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

Abstract

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.

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

Copyright © 2014 Elsevier Ltd. All rights reserved.

Figures

Figure 1

A. Phylogenetic relationships among BCG strains. A. Maximum likelihood phylogeny based on 144 variable common nucleotide positions across 12 Mycobacterium bovis BCG genome sequences. The tree is rooted with M. bovis Ravenel SRR022532. Node support after 1000 bootstrap replications is indicated in black. Prior dates used to calibrate the phylogeny for Bayesian coalescent analysis are indicated in grey. B. The stacked bars show the proportion of missing M tuberculosis T cell epitopes in the 3 BCG groups identified in panel A and delimited by the dashed lines. Group 1: BCG Russia and Japan. Group 2: BCG Australia, Connaught, Copenhagen, Denmark, Glaxo, Mexico, Phipps, Prague and Tice. Group 3: BCG Pasteur

Figure 2

Conservation of M. tuberculosis T cell epitopes in BCG strains. A. dN/dS in various gene classes of BCG strains. The calculation was done after comparing each of the 12 BCG genomes to the inferred most recent common ancestor of MTBC. This shows the dN/dS in 1) non-redundant epitope regions, 2) non-epitope regions of antigens, 3) M. tuberculosis essential genes, 4) M. tuberculosis non-essential genes. B. Comparison between the number of synonymous and non-synonymous SNPs found in M. tuberculosis non-overlapping T cell epitope regions in BCG and random sequences of same size selected from the rest of BCG genomes. The graph shows that T cell epitope regions in BCG are less affected by SNPs than expected by chance (95% confidence interval in the random replicates is indicated as error bars).