Limits...
Novel genome polymorphisms in BCG vaccine strains and impact on efficacy.

Leung AS, Tran V, Wu Z, Yu X, Alexander DC, Gao GF, Zhu B, Liu J - BMC Genomics (2008)

Bottom Line: Moreover, we have uncovered various polymorphisms in the phoP-phoR locus.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada. andreas.leung@utoronto.ca

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553098&req=5

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.

Mentions: Although the genomic profiles of BCG-Birkhaug and BCG-Sweden are similar, we have also found that BCG-Birkhaug is distinguished by a strain-specific duplication, named DU-Birkhaug. This spans the origin of replication and is analogous to the DU1 duplication in BCG-Pasteur [21,24] (Fig. 1A). However, the borders of the DU-Birkhaug are different. Whereas DU1 encompasses 29.6 kb from Rv3910 to pknB/Rv0014, DU-Birkhaug spans a slightly different region, from trxB/Rv3913 to rodA/Rv0017c. Most of the genes in these regions are involved with DNA replication and cell division. Unlike DU1, DU-Birkhaug also appears to be in a genomic location distant to its original copy. Initial PCR-based attempts to characterize the boundaries of this duplication assumed that the second copy was nearby failed to detect a product (data not shown). As such, the genome location of DU-Birkhaug remains unknown.


Novel genome polymorphisms in BCG vaccine strains and impact on efficacy.

Leung AS, Tran V, Wu Z, Yu X, Alexander DC, Gao GF, Zhu B, Liu J - BMC Genomics (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553098&req=5

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.
Mentions: Although the genomic profiles of BCG-Birkhaug and BCG-Sweden are similar, we have also found that BCG-Birkhaug is distinguished by a strain-specific duplication, named DU-Birkhaug. This spans the origin of replication and is analogous to the DU1 duplication in BCG-Pasteur [21,24] (Fig. 1A). However, the borders of the DU-Birkhaug are different. Whereas DU1 encompasses 29.6 kb from Rv3910 to pknB/Rv0014, DU-Birkhaug spans a slightly different region, from trxB/Rv3913 to rodA/Rv0017c. Most of the genes in these regions are involved with DNA replication and cell division. Unlike DU1, DU-Birkhaug also appears to be in a genomic location distant to its original copy. Initial PCR-based attempts to characterize the boundaries of this duplication assumed that the second copy was nearby failed to detect a product (data not shown). As such, the genome location of DU-Birkhaug remains unknown.

Bottom Line: Moreover, we have uncovered various polymorphisms in the phoP-phoR locus.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada. andreas.leung@utoronto.ca

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.

Show MeSH
Related in: MedlinePlus