Construction of a subunit vaccine based on Mycobacterium tuberculosis EsxE and evaluation of its immunogenicity and ex vivo mycobacterial growth inhibitory activity.

Tuberculosis (TB) remains one of the world's deadliest infectious diseases and is the leading infectious disease killer globally. Bacillus Calmette-Guérin (BCG) is currently the only licensed vaccine for TB prevention, but it has limitations in preventing latent infection and TB reactivation. To overcome these limitations, this study selected the secreted antigen Rv3904c (EsxE) to construct the recombinant prokaryotic expression plasmid pET21b-Rv3904c, and the recombinant protein rEsxE was expressed and purified. The antigen specificity of rEsxE was further confirmed by measuring cytokine levels released from peripheral blood cells of Mycobacterium tuberculosis-infected individuals upon rEsxE stimulation using a chemiluminescence assay. The results showed that rEsxE stimulated M. tuberculosis-infected individuals to produce high levels of Th1-type cytokines. Mice were immunized with rEsxE formulated in MnJ adjuvant, and serum specific antibodies, cytokine levels secreted by splenocytes, and the numbers of polyfunctional T cells in the spleen were assessed. The results demonstrated that the BCG prime-rEsxE/MnJ boost strategy induced the highest levels of specific IgG antibodies, with an IgG2c/IgG1 ratio greater than 1, indicating a Th1-skewed immune bias. Upon stimulation with rEsxE or BCG-PPD, splenocytes from this group secreted significantly higher levels of Th1-type cytokines (IFN-γ, TNF-α, and IL-2) than those from the BCG alone and rEsxE/MnJ groups. Flow cytometry analysis revealed that the BCG+rEsxE/MnJ group had the highest numbers of IFN-γ⁺/IL-2⁺ double-positive CD4⁺ and CD8⁺ T cells in the spleen. The in vitro mycobacterial growth inhibition assay provided additional evidence that the prime‑boost regimen augments the capacity of splenocytes to restrict mycobacterial growth. Taken together, these findings demonstrate that rEsxE is immunogenic and that the BCG prime‑rEsxE/MnJ boost strategy elicits robust Th1‑biased immune responses as well as ex vivo mycobacterial growth inhibitory function, thereby supporting the further development of this approach as a candidate tuberculosis subunit vaccine.