Mapping the phylogenyand lineage history of geographically distinct BCG vaccine strains
Introduction:  The  Bacillus  Calmette–Guérin  (BCG)  vaccine  has  been  in  use  forprevention  of tuberculosis  for  overa  centuryand remains  the only  widely  available tuberculosisvaccineand  its protective efficacy has varied across geographical regions. Since it was developed, the BCG strain has been shared acrossdifferent laboratories around the world, where use of differing culture methodsresulted ingenetically distinctstrainsover time. Hypothesis/gap   statement: Whilst   differing BCG vaccine   efficacy   around   the   worldis   well documented,  and  whilst  the  reasons  for  this  may  be  multifactorial,  it  has  been  hypothesised that genetic differencesin BCG vaccine strains contribute to this variation. Methods: Isolates  from  an  historic  archive  of  lyophilised  BCG  strainswereregrown,DNA  was extractedandthen whole  genome sequenced  usingOxford  Nanopore  Technologies.  The  resulting whole genome data were plotted on a dendrogramand analysed to identify presence or absence of regions of difference and SNPs relating to virulence, growth and cell wall structure.Results: Of  50strainsavailable,  36  were  revived  in  culture  and  38  were sequenced. Morphology differed between the strains distributedbefore  and after 1934. There was  phylogenetic association amongst  certain  geographically  classifiedstrains, 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, SNPs in sigK, plaA, mmaA3and eccC5were associated with early strains.Discussion:WhilstBCG-Russia,  BCG-Japan  and  BCG-Danish  showed strong geographicalisolate clustering, the  late  strains  distributed  after  1934,  includingBCG-Pasteur,  showed more  variation.Awide  range  of  SNPs were  seen within  geographically  classifiedstrains, and as much  intra-strain variation  as  between  strainvariationwas  seen. Thedate  of  distribution  from  the  original  Pasteur laboratory(earlypre-1927 or latepost-1927) gave the strongest association withgenetic differences in  regions of  difference  and  virulence-related  SNPs,  suggesting  early  strains may be  responsible  for better vaccine efficacy.