Abstract

The bacillus Calmette-Guérin (BCG) vaccine has been in use for prevention of tuberculosis for over a century. It remains the only widely available tuberculosis vaccine and its protective efficacy has varied across geographical regions. Since it was developed, the BCG vaccine strain has been shared across different laboratories around the world, where use of differing culture methods has resulted in genetically distinct strains over time. Whilst differing BCG vaccine efficacy around the world is well documented, and the reasons for this may be multifactorial, it has been hypothesized that genetic differences in BCG vaccine strains contribute to this variation. Isolates from an historic archive of lyophilized BCG strains were regrown, DNA was extracted and then whole-genome sequenced using Oxford Nanopore Technologies. The resulting whole-genome data were plotted on a phylogenetic tree and analysed to identify the presence or absence of regions of difference (RDs) and single-nucleotide polymorphisms (SNPs) relating to virulence, growth and cell wall structure. Of 50 strains available, 36 were revived in culture and 39 were sequenced. Morphology differed between the strains distributed before and after 1934. There was phylogenetic association amongst certain geographically classified strains, most notably BCG-Russia, BCG-Japan and BCG-Danish. RD2, RD171 and RD713 deletions were associated with late strains (seeded after 1927). When mapped to BCG-Pasteur 1172, the SNPs in sigK, plaA, mmaA3 and eccC5 were associated with early strains. Whilst BCG-Russia, BCG-Japan and BCG-Danish showed strong geographical isolate clustering, the late strains, including BCG-Pasteur, showed more variation. A wide range of SNPs were seen within geographically classified strains, and as much intra-strain variation as between-strain variation was seen. The date of distribution from the original Pasteur laboratory (early pre-1927 or late post-1927) gave the strongest association with genetic differences in regions of difference and virulence-related SNPs, which agrees with the previous literature.