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

Takayuki Wada, Fumito Maruyama, Tomotada Iwamoto, Shinji Maeda, Taro Yamamoto, Ichiro Nakagawa, Saburo Yamamoto, Naoya Ohara, Takayuki Wada, Fumito Maruyama, Tomotada Iwamoto, Shinji Maeda, Taro Yamamoto, Ichiro Nakagawa, Saburo Yamamoto, Naoya Ohara

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.

Figures

Figure 1. A historical record of recent…
Figure 1. A historical record of recent lots of BCG Tokyo analysed in this study.
Seven rectangle boxes correspond to BCG lots that were subjected to deep sequencing. Arrows indicate culturing processes to establish or produce descendant lots from each seed lot.
Figure 2. Heterogeneity of Type I and…
Figure 2. Heterogeneity of Type I and II subpopulations in each BCG lot was monitored based on the RD16 region.
(A) Agarose gel electrophoresis of PCR products of the RD16 region. (B) Ratios of intact/deleted RD16 in each lot were calculated from the copy numbers derived from quantitative real-time PCR. The Y-axis shows the percentage of respective lots that were of the Type II subpopulation. The error bars indicate the propagated standard deviation (±2σ) of respective ratios.
Figure 3. Percentage of covered nucleotides of…
Figure 3. Percentage of covered nucleotides of the reference genome sequence (NC_012207, 4,171,711 bp) mapped with short reads from seven seed/commercial lots of Tokyo-172 by Bowtie2 (incorporated in Breseq v0.24rc6).
The X-axis shows the depth of reads covering the reference genome, and the Y-axis shows the % of the reference sequence mapped with the depth. Short reads were obtained twice (A,B) from the same lots and used for subsequent analysis.

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