Divergence of immunologic and protective responses of different BCG strains in a murine model

The ongoing evolution of BCG after its introduction in 1921 resulted in strains that differ genetically and phenotypically. Based on a genomic deletion (Region of Difference 2 or RD2) that occurred between 1927 and 1931, BCG strains can be sub-classified by the presence or absence of RD2. The existence of other mutations that distinguish BCG strains precludes simple comparison of RD2-positive and RD2-negative BCG strains to determine the importance, if any, of RD2 for vaccine protection. In this study, we have compared the RD2-containing BCG Russia, BCG Pasteur (which is a natural mutant for RD2), and BCG Pasteur complemented with RD2-genes Rv1979c-Rv1982 through various in vitro and in vivo assays of immunogenicity and protection. We determined that the presence of RD2 did not affect vaccine persistence, but lead to increased immunogenicity, as measured by ELISpot. Additionally, T-cells from animals immunized with BCG Russia and BCG Pasteur::Rv1979c-82 were more effective at killing Mycobacterium tuberculosis in macrophages than T-cells from animals immunized with BCG Pasteur. In a mouse vaccine-challenge model, the presence of RD2 had no effect on pulmonary TB, as measured by M. tuberculosis burden and degree of histopathology, at 4, 8 or 12 weeks post-infection. The presence of RD2 was however associated with decreased dissemination of M. tuberculosis to the spleen. Together, our data demonstrated that the loss of RD2 resulted in decreased immunogenicity but did not affect protection against pulmonary TB, indicating a dissociation between these phenotypes associated with BCG vaccination.