Bacille Calmette–Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the only vaccine available for tuberculosis (TB) control. However, BCG is not an ideal vaccine and has two major limitations: BCG exhibits highly variable effectiveness against the development of TB both in pediatric and adult populations and can cause disseminated BCG disease in immunocompromised individuals. BCG comprises a number of substrains that are genetically distinct. Whether and how these genetic differences affect BCG efficacy remains largely unknown. In this study, we performed comparative analyses of the virulence and efficacy of 13 BCG strains, representing different genetic lineages, in SCID and BALB/c mice. Our results show that BCG strains of the DU2 group IV (BCG-Phipps, BCG-Frappier, BCG-Pasteur, and BCG-Tice) exhibit the highest levels of virulence, and BCG strains of the DU2 group II (BCG-Sweden, BCG-Birkhaug) are among the least virulent group. These distinct levels of virulence may be explained by strain-specific duplications and deletions of genomic DNA. There appears to be a general trend that more virulent BCG strains are also more effective in protection against Mycobacterium tuberculosis challenge. Our findings have important implications for current BCG vaccine programs and for future TB vaccine development.

Comparative genome analyses have revealed a number of regions of difference (RD) among mycobacterial species. The functional consequences of most of these genome variations have not been studied. RD4, which encompasses Rv1506c-Rv1516c of Mycobacterium tuberculosis (M. tb) H37Rv, is absent in the closely related Mycobacterium bovis and M. bovis Bacille Calmette-Guérin (BCG). On the other hand, we previously found that Mycobacterium marinum has an extended RD4 which includes a number of genes involved in the biosynthesis of lipooligosaccharides (LOSs). As such, there appears to be a gradual decay of RD4 in mycobacterial genomes in the order of M. marinum, M. tb, and M. bovis (including BCG). To understand the potential effect of RD4 on mycobacterial virulence, in this study, we cloned the entire (Rv1501–1516c) and partial (Rv1501–1508c) RD4 into an integrating vector. These constructs were introduced to M. bovis BCG and M. marinum and the virulence of the RD4 knock-in strains were evaluated in the SCID mice and zebrafish infection models, respectively. BCG containing the entire RD4 exhibited similar levels of virulence to the parental strain but BCG containing partial RD4 (Rv1501–Rv1508c) was more attenuated. Similarly, zebrafish infection experiments showed that addition of partial RD4 also appeared to attenuate the virulence of M. marinum. However, M. marinum containing entire RD4 was more virulent than the wild type strain. Interestingly, BCG strains containing the entire or partial RD4 exhibited better protection of zebrafish against M. marinum challenge than the parental BCG. Taken together, our data suggest that RD4 plays a role in mycobacterial virulence and that RD4 knock-in BCG strains confer improved protection. Our study has provided new insights into the biological function of RD4 and evolution of mycobacterial genomes.

One strategy to develop the next generation of tuberculosis vaccines is to construct subunit vaccines based on T cell antigens. In this study, we have evaluated the vaccine potential of a fusion protein combining EsxB, EsxD, EsxG, EsxU, and EsxM of Mycobacterium tuberculosis (M. tb). This recombinant protein, named BM, was expressed in and purified from Escherichia coli. Immunization of C57BL/6 mice with purified BM protein formulated in Freund's incomplete adjuvant induced the production of Th1 cytokines (IFN-γ, TNF, and IL-2) and multifunctional CD4+ T cells. Vaccination of BALB/c mice with BM protein followed by intravenous challenge with Mycobacterium bovis BCG resulted in better levels of protection than the two leading antigens, Ag85A and PPE18. Taken together, these results indicate that BM is a protective antigen. Future studies to combine BM with other antigens and evaluate its effectiveness as a booster of BCG or as a therapeutic vaccine are warranted.