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.
Highlights
Mycobacterium bovis BCG (Bacillus Calmette–Guérin) was generated over a century ago for protection against Mycobacterium tuberculosis (Mtb) and is one the oldest vaccines still in use [1]
Previous and current studies have shown that fermentation of M. bovis BCG is feasible and that BCG vaccine production in liquid culture affords an equivalent level of protection to that grown through pellicle-production [13,16]
Master stocks of Mycobacterium bovis BCG (Bacillus of Calmette and Guérin) Danish strain 1331 (BCG) were prepared by spreading M. bovis BCG from an SSI vaccine stock vial (Staten Serum Institute, Copenhagen, Denmark) onto Middlebrook 7H10 supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC) and incubating for three weeks at 37 ◦C
Summary
Mycobacterium bovis BCG (Bacillus Calmette–Guérin) was generated over a century ago for protection against Mycobacterium tuberculosis (Mtb) and is one the oldest vaccines still in use [1]. It has been shown that more efficient culture methods, such as shaking cultures in batch flasks or batch fermentation, consistently produced a high titre of M. bovis BCG and provided comparable protection in animals, thereby posing a viable alternative to current BCG production methods [13,16]. Previous and current studies have shown that fermentation of M. bovis BCG is feasible and that BCG vaccine production in liquid culture affords an equivalent level of protection to that grown through pellicle-production [13,16]. We recently found that BCG vaccine produced by fermenter-growth gave protection to European badgers (Meles meles) against M. bovis infection [17] This provided a foundation to further explore which fermentation conditions and storage conditions give the most favourable outcomes in terms of the yield of viable bacilli and stability of BCG. Samples taken throughout the culture were stored in different cryoprotectants at different temperatures to assess the effect of these combined conditions on bacterial titres
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