M. Bovis BCG-1 (Russia) Sub-Strain Genome Stability Investigation Within the Entire Production Process

Abstract

Aim. The aim of the current study was to analyze the genome structure of the M. bovis BCG-1 (Russia) sub-strain, used for the vaccine production, as well as its genome stability within the entire production process. Materials and methods. Whole genome sequencing and M. bovis BCG-1 (Russia) working seed lot and for the last production passage of the sub-strain cultivation from a number of the vaccine batches. Additionally, VNTR sequences of 24 locus analyses, RD patterns comparison, as well as spoligotyping were performed. Results. The whole genome sequence of the M. bovis BCG-1 (Russia) working seed lot was assembled, annotated and deposited to GenBank. On the basis of DU2- and RD-regions analyzes M. bovis BCG-1 (Russia) sub-strain was confirmed to be belonged to BCG Russia strains of DU2-I group. Whole genome sequencing followed by comparative analysis of RD patterns and SNPs confirmed the stability of the vaccine sub-strain genome from the working seed lot to a number of the vaccine batches obtained within the two-years period. VNTR profile and spoligopattern exactly matched the M. bovis BCG-1 (Russia). Conclusion. Thus the M. bovis BCG-1 (Russia) sub-strain genome identity and stability have been studied and demonstrated. The obtained result confirmed the vaccine production process consistency.

Complete Genome Sequence, Genome Stability and Phylogeny of the Vaccine Strain Mycobacterium bovis BCG SL222 Sofia

“…The BCG phylogeny based on the nucleotide substitution rate among the BCG sub-strains with known complete genome sequence is presented in Figure 2 . The reported data in Figure 2 are supported by historical [ 1 ], deletions mapping [ 27 , 28 ], and sequencing data [ 25 ].…”

“…DU2 duplication with four copies of marker gene sigH and other repeated regions (phoP) was found in the genome of the Bulgarian BCG sub-strain. DU2 is the same duplication found in the BCG-I genome in previous observations and is related to the catabolic activity of some enzymes [ 8 , 25 , 27 ]. We confirm the presence of the full sequence of RD14 of 9073 bp (including leuA repeat region of 1517 bp), which in some BCG sub-strains, like BCG-Pasteur is absent.…”

“…RD1 (Region of Difference) was deleted in all BCG sub-strains including BCG SL222 Sofia. The unique deletion BCG-I RDRussia of 1603 bp (Rv3697c to Rv3698) according to the location in the M. tuberculosis H37Rv genome [ 3 , 25 ] was also present in BCG SL222 Sofia [ 26 ]. The RDRussia deletion was 1608 bp as compared to the reference M. bovis genome sequence LT708304.…”

First insight into the whole-genome sequence variations in Mycobacterium bovis BCG-1 (Russia) vaccine seed lots and their progeny clinical isolates from children with BCG-induced adverse events

“…The G variant was previously reported in the glnD in BCG-1 (Russia) current seed lot 368 and some earlier vaccine products [ 35 ], though alternative variant A was detected in two other BCG-1 (Russia) 368 complete genomes [ 13 , 36 ]. The stability of variant A in the glnD gene of the BCG-1 (Russia) 368 working seed lot (GenBank: CP013741) and consistent vaccine batches within 2 years was confirmed by WGS [ 36 , 37 ]. Noteworthy, all three complete genomes of BCG-1 (Russia) were sequenced independently from the same generation 368 (“shch”), which is still in use since 2008 for the BCG vaccine production by two Russian manufactures: Medgamal (Moscow) and Microgen (Stavropol).…”

“…The complete genome sequences of the current M. bovis BCG-1 (Russia) 368 (“shch”) freeze-dried seed lot and its working seed lot used for vaccine production by one of the Russian manufacturers are available in GenBank (NCBI) under accession numbers CP011455 [ 13 ], CP009243 [ 35 ], and CP013741 [ 36 ]. The genome constancy of M. bovis BCG-1 (Russia) 368 within the entire vaccine production flow (from working seed lot, the last production passage to final vaccine product) was confirmed by spoligotyping, 24 loci VNTR-typing, and WGS [ 36 , 37 ]. However, so far, there is a lack of data on the contribution of BCG genomic diversity to the viability, fitness, and residual virulence of vaccine populations in vivo in the context of the development of BCG-induced adverse events.…”