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Vaccine. 2014 Oct 14;32(45):5998-6004. doi: 10.1016/j.vaccine.2014.07.113. Epub 2014 Sep 6.

Impact of in vitro evolution on antigenic diversity of Mycobacterium bovis bacillus Calmette-Guerin (BCG).

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Department of Medicine, Division of Infectious Diseases, New York University School of Medicine, 522 First Avenue, Smilow 901, New York, New York, 10016, USA.
Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland.
University of Basel, Petersplatz 1, Basel 4003, Switzerland.
Center for Health Informatics and Bioinformatics, New York University Langone Medical Center, New York, NY, USA.
Departments of Microbiology and Pathology, New York University School of Medicine, 522 First Avenue, Smilow 901, New York, New York, 10016 USA.
Contributed equally


Mycobacterium bovis bacillus Calmette-Guerin (BCG), the only vaccine currently used against tuberculosis, is an attenuated derivative of M. bovis that has been propagated in vitro for more than 40 years. We have previously reported that the experimentally-verified human T cell epitopes of the M. tuberculosis complex (MTBC) are the most conserved elements of the genome; whether immune recognition is the force driving the conservation of epitopes in the MTBC is unknown. Therefore, we sequenced the genomes of 12 BCG strains to determine whether T cell epitopes were under selection pressure during BCG in vitro evolution. We constructed a genome-wide phylogeny and refined the previously-determined BCG phylogeny. Notably, we identified a new cluster between BCG Japan and BCG Russia, and repositioned the relationships of several strains within the lineage. We also compared the sequence diversity of 1530 experimentally verified human T cell epitopes in the BCG vaccines with those in the MTBC. We found 23% of the known T cell epitopes are absent, and that the majority (82%) of the absent epitopes in BCG are contained in 6 proteins encoded in 2 regions of difference (RD) unique to BCG strains. We also found that T cell epitope sequences in BCG are more conserved than non-epitope sequences in the same gene. Finally, we find evidence that epitope sequence variation in BCG potentially affects human T cell recognition. These findings provide new insight into sequence variation in a slow-growing bacterium closely related to the MTBC that has been subjected to prolonged passage outside of a mammalian host, and indicate little difference in the extent of variation in vivo and in vitro.


In vitro evolution; Mycobacterium bovis bacillus Calmette-Guerin (BCG); Phylogenetic diversity; T cell epitopes

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