A novel approach to determine the value of ESAT-6 as an antigen in M. tuberculosis infection and vaccines - Project Summary/Abstract Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) remains a major global health problem, and the only licensed vaccine, Bacille Calmette-Guérin (BCG), lacks sufficient efficacy to control the TB pandemic. Therefore, development of effective TB vaccines is a high priority for controlling global TB. Since CD4 T cells are known to be important for protective immunity to TB, they have been the focus of TB vaccine development. Regardless of the mechanism, to be effective, vaccines must contain one or more antigens that can induce protective immune responses; not all antigens possess this potential, and some antigens can induce detrimental immune responses. Of ~4,000 Mtb proteins, ESAT-6 is among the most immunogenic, as it induces robust CD4 T cell responses in response to infection in multiple host species including humans, mice, and cattle. Consequently, ESAT-6 has been assumed be a valuable vaccine antigen, and it is included in multiple TB vaccines at various stages of development and testing. However, vaccines that contain ESAT-6 combined with other antigens have only modest efficacy even in animal models, and T cell responses to ESAT-6 in Mtb-exposed humans do not distinguish those with latent TB from those with active TB. These results are consistent with ESAT-6 acting as a decoy antigen that does not induce protective immunity to Mtb, and that may even interfere with optimal immunity. Determining if ESAT-6 serves a decoy antigen is important for vaccine design, since vaccination might enhance a decoy effect and limit vaccine efficacy. However, understanding the protective value of ESAT-6 and testing the decoy hypothesis have been impeded by the fact that ESAT-6 is also essential for Mtb virulence. Since traditional approaches have not been able to dissociate the role of ESAT-6 as a T cell antigen and its role in virulence, we took an innovative approach and generated transgenic mice whose CD4 T cells selectively lack the ability to respond to ESAT-6. We developed a novel strategy to express the I-Ab CD4 T cell epitope of ESAT-6 in the thymus so it is recognized as self during T cell development and causes deletion of ESAT-6-responsive CD4 T cells. We generated the mice and confirmed that they do not develop CD4 T cell responses to ESAT-6 during Mtb infection. With these (‘ESAT-6 tolerant’, or ‘6T’) mice, we can now determine the value of ESAT-6 as a CD4 T cell antigen without perturbing the function of ESAT-6 as a cell autonomous virulence determinant. In this project, we propose to determine whether CD4 T cell responses to ESAT-6 are beneficial, detrimental, or neutral in Mtb infection; we will determine whether ESAT-6 is a decoy antigen that alters recognition of other Mtb antigens; and we will test the hypothesis that ESAT-6 contributes to vaccine efficacy through mechanisms other than by serving as a T cell immunogen. Our findings will be valuable for guiding antigen selection in new TB vaccines and will either increase confidence that ESAT-6 is a valuable TB vaccine antigen or provide evidence that alternative antigens should be prioritized. The ESAT-6-tolerant mice we generated may also provide a new model for novel vaccine antigen discovery.
