Novel genome polymorphisms in BCG vaccine strains and impact on efficacy

Abstract

Bacille Calmette-Guérin (BCG) is an attenuated strain of Mycobacterium bovis currently used as a vaccine against tuberculosis. Global distribution and propagation of BCG has contributed to the in vitro evolution of the vaccine strain and is thought to partially account for the different outcomes of BCG vaccine trials. Previous efforts by several molecular techniques effectively identified large sequence polymorphisms among BCG daughter strains, but lacked the resolution to identify smaller changes. In this study, we have used a NimbleGen tiling array for whole genome comparison of 13 BCG strains. Using this approach, in tandem with DNA resequencing, we have identified six novel large sequence polymorphisms including four deletions and two duplications in specific BCG strains. Moreover, we have uncovered various polymorphisms in the phoP-phoR locus. Importantly, these polymorphisms affect genes encoding established virulence factors including cell wall complex lipids, ESX secretion systems, and the PhoP-PhoR two-component system. Our study demonstrates that major virulence factors are different among BCG strains, which provide molecular mechanisms for important vaccine phenotypes including adverse effect profile, tuberculin reactivity and protective efficacy. These findings have important implications for the development of a new generation of vaccines.

Figure 1

Novel duplications identified in BCG-Birkhaug and BCG-Tice by NimbleGen tiling array. Sections of the ratio plot are shown. The ratio of the reference (M. tb H37Rv) probe intensity (Cy5) was divided by the test (BCG strain) probe intensity (Cy3). Reference probes and test probes that do not span a mutation should represent full-length perfect match hybridization, and thus should have similar intensities, with a reference/test ratio near 1. If the test genome contains an amplification event (increased copy number when compared to the reference), then the reference/test ratio will shift below 1. (A) Novel duplication (DU-Birkhaug) identified in BCG-Birkhaug, which is analogous to the DU-Pasteur (DU1) but has different borders. The same genomic region of BCG-Sweden, which is closely related to BCG-Birkhaug, is shown for comparison. (B) Novel duplication (DU-Tice) identified in BCG-Tice. Three other BCG strains belonging to the same group (DU2-IV) are shown for comparison. (C) The precise border of DU-Tice is mapped by PCR amplification using primers specific to the junction. The two copies are immediately adjacent to each other and overlap by 1 bp.

Figure 2

IS6110 insertion in the phoP promoter in BCG-Russia, -Moreau, and -Japan. (A) Schematic representation of the phoP-phoR locus with IS6110 inserted in an inverse orientation 18 bp upstream from phoP start codon. (B) Nucleotide sequence surrounding IS6110. The IS6110 sequence is boxed. The GAA direct repeats flanking the IS6110 insertion site is underlined and in boldface. The ATG start codons of phoP and phoR are indicated by arrows and in boldface.

Figure 3

Refined genealogy of BCG vaccines. The genealogy is modified from a previous model [24]. Genetic markers identified in this work are highlighted.