The BCG live attenuated TB vaccine is one of the oldest and most widely used vaccines in human medicine. Each year, BCG vaccines are administered to over 100 million newborns (i.e. 75% of all newborns on the planet). In an effort to further standardize the vaccine production and to prevent severe adverse reactions related to BCG vaccination, three substrains, i.e. Danish 1331, Tokyo 172-1 and Russian BCG-1 were established as the WHO reference strains in 2009 and 2010. Of these, the BCG Danish 1331 strain is the most frequently used one, and it also serves as a basis of most current “next-generation” engineering efforts to improve the BCG vaccine or to use it as a “carrier” for antigens of other pathogens. Complete genome elucidation of BCG strains is challenging by the occurrence of large genome segment duplications and a high GC content. Therefore, only partial BCG Danish, which hinders further standardization efforts.
By combining second (Illumina) and third (PacBio) generation sequencing technologies and an integrated bioinformatics workflow we have for the first time fully assembled the BCG Danish 1331 (07/270) strain genome sequence. Two large tandem chromosomal duplications characterize the BCG strains; the DU2 and DU1. To demonstrate how this genome analysis methodology contributes to full characterization of improved BCG-derived engineered vaccines, we applied it to a knock-out mutant (KO) for the sapM secreted acid phosphatase, located in the analytically challenging long duplication region DU2. The availability of the complete reference genome for BCG Danish 1331 as well as the associated genome analysis workflow, now permits full genomic characterization of (engineered) TB vaccine strains, which should contribute to more consistent manufacturing of this highly cost-effective vaccine that protects the world’s newborns from disseminated TB.
Reference genome for the WHO reference strain for Mycobacterium bovis BCG Danish, the present tuberculosis vaccine
bioRxiv doi: https://doi.org/10.1101/513408
Katlyn Borgers, Jheng-Yang Ou, Po-Xing Zheng, Petra Tiels, Annelies Van Hecke, Evelyn Plets, Gitte Michielsen, Nele Festjens, Nico Callewaert, Yao-Cheng Lin
- M. bovis BCG Danish genome
- M. bovis sapM KO genome
- Copy number variation comparing with M. tuberculosis H37Rv
Public repoditory – raw reads, genome assemblies and annotation