Optimisation of Mycobacterium bovis BCG Fermentation and Storage Survival

Abstract

Mycobacterium bovis Bacillus Calmette–Guérin (M. bovis BCG) was generated over a century ago for protection against Mycobacterium tuberculosis (Mtb) and is one the oldest vaccines still in use. The BCG vaccine is currently produced using a pellicle growth method, which is a complex and lengthy process that has been challenging to standardise. Fermentation for BCG vaccine production would reduce the complexity associated with pellicle growth and increase batch to batch reproducibility. This more standardised growth lends itself to quantification of the total number of bacilli in the BCG vaccine by alternative approaches, such as flow cytometry, which can also provide information about the metabolic status of the bacterial population. The aim of the work reported here was to determine which batch fermentation conditions and storage conditions give the most favourable outcomes in terms of the yield and stability of live M. bovis BCG Danish bacilli. We compared different media and assessed growth over time in culture, using total viable counts, total bacterial counts, and turbidity throughout culture. We applied fluorescent viability dyes and flow cytometry to measure real-time within-culture viability. Culture samples were stored in different cryoprotectants at different temperatures to assess the effect of these combined conditions on bacterial titres. Roisin’s minimal medium and Middlebrook 7H9 medium gave comparable, high titres in fermenters. Flow cytometry proved to be a useful tool for enumeration of total bacterial counts and in the assessment of within-culture cell viability and cell death. Of the cryoprotectants evaluated, 5% (v/v) DMSO showed the most significant positive effect on survival and reduced the negative effects of low temperature storage on M. bovis BCG Danish viability. In conclusion, we have shown a reproducible, more standardised approach for the production, evaluation, and storage of high titre, viable, BCG vaccine.

RNA Microarray-Based Comparison of Innate Immune Phenotypes between Human THP-1 Macrophages Stimulated with Two BCG Strains

“…The THP-1 cell line is an alternative study model that serves as a support to understand various cellular responses without assuming that the results are the same as those of macrophages obtained live from patients [20]. The spectrophotometric absorbance values observed for each strain over time, which were used to estimate the log phase for maximal growth, were similar to those reported by other authors examining different BCG strains [29,30]. The differential amount of intracellular bacteria we found over time could be associated with the phenotypic characteristics of each strain and could explain both the phagocytosis process and the immune response [23][24][25]31].…”

After 100 Years of BCG Immunization against Tuberculosis, What Is New and Still Outstanding for This Vaccine?

“…Among potential explanations for these variations, manufacturing processes that are difficult to standardize have been suggested. There, production using fermentation and defined chemical media have been shown to affect viability [15]. What other consequences that a change in the method of manufacturing BCG would have in terms of efficacy of protection remain to be determined.…”

Comparison of Lowenstein Jensen Media and Ogawa Media Usage for Viability Test of BCG Vaccine Pasteur P11732 and Russian (Moscow) – 384 sub-strains

“…Solid mediums containing eggs, such as LJ and Ogawa, are the least expensive within these mediums yet can provide reliable results. It has been demonstrated that production through fermentation and specific chemical medium affects viability (2). Consequently, data regarding the influence of culture medium on the growth of Mycobacterium bovis (M. bovis) is still required for optimum quality control of BCG vaccines.…”