Multivalent DNA vaccines induce potent cellular responses and prolong BCG-mediated control of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) remains a leading public health concern worldwide. While Bacillus Calmette-Guerin (BCG) vaccination is effective at protecting infants/children, efficacy wanes over time. Advancements in synthetic DNA technology have resulted in the delivery of structurally complex antigens that elicit robust, multifaceted cell-mediated immunity in humans. Using this platform, we evaluated the comparative immunogenicity and protective capacity of four multivalent plasmid DNA (pDNA) constructs that together encode a diverse set of 23 potential TB antigens. All cassettes were immunogenic in both naive and BCG-primed CB6F1 mice, resulting in robust antigen-specific polyfunctional CD4+ and CD8+ T cell populations secreting primarily IFNγ+/TNF-α+. In BCG-primed mice, several pDNA constructs conferred modest improvement in bacterial control following Mtb challenge. However, animals boosted with the leading construct, pESX exhibited significantly lower bacterial loads and prolonged protection in the lungs compared to BCG-vaccinated control groups. Furthermore, bacterial burdens inversely correlate with the magnitude of numerous vaccine-induced T cell populations. The pESX-mediated efficacy was mapped to the EsxR antigen, which is sufficient to suppress lung bacterial load in this mouse model. These findings provide insight into the relative cellular immunogenicity of novel vaccine candidates and support the critical role of broad, polyfunctional T cells in the long-term control of Mtb.