Characterization of the innate killing evasion (ike) genes of Mycobacterium and application to Mycobacterium tuberculosis vaccines

Abstract

Mycobacterium tuberculosis contains a specialized secretion system encoded partly by genes in RD1 (Region of Difference 1), the deletion of which is the primary attenuating mutation of BCG. RD1 encodes components of a secretion system known as ESX-1. M. tuberculosis has 4 other homologs of ESX-1 (ESX-2 through ESX-5), and ESX-3 (genes Rv0282 to Rv0292) is the only homolog that is conserved in all mycobacterial strains examined. The genes Rv0282 to Rv0292 excluding Rv0287 and Rv0288 (esat-6 and cfp-10) were confirmed to be essential in M. tuberculosis for growth in culture. However, the genes in M. smegmatis paralogous to Rv0282 to Rv0292 could be deleted without having an impact on viability. In a high dose intravenous challenge model of M. smegmatis, immuno-compentent and immuno-compromised mice die rapidly from parental infection, whereas the Esx-3 deletion does not kill mice and is rapidly cleared from all organs with minimal tissue destruction.;Furthermore, the Esx-3 deletion is highly attenuated in a variety of immune deficient mice, excluding myD88-/- mice. Based on these data, we concluded that the Esx-3 genes were required to evade killing by innate immunity and henceforth designated this region as ike (innate killing evasion). Upon further investigation, it was shown that the ike deletion elicits a rapid and robust TH 1 serum cytokine response in infected mice, and infection in bone marrow derived macrophage cell lines demonstrates that this region delays phagosome maturation.;The ike locus of M. tuberculosis ( Rv0282 to Rv0292) was introduced into the M. smegmatis Deltaike strain, generating a strain referred to as IKEPLUS. Upon infection, the M. tuberculosis genes do not complement the ike deletion in M. smegmatis , and in fact is attenuated in mouse infection models. In a low dose aerosol challenge model of M. tuberculosis, it was found that IKEPLUS protected as well as BCG at 30 days post infection. Amazingly, in an intravenous challenge model, IKEPLUS elicited bactericidal immunity from M. tuberculosis challenge, providing an astounding 3-log reduction of bacterial burden whereas BCG did not. This protection is dependent on CD4+ MHC-II restricted central memory T cells.;Our demonstration that IKEPLUS produces bactericidal activity against M. tuberculosis is encouraging as it suggests that the inclusion of additional protective antigens of M. tuberculosis could lead to an even more effective anti-Tuberculosis vaccine. Overall we have identified a novel gene set that is involved in evasion of innate and subsequently effective adaptive immunity. Ultimately, the Deltaike strain represents a novel live and safe vaccine vector for eliciting therapeutic TH1 immunity.