T cells plays important role in controlling the infection. Previous research findings have suggested that many immunodominant antigens are not similarly immunogenic. We attempt to discover novel antigen that can be developed as a potential vaccine candidate. Low dose Mycobacterium tuberculosis (Mtb) infection in Cynomolgus macaque results in human like granuloma formation. It is comprised of T cells, B cells, NK cells, and several other cells. Upon FDG-PET scan it has been shown that some of these granulomas are permissive or progressive in nature and some are restrictive or controlling.

Our aim is to discover Mtb antigens that can be recognized by the T cells present in the granuloma. To investigate our hypothesis, we have infected Cynomolgus macaque with low dose of Mtb. T cells were isolated from the low and high burden granuloma. They were subjected to scRNA sequencing. Using bioinformatic tools and algorithms we have identified high value TCRs from controlling granulomas. The TCRs were reconstructed and transfected in TCR deficient Jurkat cells with NFAT promoter and luciferase reporter gene. The Mtb ORFeome library was cloned and expressed in E. coli. For antigen preparation, IPTG induced recombinant E. coli were killed by Peracetic acid or heat-killed and presented to the high value TCRs expressed in the reporter Jurkat cells by autologous BLCLs. The recognition was measured using luciferase activity.

The selected unique TCRs show remarkable luciferase activity in response to PMA and Ionomycin stimulation. We believe that the antigen recognized by these TCRs will be equally immunogenic and can be developed as the new target for the vaccine against tuberculosis.

Supported by BAA-NIAID-NIHAI201700104

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