Some of the adverse side-effects such as leukocytosis, hyperinsulinemia, hypoglycemia and sensitization to histamine, caused by diphtheria, tetanus and whole cell pertussis (DTwP) vaccines are related to the presence of non-inactivated pertussis toxin (PTx) residues (NiPTxR). The CHO cell clustering assay is an in vitro assay to measure NiPTxR in DTwP vaccines based on the ability of active PTx to cause cellular clustering. To study the biochemical mechanism involved in the clustering effect in CHO cells induced by PTx and by two DTwP vaccines, the levels of total cyclic cAMP were measured and compared to those obtained after treatment with cholera toxin (CTx) able to induce CHO cells elongation instead of cell clustering. Our results showed an increment of cAMP levels by CTx and total cell elongation in CHO cells. However, changes in cAMP levels were not associated with the total clustering induced by PTx or by DTwP vaccines. The high correlation seen between the levels of NiPTxR in the DTwP vaccines determined by the in vivo lethal histamine sensitization (HIST) assay and the in vitro CHO cell clustering assay indicated that the latter could be a suitable alternative test to HIST assay for the toxicological approval and release of batches of DTwP vaccines in their final formulation for human use in accordance with the application of the 3R's principle.

白喉、破伤风和全细胞百日咳 (DTwP) 疫苗引起的一些不良副作用，如白细胞增多、高胰岛素血症、低血糖和对组胺过敏，与非灭活百日咳毒素 (PTx) 残留物 (NiPTxR) 的存在有关. CHO 细胞聚类分析是一种体外基于活性 PTx 引起细胞聚集的能力，测定 DTwP 疫苗中的 NiPTxR。为了研究由 PTx 和两种 DTwP 疫苗诱导的 CHO 细胞聚集效应所涉及的生化机制，测量了总环 cAMP 的水平，并将其与用能够诱导 CHO 细胞伸长的霍乱毒素 (CTx) 处理后获得的水平进行比较细胞聚类。我们的结果显示通过 CTx 和 CHO 细胞中的总细胞伸长率增加了 cAMP 水平。然而，cAMP 水平的变化与 PTx 或 DTwP 疫苗诱导的总聚类无关。通过体内致死组胺致敏 (HIST) 试验和体外试验确定的 DTwP 疫苗中 NiPTxR 水平之间存在高度相关性 CHO 细胞聚类分析表明，根据 3R 原则的应用，后者可以作为 HIST 分析的合适替代测试，用于毒理学批准和释放最终用于人类使用的 DTwP 疫苗批次。