The whole genome sequence of Polish vaccine strain Mycobacterium bovis BCG Moreau

Abstract

Currently, tuberculosis immunoprophylaxis is based solely on Bacillus Calmette-Guérin (BCG) vaccination, and some of the new potential tuberculosis vaccines are based on the BCG genome. Therefore, it is reasonable to analyze the genomes of individual BCG substrains. The aim of this study was the genetic characterization of the BCG-Moreau Polish (PL) strain used for the production of the BCG vaccine in Poland since 1955. Sequencing of different BCG lots showed that the strain was stable over a period of 59 years. As a result of comparison, BCG-Moreau PL with BCG-Moreau Rio de Janeiro (RDJ) 143 single nucleotide polymorphisms (SNPs) and 32 insertion/deletion mutations (INDELs) were identified. However, the verification of these mutations showed that the most significant were accumulated in the BCG-Moreau RDJ genome. The mutations unique to the Polish strain genome are 1 SNP and 2 INDEL. The strategy of combining short-read sequencing with long-read sequencing is currently the most optimal approach for sequencing bacterial genomes. With this approach, the only available genomic sequence of BCG-Moreau PL was obtained. This sequence will primarily be a reference point in the genetic control of the stability of the vaccine strain in the future. The results enrich knowledge about the microevolution and attenuation of the BCG vaccine substrains.

Importance: The whole genome sequence obtained is the only genomic sequence of the strain that has been used for vaccine production in Poland since 1955. Sequencing of different BCG lots showed that the strain was stable over a period of 59 years. The comprehensive genomic analysis performed not only enriches knowledge about the microevolution and attenuation of the BCG vaccine substrains but also enables the utilization of identified markers as a reference point in the genetic control and identity tests of the stability of the vaccine strain in the future.

Keywords: BCG Moreau; BCG Moreau PL; BCG vaccine; complete genomic sequence; next-generation sequencing; tuberculosis.

FIG 1

Circular representation of general genomic features of M. bovis BCG Moreau PL using Proksee (https://proksee.ca) (15). The scale is shown in megabases on the black central circle. The next circle shows the guanine-cytosine content (GC content). The two outer violet circles show forward and reverse strand coding DNA sequence (CDS), respectively. Some genes (violet), tRNA (orange), rRNA (light blue), and tmRNA (red) are pointed in the outer violet circle with the Proksee’s default parameters.

FIG 2

Schematic diagram of the two largest deletions identified in the M. bovis BCG Moreau PL genome relative to the M. bovis BCG Moreau RDJ reference genome. Genes associated with mycobacterial virulence are colored in green.

FIG 3

Diagram of the BCG microevolution, including the year a given substrain was obtained and the most important genetic changes (16–18, 27–29).

FIG 4

Diagram of the production of TB vaccines in Poland. The material used for the tests is marked in green.