疫方案静脉注射给兔子时，由大肠杆菌O26:B6和鼠伤寒沙门氏菌C5制备的菌影，以25 ng kg-1的剂量可诱导针对细菌细胞或其相应脂多糖（LPS）的剂量依赖性抗体反应。比较等量的菌影和抗生素处理的全细胞时，未观察到兔子免疫反应之间的差异。结果表明，菌影具有活细胞的所有抗原特性。按照美国药典推荐的测试方法，当剂量小于250 ng kg-1的大肠杆菌O26:B6菌影以及剂量高达250 ng kg-1的鼠伤寒沙门氏菌C5菌影时，兔子未出现明显发热反应。这些发现与细胞培养实验相关，在细胞培养实验中，RAW小鼠巨噬细胞培养物释放肿瘤坏死因子α（TNFα）和前列腺素E2（PGE2）需要100 ng ml-1的菌影剂量。然而，通常用作LPS标准品的马流产沙门氏菌游离LPS，以1 ng ml-1的剂量即可刺激RAW细胞合成TNFα和PGE2。通过标准鲎试剂法和2-酮-3-脱氧辛酸测定法分析，我们的细菌制剂的内毒素活性与刺激RAW小鼠巨噬细胞培养物释放PGE2和TNFα的能力以及兔子的内毒素反应相关。可以得出结论，这些体外系统可作为细菌疫苗制剂，特别是菌影制剂的简易预测测试系统。
