Conclusions Subunit vaccines such as H4/IC31, H56/IC31 , ID93/GLA-SE and M72/AS01E are currently in clinical trials. by inhibiting host defense mechanisms, including phagosome maturation, by secretion of inflammatory cytokines and antigen presentation Avibactam . In early infection, an understanding of the mechanism of T-cell evasion, including impaired or misregulated dendritic cell maturation and delayed priming of CD4+ T cells [4,5,6], is essential in the design of vaccines for successful antigen activation of CD4+ T cells. In the lungs, na?ve T cells that encounter mycobacterial antigens induce various CD4+ T-cell responses through DCs, which bridge innate and adaptive immunity. After lung DCs endocytose antigens and migrate to lymphoid organs, they initiate T-cell differentiation in the lung-draining lymph nodes . The migration of DCs is promoted by interleukin (IL)-12p40 and limited by IL-10 [8,9]. There are many subsets of CD4+ T cells whose development is triggered by DCs, such as T-helper 1 (Th1), Th2, Th17, and regulatory T cells (Tregs), which cooperate or interfere with each other to control the balance between active and latent TB . In particular, antigen-specific CD4+ Th1 cells play a protective role against TB by producing cytokines such as interferon (IFN)- or tumor necrosis factor (TNF)-, which Avibactam contribute to the recruitment and activation of innate immune cells such as monocytes . A study of infectious progression in mice lacking IFN- showed that Th1-polarized CD4+ T cells are more important than Th2-immune response for the control of TB [12,13]. However, although many studies have identified the function of CD4+ T cells against TB, the role of T cells in host immunity is not defined. There are several CD4+ T cell effector subtypes, from activated T cells that only produce IL-2 or IFN- to multifunctional cells expressing IL-2, IFN-, and TNF- . The role of multifunctional T cells is closely related to the regulation of TB infection and protection [9,15,16]. Since the development of these effector T cells initially requires DC activation and migration to the lymph nodes, it is necessary to regulate the activity of DCs to exert protective immunity against TB. Many studies have focused on the development of multi-subunit vaccines using viral vectors or adjuvants. Although diverse mycobacterial antigens have been investigated for development Avibactam of TB vaccine, only a few proteins have been selected as vaccine candidates to enter human clinical trials. Among them, antigen 85 complex and ESAT6 are most commonly used [17,18,19]. However, MVA85A did not elicit significant BCG-prime boosting effect in human trial , which has led to search a new vaccine target and develop the diverse strategies for vaccine research. Therefore, it is important to search other antigens for replacing T cell-stimulating antigens and enhancing their activity. Several PLA2G10 mycobacterial antigens are reported to induce DC maturation and Th1 polarization [21,22,23], but their vaccine potential against TB is yet to explored. We previously reported a novel DC-activating protein, Rv2299c. Rv2299c-matured DCs induce Th1 differentiation with bactericidal activity, and in particular, the fusion of Rv2299c to ESAT6 enhances the immunoreactivity and BCG prime-boosting efficacy of ESAT6 , whereas Rv2299c alone does not confer a significant protective effect. Based on these data, we hypothesized that the DC-activating protein Rv2299c could enhance the protective immunity of other vaccine candidates comprising T-cell-stimulating antigens. In this study, to prove our hypothesis, we selected the fusion protein Ag85B-ESAT6, a well-known TB vaccine candidate, as the fusion partner of Rv2299c. We found that the inclusion of Rv2299c to the Ag85B-ESAT6 fusion protein leads to an increase in the immune reactivities and protective efficacy of Ag85B-ESAT6. In addition, a higher frequency of multifunctional CD4+ T cells was found in mice vaccinated with Rv2299c-Ag85B-ESAT6 than in Ag85B-ESAT6. Thus, our results provide a new strategy for the design of vaccine candidates based on DC-activating antigens. 2. Results 2.1. Rv2299c-Ag85B-ESAT6 Protein Induces DC Maturation The recombinant Rv2299c-Ag85B-ESAT6 fusion protein (RAE6) was constructed in the pET22b vector (Figure 1a). The fusion protein was produced in BL21 cells and purified under endotoxin-free conditions. After purification and dialysis, the purity of the recombinant protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.