Construction, expression, and immunogenicity analysis of a fusion gene based on the PE/PPE family in combination with adjuvants.

Tuberculosis (TB) remains the leading global cause of death from a single infectious agent and a major fatal infectious disease worldwide. However, the Bacillus Calmette-Guérin (BCG) vaccine-currently the only licensed TB vaccine in clinical practice-has critical limitations in preventing latent infection and disease reactivation, failing to deliver comprehensive protective immunity. Given these gaps, targeting Mycobacterium tuberculosis (M.tb) antigens with strong immunogenic potential is a key strategy for next-generation TB vaccine development. The pathogenic M.tb harbors the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motif gene families, named for their N-terminal conserved proline (P) and glutamic acid (E) residues, which are increasingly recognized as promising vaccine targets due to their roles in host-pathogen interaction and immune modulation. To address BCG's limitations, this study selected the PE/PPE family members Rv0832 and Rv3653 for full-length gene synthesis to construct a fusion antigen designated ZMM6, which was expressed using the pET-28a vector. The immunogenicity of the ZMM6 subunit vaccine was then evaluated in M.tb-infected human cohorts and animal models. The results showed that the fusion protein ZMM6 induced peripheral blood mononuclear cells (PBMCs) from M. tb-infected individuals to secrete high levels of IFN-γ, IL-2, and IL-6 cytokines. Moreover, the levels of cytokine secretion induced by the fusion protein were higher than those induced by the individual subunit proteins, suggesting that the fusion protein possesses higher immunogenicity. In mice immunized with the protein vaccine (adjuvant formulation) ZMM6/MnJ, high titers of various IgG subclass antibodies were elicited, with the immune response skewed toward a Th1-type profile, accompanied by robust induction of multiple Th1-type cytokines. Flow cytometry (FCM) analysis further revealed a significant increase in CD4+IFN-γ+ single-positive T cells in ZMM6/MnJ-immunized mice. Collectively, these findings demonstrate that the protein vaccine (adjuvant formulation) ZMM6/MnJ exhibits strong immunogenicity.