■针对C型产气荚膜梭菌的兽医疫苗需要进行无毒性测试,根据全世界药典的规定。这种毒性测试需要在多个制造步骤中进行,并且依赖于涉及严重动物痛苦的过时的小鼠测试。C型产气荚膜梭菌产生几种毒素,其中β-毒素是引起疾病的主要成分。这里,我们描述了一种新的基于细胞的体外测定法的成功开发,该测定法可以解决β毒素的特定毒性。
■基于细胞的测定的开发遵循针对Cl开发的体外测试的原理。败血症疫苗,这是基于Vero细胞。我们筛选了四种细胞系,并选择了THP-1细胞系,它被证明是对β毒素活性最特异和最敏感的,与市售方法组合以测定细胞活力(MTS测定)作为读数。
■估计当前的动物试验检测到Cl的100-1000倍稀释度。产气荚膜C型非灭活抗原。当用活性Cl测试时。产气荚膜C型抗原制备,来自商业疫苗生产过程,我们的THP-1细胞为基础的试验能够检测毒素活性从未稀释到超过10000倍稀释,显示在大约1000和10000倍稀释之间的线性范围。用中和抗体和缺乏对Cl的反应证实了对β-毒素的测定特异性。产气荚膜菌培养基。此外,试验参数表现出良好的可重复性。
■这里,我们已经证明了基于THP-1细胞的兽药Cl毒性试验的概念证明。C型产气荚膜疫苗,适用于所有疫苗生产步骤。该结果代表了该兽用梭菌抗原替代基于动物的毒性测试的重要步骤。作为下一步,为了在疫苗质量控制中正式实施试验,必须在商业生产环境中评估试验的灵敏度和可重复性以及方法的验证。
UNASSIGNED: Veterinary vaccines against Clostridium perfringens type C need to be tested for absence of toxicity, as mandated by pharmacopoeias worldwide. This toxicity testing is required at multiple manufacturing steps and relies on outdated mouse tests that involve severe animal suffering. Clostridium perfringens type C produces several toxins of which the β-toxin is the primary component responsible for causing disease. Here, we describe the successful development of a new cell-based in vitro assay that can address the specific toxicity of the β-toxin.
UNASSIGNED: Development of the cell-based assay followed the principle of in vitro testing developed for Cl. septicum vaccines, which is based on Vero cells. We screened four cell lines and selected the THP-1 cell line, which was shown to be the most specific and sensitive for β-toxin activity, in combination with a commercially available method to determine cell viability (MTS assay) as a readout.
UNASSIGNED: The current animal test is estimated to detect 100 - 1000-fold dilutions of the Cl. perfringens type C non-inactivated antigen. When tested with an active Cl. perfringens type C antigen preparation, derived from a commercial vaccine manufacturing process, our THP-1 cell-based assay was able to detect toxin activity from undiluted to over 10000-fold dilution, showing a linear range between approximately 1000- and 10000-fold dilutions. Assay specificity for the β-toxin was confirmed with neutralizing antibodies and lack of reaction to Cl. perfringens culture medium. In addition, assay parameters demonstrated good repeatability.
UNASSIGNED: Here, we have shown proof of concept for a THP-1 cell-based assay for toxicity testing of veterinary Cl. perfringens type C vaccines that is suitable for all vaccine production steps. This result represents a significant step towards the replacement of animal-based toxicity testing of this veterinary clostridial antigen. As a next step, assessment of the assay\'s sensitivity and repeatability and validation of the method will have to be performed in a commercial manufacturing context in order to formally implement the assay in vaccine quality control.