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 1,530 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.

MTBVAC from discovery to clinical trials in tuberculosis-endemic countries

“…The majority (82%) of these absent epitopes are contained in 6 antigenic proteins encoded in RD1 and RD2 deleted in BCG strains. These and related studies further suggested that epitope sequence variation in BCG potentially affects human T cell recognition that could result in ineffective priming of the host immune system [16,17,[50][51][52].…”

“…It is generally well assumed that T cell epitopes are evolutionarily conserved in the MTBC [48,49]. Copin et al [50] compared the sequence diversity of 1530 experimentally verified human T cell epitopes and found that 23% of the known MTBC T cell epitopes are absent in BCG. The majority (82%) of these absent epitopes are contained in 6 antigenic proteins encoded in RD1 and RD2 deleted in BCG strains.…”

“…In newborn C57BL/6 mice, MTBVAC vaccination induced differential immune response to Ag85B compared to BCG vaccination, as measured by IFNγ secretion in splenocytes [71]. A plausible explanation for these differential immune responses to Ag85B between MTBVAC (or SO2) and BCG could be due to an acquired point mutation in the fbpB gene in all BCG sub-strains, which results in an unstable protein [50]. Interestingly, in addition to the rich antigenspecific repertoire inherent of MTBVAC, recent data by our group observed that the PhoP-dependent ncRNA mcr7 leads to an increased activity of the Tat-C secretion system resulting in greater secretion of Ag85 complex proteins in MTBVAC [44,59].…”

Breaking Transmission with Vaccines: The Case of Tuberculosis

“…The genome sequences of different BCG strains were compared to determine T-cell epitope conservation (47). It was found that among the 1,530 human T-cell epitopes, 23% of them are absent in the BCG vaccines.…”

“…It was found that among the 1,530 human T-cell epitopes, 23% of them are absent in the BCG vaccines. The majority of the absent epitopes in BCG are contained in three proteins: ESAT-6, CFP-10 (10-kDa culture filtrate protein), and PPE68 (cell envelope protein from PPE family that contributes to M. tuberculosis maintenance of infection) all of them encoded in the RD1 region absent in BCG (47). If these epitopes are necessary to complete the pulmonary cycle of M. tuberculosis, we could hypothesize that vaccine candidates containing epitopes absent in BCG could confer immunity to protect against pulmonary TB.…”