Mechanistic investigation of differential T cell responses to distinct Mycobacterium tuberculosis antigens

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Abstract Tuberculosis (TB) is a major global health threat and the only licensed TB vaccine, Bacille Calmette-Guérin (BCG), is inadequate: despite widespread use of BCG, there were 9.9 million new TB cases globally in 2020. Thus, improved vaccines against Mtb are urgently needed. T cell responses remain the primary goal of TB vaccines, as CD4 T cells are necessary to control Mtb in humans and animal models. T cell vaccines have been thus far unsuccessful in preventing infection, but the recent clinical trial of M72-AS01E demonstrated ~50% efficacy as a Prevention of Disease (POD) vaccine. Since TB is transmitted by those with active disease, which is characterized by inflammation and immunopathology, a vaccine that reduces or prevents active disease and immunopathology can have a large impact on the global problem of TB. However, the most optimal antigen target(s) for TB vaccines are not defined, and the lack of evolutionary variation in the known antigens of Mtb suggests that T cell recognition of those antigens is not detrimental to the pathogen or beneficial to the host. Through a comprehensive analysis of genomes from 216 phylogenetically diverse Mtb, my advisor and his colleagues discovered a distinct subset of Mtb antigens that are sequence variable and exhibit evidence of evolutionary selection pressure. In a screen of DNA vaccines encoding these sequence variable antigens, I discovered one (encoding 4 sequence variable antigens) that alters immunopathology and inflammatory Th1 responses in mice subsequently challenged with Mtb, without reducing lung bacterial burdens. Additional studies in our laboratory have demonstrated distinct CD4 T cell effector responses to these same antigens in humans. From these and other results, we hypothesize that a functionally distinct T cell response to one or more of our vaccine antigens is responsible for altered immunopathology in vaccinated mice after challenge. The objective of this proposal is to characterize the cellular responses in vaccinated mice after challenge and identify the mechanism that accounts for altered immunopathology. I will characterize the cellular response using spectral flow cytometry and immunofluorescence microscopy. I will then characterize the vaccine-specific T cell response using peptide:MHC tetramers and single cell RNA sequencing to identify potential mechanisms mediating the altered immunopathology response. Finally, I will identify the underlying mechanism(s) by performing adoptive transfers of sorted T cells to identify the phenotype of the T cells that alter immunopathology during infection. This work will identify differential T cell responses to distinct Mtb antigens and demonstrate a role of immunoregulation in mediating a vaccine response to Mtb infection. The findings will provide an understanding of T cell responses to Mtb antigens that can modulate immunopathology. This will provide a framework of vaccine-induced immunoregulatory responses to inform development of POD vaccines for Mtb.