Friday, May 9, 2025
5/9/2025
Mechanisms of a therapeutic DNA vaccine targeting Mycobacterium tuberculosis persisters - PROJECT SUMMARY Host status plays a major role in tuberculosis (TB) disease outcomes, with many efforts to develop host-directed therapies to boost immune response. It is unknown how these strategies will perform in people living with HIV/AIDS (PLWHA) who are at increased risk of adverse TB treatment outcomes. Persistent Mycobacterium tuberculosis (Mtb), which obligates prolonged TB treatment, highly expresses the stringent response protein RelMtb. One attractive host-directed therapeutic strategy is boosting RelMtb-specific cellular responses. We developed a novel therapeutic DNA TB vaccine that promotes immune responses to RelMtb, which are further enhanced by fusion with a chemokine gene, Macrophage Inflammatory Protein-3 alpha (MIP-3α), that targets relMtb to immature dendritic cells (DCs). Our data show that an intranasal (IN) or intramuscular (IM) DNA vaccine expressing MIP-3α/relMtb (“fusion” vaccine) demonstrated greater adjunctive therapeutic efficacy when combined with the first-line TB treatment in Mtb-infected mice as compared to IM vaccination with relMtb alone (“non-fusion” vaccine). This proposal will dissect the mechanisms of this novel therapeutic strategy, specifically with IN administration which had the highest efficacy. We will also test whether this strategy is likely to perform in PLWHA, analyzing whether RelMtb-specific immune responses in PLWHA can predict therapeutic TB outcomes. Aim 1 will investigate the molecular mechanism by which the IN administration potentiates anti-TB drugs, while Aim 2 will focus on the role of DCs in the adjunctive therapeutic efficacy of the fusion vaccination strategy. Aim 3 will test the translational potential of the RelMtb-specific T-cell immunity in PLWHA. In Aim 1, we test if IN vaccine administration enhances the host response to Mtb infection through increased IL-17A secretion. We will perform single-cell transcriptomics on cells from murine lungs to confirm the upregulation of IL-17A response pathways in the IN arm, followed by Mtb-infected IL-17A knockout murine experiments. In Aim 2, we hypothesize that the IN fusion vaccine leads to more efficient systemic and local T-cell maturation, activation, and differentiation through enhanced DC activation compared to the IN non-fusion vaccine. Aim 2a will assess differences in DC activation by flow cytometry, DC and T-cell co-localization in murine tissues, and differences in T-cell activation/differentiation using co-culture experiments with DCs derived from each vaccination group. In Aim 2b, we will adoptively transfer DCs from mice immunized with either vaccine into Mtb-infected mice concurrently with TB treatment to compare the adjunctive therapeutic efficacy. In Aim 3, we hypothesize that RelMtb-specific Th1/Th17 cell responses in peripheral blood mononuclear cells derived from HIV-infected and uninfected patients receiving treatment for pulmonary TB, correlate inversely with sputum culture conversion and TB recurrence rates, independently of HIV status. The proposed studies will uncover critical details of the mechanism of a novel therapeutic vaccination approach against TB with important implications in PLWHA and help the PI to gain significant expertise in TB/HIV immunology.
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