Optimisation of Mycobacterium bovis BCG Fermentation and Storage Survival
Open Access
- 21 September 2020
- journal article
- research article
- Published by MDPI AG in Pharmaceutics
- Vol. 12 (9), 900
- https://doi.org/10.3390/pharmaceutics12090900
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.Keywords
Funding Information
- Department for Environment, Food and Rural Affairs, UK Government (SE3247)
This publication has 44 references indexed in Scilit:
- Quality control and safety assessment of BCG vaccines in the post-genomic eraBiotechnology & Biotechnological Equipment, 2014
- Non-Replicating Mycobacterium tuberculosis Elicits a Reduced Infectivity Profile with Corresponding Modifications to the Cell Wall and Extracellular MatrixPLOS ONE, 2014
- Protection by BCG Vaccine Against Tuberculosis: A Systematic Review of Randomized Controlled TrialsClinical Infectious Diseases, 2013
- Randomized Trial of BCG Vaccination at Birth to Low-Birth-Weight Children: Beneficial Nonspecific Effects in the Neonatal Period?The Journal of Infectious Diseases, 2011
- Drying a tuberculosis vaccine without freezingProceedings of the National Academy of Sciences, 2007
- Compiling a Molecular Inventory for Mycobacterium bovis BCG at Two Growth Rates: Evidence for Growth Rate-Mediated Regulation of Ribosome Biosynthesis and Lipid MetabolismJournal of Bacteriology, 2005
- Thermal Stability of VaccinesJournal of Pharmaceutical Sciences, 2003
- The use of fluorogenic esters to detect viable bacteria by flow cytometryJournal of Applied Bacteriology, 1994
- Multiple comparison of dried BCG vaccines: stability at 37°C and persistence of strains in the mouse spleenVaccine, 1984
- The estimation of the bactericidal power of the bloodEpidemiology and Infection, 1938