Deep sequencing analysis of the heterogeneity of seed and commercial lots of the bacillus Calmette-Guérin (BCG) tuberculosis vaccine substrain Tokyo-172

Abstract

BCG, only vaccine available to prevent tuberculosis, was established in the early 20th century by prolonged passaging of a virulent clinical strain of Mycobacterium bovis. BCG Tokyo-172, originally distributed within Japan in 1924, is one of the currently used reference substrains for the vaccine. Recently, this substrain was reported to contain two spontaneously arising, heterogeneous subpopulations (Types I and II). The proportions of the subpopulations changed over time in both distributed seed lots and commercial lots. To maintain the homogeneity of live vaccines, such variations and subpopulational mutations in lots should be restrained and monitored. We incorporated deep sequencing techniques to validate such heterogeneity in lots of the BCG Tokyo-172 substrain without cloning. By bioinformatics analysis, we not only detected the two subpopulations but also detected two intrinsic variations within these populations. The intrinsic variants could be isolated from respective lots as colonies cultured on plate media, suggesting analyses incorporating deep sequencing techniques are powerful, valid tools to detect mutations in live bacterial vaccine lots. Our data showed that spontaneous mutations in BCG vaccines could be easily monitored by deep sequencing without direct isolation of variants, revealing the complex heterogeneity of BCG Tokyo-172 and its daughter lots currently in use.

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

“…Previously, spontaneous heterogeneity of BCG seed lots and commercial vaccines during vaccine production determined by variant calling was analyzed in the Tokyo-172 vaccine strain [32]. In line with the published data, we observed a synonymous A2139G single nucleotide base substitution (position 3,175,529, "-" chain) in BOVR_15,090, i.e., the glnD (ortholog Rv2918c in M. tuberculosis H37Rv) gene, without Ala replacement in position 713 of 808 amino acids in the conservative domain of the PII uridylyltransferase protein in all genomes studied.…”

“…Interestingly, allele G was recorded in the genomes of BCG str. Tokyo 172 which represents a well-defined Type I subpopulation of BCG Tokyo-172, the closest to BCG Russia (GenBank: NZ_CUWO01000001) and Russia ATCC 35740, members of presumably monophyletic "early" substrain group of BCG [10,30,32]. The G variant was also common in the reference sequences of some other BCG strains, e.g., Pasteur 1173P2, Danish 1331, ATCC 35743 (Tice), Moreau RDJ, used worldwide for vaccine production and research purposes.…”

“…The population of the sequenced seed lot 368 was almost homogenous containing a single base deletion (69% reads) in methionine aminopeptidase ( mapA ) gene compared to the previously reported complete genomes of BCG-1 (Russia) current-generation 368 [ 13 , 35 , 36 ] and BCG Tokyo 172 type 1 [ 32 ]. This frameshift leading to protein truncation was inherited in the descendant isolate 577 (47% reads), also bearing an isolate-specific allele (43% reads) in the ATP synthase subunit beta gene ( atpD ).…”

“…During the last decades, the whole-genome sequencing (WGS) was applied to assess the genome stability of BCG vaccine strains from different culture collections and to evaluate the impact of minor sequence variations occurring in seed and commercial lots on the efficacy of BCG-vaccines used in different countries [30][31][32][33][34]. Recently, sequence variations were identified in clinical isolates from two infants with BCGinduced disease compared with the sub-cultured M. bovis BCG Moscow strain (Serum Institute of India, India) in South Africa [15].…”

Complete Genome Sequence of Mycobacterium bovis Strain BCG-1 (Russia)

“…It belongs to early strains and is one of the most widely used strains in the world ( 2 ). Though most of the described differences among BCG strains refer to the period before the worldwide use of lyophilized vaccine, Wada et al ( 3 ) have shown that mutations in vaccine strains obtained by various vaccine manufacturers from a single cell source over time reach detectable levels. Because of that, whole-genome sequencing and comparison of all BCG Russia strains are extremely important.…”