Genome plasticity of BCG and impact on vaccine efficacy

Abstract

To understand the evolution, attenuation, and variable protective efficacy of bacillus Calmette-Guérin (BCG) vaccines, Mycobacterium bovis BCG Pasteur 1173P2 has been subjected to comparative genome and transcriptome analysis. The 4,374,522-bp genome contains 3,954 protein-coding genes, 58 of which are present in two copies as a result of two independent tandem duplications, DU1 and DU2. DU1 is restricted to BCG Pasteur, although four forms of DU2 exist; DU2-I is confined to early BCG vaccines, like BCG Japan, whereas DU2-III and DU2-IV occur in the late vaccines. The glycerol-3-phosphate dehydrogenase gene, glpD2, is one of only three genes common to all four DU2 variants, implying that BCG requires higher levels of this enzyme to grow on glycerol. Further amplification of the DU2 region is ongoing, even within vaccine preparations used to immunize humans. An evolutionary scheme for BCG vaccines was established by analyzing DU2 and other markers. Lesions in genes encoding sigma-factors and pleiotropic transcriptional regulators, like PhoR and Crp, were also uncovered in various BCG strains; together with gene amplification, these affect gene expression levels, immunogenicity, and, possibly, protection against tuberculosis. Furthermore, the combined findings suggest that early BCG vaccines may even be superior to the later ones that are more widely used.

Fig. 1.

Circular representation of the M. bovis BCG Pasteur chromosome. The scale is shown in megabases in the outer black circle. Moving inward, the next two circles show forward and reverse strand CDS, respectively, with colors representing the functional classification (red, replication; light blue, regulation; dark blue, virulence; light green, hypothetical protein; dark green, cell wall and cell processes; orange, conserved hypothetical protein; cyan, IS elements; yellow, intermediate metabolism; gray, lipid metabolism; purple, PE/PPE). The following two circles show forward and reverse strand pseudogenes (colors represent the functional classification), the next circle shows RD (black) and DU (red), followed by the G+C content, and finally the GC skew (G-C)/(G+C) plotted by using a 10-kb window. For more details see SI Table 2.

Fig. 2.

Mapping duplications in BCG strains. DU1 is confined to M. bovis BCG Pasteur as shown by Southern blotting of HindIII restriction digests of various BCG vaccines and hybridization with a probe for the oriC region. Note that the difference in size of the fragment hybridizing in M. tuberculosis (M. tub.) H37Ra is due to an IS6110 insertion in the fragment.

Fig. 3.

Genomic variations occur in vaccine preparations intended for human use. Variation in the DU2 region revealed by Southern blotting of AsnI restriction digests and hybridization with a probe for the 3,686-kb region. Note that the AsnI sites in the DU2 region are outside the duplicated region and that additional hybridizing bands are due to ongoing amplification events, such as triplications.

Fig. 4.

Scheme showing the appearance of DU2-I through DU2-IV. (A) Duplicated regions use a color scheme, and each duplication is boxed. Genomic coordinates based on M. tuberculosis H37Rv are indicated together with the positions of junctions (JDU2-I–JDU2-IV). (B). Identification of genes present in the region common to DU2-I through DU2-IV. Note the color scheme of A also applies to B.

Fig. 5.

Refined genealogy of BCG vaccines. The scheme shows the position of genetic markers identified in this work, RD markers, some strain-specific deletions, and the distribution of vaccines into the four groups. Details of primers used for differentiation are listed in SI Table 4.