Development of a multiplex-based immunoassay for the characterization of diphtheria, tetanus and acellular pertussis antigens in human combined DTaP vaccines.

Routine batch quality testing before vaccine release, notably for potency evaluation, still relies on animal use for several animal and human vaccines. In this context, the VAC2VAC project is a public-private consortium of 22 partners funded by EU whose the main objective is to reduce the number of animal used for batch testing by developing immunoassays that could be implemented for routine potency assessment of vaccines. This paper focused on the development of a Luminex-based multiplex assay to monitor the consistency of antigen quantity and quality throughout the production process of DTaP vaccines from two human vaccine manufacturers. Indepth characterized monoclonal antibody pairs were used for development and optimization of the Luminex assay with non-adsorbed and adsorbed antigens and with complete vaccine formulations from both manufacturers. The multiplex assay demonstrated good specificity, reproducibility and absence of cross-reactivity. Analysis of over and underdosed formulations, heat and H2O2-degraded products as well as batch to batch consistency of vaccines from both manufacturers brought the proof of concept for a future application of the multiplex immunoassay as a useful tool in the frame of DTaP vaccine quality control.

Production of Prodiginines Is Part of a Programmed Cell Death Process in Streptomyces coelicolor.

Actinobacteria are prolific producers of antitumor antibiotics with antiproliferative activity, but why these bacteria synthetize metabolites with this bioactivity has so far remained a mystery. In this work we raised the hypothesis that under certain circumstances, production of antiproliferative agents could be part of a genetically programmed death of the producing organism. While programmed cell death (PCD) has been well documented when Streptomyces species switch from vegetative (nutrition) to aerial (reproduction) growth, lethal determinants are yet to be discovered. Using DNA-damaging prodiginines of Streptomyces coelicolor as model system, we revealed that, under certain conditions, their biosynthesis is always triggered in the dying zone of the mycelial network prior to morphological differentiation, right after an initial round of cell death. The programmed massive death round of the vegetative mycelium is absent in a prodiginine non-producer (ΔredD strain), and mutant complementation restored both prodiginine production and cell death. The redD null mutant of S. coelicolor also showed increased DNA, RNA, and proteins synthesis when most of the mycelium of the wild-type strain was dead when prodiginines accumulated. Moreover, addition of the prodiginine synthesis inhibitors also resulted in enhanced accumulation of viable filaments. Overall, our data enable us to propose a model where the time-space production of prodiginines is programmed to be triggered by the perception of dead cells, and their biosynthesis further amplifies the PCD process. As prodiginine production coincides with the moment S. coelicolor undergoes morphogenesis, the production of these lethal compounds might be used to eradicate the obsolete part of the population in order to provide nutrients for development of the survivors. Hence, next to weapons in competition between organisms or signals in inter- and intra-species communications, we propose a third role for antibiotics (in the literal meaning of the word 'against life') i.e., elements involved in self-toxicity in order to control cell proliferation, and/or for PCD associated with developmental processes.