A novel mi3 VLP nanoparticle vaccine displaying ESAT-6 -PPE57 -PPE68 -EsxV antigens exhibits superior immunogenicity and Mycobacterium tuberculosis growth inhibition

Tuberculosis (TB) is an ancient disease caused by Mycobacterium tuberculosis (Mtb), and vaccination represents the most effective strategy for preventing and controlling TB infection. In this study, we screened 200 antigens distributed across different infection stages/types in the Mtb genome. Using IEDB and VaxiJen v2.0 algorithm, we identified 10 Mtb-specific T cell target antigens. Further selection based on IFN-γ expression levels in PBMC from QFT⁺ and QFT^- individuals after stimulation with the 10 antigens, led to the identification of ESAT-6, PPE57, PPE68, and EsxV as four candidate antigens. These antigens were displayed on the surface of mi3 virus-like particles (VLP) via the Spycatcher/SpyTag system and formulated with adjuvant AS01E to construct a multivalent VLP nanoparticle vaccine (EPPE+mi3/AS01E). The immunogenicity of this vaccine was evaluated in C57BL/6 mice following a three-dose immunization schedule, and its protective efficacy was assessed using the mycobacterial growth inhibition assay (MGIA). Our results demonstrated that EPPE+mi3/AS01E elicited robust Th1, Th2, and Th17 cellular immune responses, as well as potent antigen-specific humoral immune responses. It significantly enhanced the capacity of splenic lymphocytes to inhibit the in vitro growth of Mtb H37Rv, outperforming both the subunit vaccine (EPPE/AS01E) and BCG alone. When administered as a BCG booster, EPPE+mi3/AS01E synergistically enhanced the immunological response and protective efficacy conferred by BCG. Collectively, the mi3 VLP multi-antigen nanovaccine developed in this study demonstrated strong immunogenicity and significant inhibition of Mtb growth, highlighting it as a promising vaccine candidate for further development.