Stoddard Mark B, Pinto Valerian, Keiser Paul B, Zollinger Wendell
Department of Bacterial and Rickettsial Diseases, WRAIR, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, USA.
Clin Vaccine Immunol. 2010 Jan;17(1):98-107. doi: 10.1128/CVI.00342-09. Epub 2009 Nov 18.
Bacterial endotoxin interacts with the human immune system via complex immunological pathways. The evaluation of endotoxicity is important in the development of safe vaccines and immunomodulatory therapeutics. The Limulus amebocyte lysate (LAL) assay is generally accepted by the FDA for use for the quantification of lipopolysaccharide (LPS), while the rabbit pyrogen test (RPT) is used to estimate pyrogenicity during early development and production. Other in vitro assays, such as cytokine release assays with human whole blood (WB) or peripheral blood mononuclear cells (PBMCs), have also been used and may better estimate the human immunological response to products containing novel LPS molecules. In this study, WB and PBMC interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) release assays were used to estimate the endotoxic activities of purified LPS and native outer membrane vesicle (NOMV) vaccines derived from wild-type (hexa-acylated lipid A) and genetically detoxified (penta- and tetra-acylated lipid A) group B Neisseria meningitidis. A method for quantification of the differences in endotoxicity observed in the WB and PBMC assays is elucidated. The LAL assay was shown to be relatively insensitive to lipid A variations, and the RPT was less sensitive than the cytokine release assay with WB. The IL-6 and TNF-alpha assays with WB but not the assays with PBMCs distinguished between vaccines containing LPS from penta- and tetra-acylated strains. The high degree of sensitivity of the WB system to LPS variations and the presumed relevance of the use of human tissues to predict toxicity in humans suggest that this assay may be particularly well suited for the safety evaluation of vaccines and therapeutics containing acylation variants of LPS.
细菌内毒素通过复杂的免疫途径与人类免疫系统相互作用。内毒素活性评估在安全疫苗和免疫调节疗法的研发中至关重要。鲎试剂（LAL）检测法被美国食品药品监督管理局（FDA）普遍认可用于定量脂多糖（LPS），而家兔热原试验（RPT）则用于在早期研发和生产过程中评估热原性。其他体外检测方法，如用人全血（WB）或外周血单核细胞（PBMCs）进行的细胞因子释放检测，也已被使用，并且可能能更好地评估人类对含有新型LPS分子的产品的免疫反应。在本研究中，采用WB和PBMC白细胞介素-6（IL-6）及肿瘤坏死因子α（TNF-α）释放检测法来评估源自野生型（六酰化脂多糖A）和基因解毒型（五酰化和四酰化脂多糖A）B群脑膜炎奈瑟菌的纯化LPS和天然外膜囊泡（NOMV）疫苗的内毒素活性。阐明了一种定量WB和PBMC检测中观察到的内毒素活性差异的方法。结果表明，LAL检测法对脂多糖A的变异相对不敏感，而RPT比用WB进行的细胞因子释放检测灵敏度低。用WB进行的IL-6和TNF-α检测，而非用PBMCs进行的检测，能够区分含有来自五酰化和四酰化菌株LPS的疫苗。WB系统对LPS变异的高灵敏度以及使用人体组织预测人类毒性的假定相关性表明该检测法可能特别适合用于评估含有LPS酰化变体的疫苗和疗法的安全性。
