Metz Bernard, Hoonakker Marieke, Uittenbogaard Joost P, Weyts Michel, Mommen Geert P M, Meiring Hugo D, Tilstra Wichard, Pennings Jeroen L A, van der Pol Leo A, Kuipers Betsy, Sloots Arjen, van den IJssel Jan, van de Waterbeemd Bas, van der Ark Arno
Institute for Translational Vaccinology (Intravacc) , P.O. Box 450, 3720 AL Bilthoven, The Netherlands.
National Institute for Public Health and the Environment , P.O. Box 1, 3720 BA Bilthoven, The Netherlands.
J Proteome Res. 2017 Feb 3;16(2):528-537. doi: 10.1021/acs.jproteome.6b00668. Epub 2016 Dec 23.
Physicochemical and immunochemical assays were applied to substantiate the relation between upstream processing and the quality of whole-cell pertussis vaccines. Bordetella pertussis bacteria were cultured on a chemically defined medium using a continuous cultivation process in stirred tank reactors to obtain uniform protein expression. Continuous culture favors the consistent production of proteins known as virulence factors. Magnesium sulfate was added during the steady state of the culture in order to diminish the expression of virulence proteins. Changes in gene expression and antigen composition were measured by microarrays, mass spectrometry and ELISA. Transcriptome and proteome data revealed high similarity between the biological triplicates demonstrating consistent cultivation of B. pertussis. The addition of magnesium sulfate resulted in an instant downregulation of the virulence genes in B. pertussis, but a gradual decrease of virulence proteins. The quantity of virulence proteins concurred highly with the potency of the corresponding whole-cell pertussis vaccines, which were determined by the Kendrick test. In conclusion, proteome analysis provided detailed information on the composition and proportion of virulence proteins present in the whole-cell preparations of B. pertussis. Moreover, proteome analysis is a valuable method to monitor the production process of whole-cell biomass and predict the product quality of whole-cell pertussis vaccines.
采用物理化学和免疫化学分析方法来证实上游加工与全细胞百日咳疫苗质量之间的关系。使用搅拌罐反应器中的连续培养工艺，在化学成分明确的培养基上培养百日咳博德特氏菌，以获得均匀的蛋白质表达。连续培养有利于持续产生被称为毒力因子的蛋白质。在培养的稳定期添加硫酸镁，以减少毒力蛋白的表达。通过微阵列、质谱和酶联免疫吸附测定法测量基因表达和抗原组成的变化。转录组和蛋白质组数据显示生物学重复样本之间具有高度相似性，表明百日咳博德特氏菌培养一致。硫酸镁的添加导致百日咳博德特氏菌中毒力基因立即下调，但毒力蛋白逐渐减少。毒力蛋白的数量与通过肯德里克试验测定的相应全细胞百日咳疫苗的效力高度一致。总之，蛋白质组分析提供了关于百日咳博德特氏菌全细胞制剂中毒力蛋白的组成和比例的详细信息。此外，蛋白质组分析是监测全细胞生物质生产过程并预测全细胞百日咳疫苗产品质量的一种有价值的方法。
