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.

Introduction

Tuberculosis (TB) remains a major global health challenge, causing an estimated 8.8 million new cases and 1.3 million deaths in 2012 [1]. In 1921, Albert Calmette and Camille Guérin developed the only vaccine currently used against TB. This vaccine, known as Bacillus Calmette-Guerin (BCG), is an attenuated derivative of Mycobacterium bovis, the causative agent of bovine TB. More than 120 million doses of BCG are administered annually in more than 100 countries [2]. Although BCG is effective in preventing disseminated TB in children [3], [4], the efficacy of BCG against pulmonary TB in adults varies from 0 to 80% [5]. Various hypotheses have been put forward to explain the low and variable efficacy of BCG. These include human genetic factors, variable exposure (and immune responses) to environmental mycobacteria, and differences in the circulating M. tuberculosis populations [6], [7], [8]. Mismatches between the antigenic composition of BCG and virulent M. tuberculosis may also contribute, but have not been systematically examined. Shortly after its original development, BCG was distributed to laboratories in multiple countries in Europe, Asia, and North- and South America for local preparation of vaccine. This process led to the diversification of BCG into distinct sub-strains. Lyophilization was introduced in 1961, which allowed long-term storage of seed stocks, with each of the BCG sub-strains named after the country, city, or laboratory where it was propagated. By that time, BCG Pasteur for example, had been subjected to 1173 passages [9]. Although it is likely that most laboratories established seed stocks shortly after lyophilization became available, detailed information is not readily available.

Several studies have compared the genomic diversity of BCG sub-strains and identified several deletions, known as regions of difference (RD), as well as tandem duplications (DU) and single nucleotide polymorphisms (SNPs) [10], [11]. This diversity has been used to construct phylogenetic trees for BCG and infer the evolutionary history of individual BCG sub-strains [12]. Whether the diversity between individual BCG strains contributes to the variable outcomes of BCG vaccine trials is unknown, but evidence from studies in humans (reviewed in [6]) and experimental animals [13] have shown that BCG strains differ in their ability to induce specific cellular immune responses.

Immunity to TB depends on T lymphocytes. Infection with M. tuberculosis induces antigen-specific T cell responses in humans [14] and mice [15]. Moreover, T cell-deficient humans, nonhuman primates, and mice are susceptible to rapidly-progressive disease [16], [17], [18]. Immunization with BCG induces antigen-specific T cell responses in humans [7], although a clear correlate of vaccine-induced immunity to human TB has not yet been identified. Activation of conventional T cells after infection or vaccination depends on recognition of specific peptides bound to MHC (HLA in human) molecules on antigen presenting cells. For a given peptide to be immunogenic, it must bind with sufficient affinity to one or more MHC/HLA proteins, of which there are numerous allelic variants. The peptide/MHC complex must then be recognized by clonotypic T cell antigen receptors in the repertoire of the host. Variation in the sequence of a peptide epitope can result in loss of recognition by T cells [19], indicating that a close match between the sequence of immunogenic peptides in a vaccine and in the natural pathogen may be necessary to induce optimal immunity.

We previously reported that 491 experimentally-verified human T cell epitopes of the M. tuberculosis complex (MTBC) were highly conserved [20], with 95% of the epitopes examined harboring no amino acid sequence variants in 21 genetically-diverse M. tuberculosis clinical strains. These findings implied that for the majority of these immune targets, antigenic variation does not contribute to immune evasion. If epitopes in M. tuberculosis are conserved by selection pressure exerted by human T cell recognition, we hypothesized that 40 years of in vitro evolution between the first derivation of BCG and its lyophilization would lead to accumulation of diversity. The goals of the present study were (1) to determine the sequence variation in a close relative of M. tuberculosis (i.e. BCG) following prolonged in vitro passage in the absence of immune selection; and (2) to define if MTBC epitopes in BCG are more diverse than in clinical MTBC isolates because of the absence of immune pressure.