Immunogenicity Enhancement via Self-Assembled mi3 Nanoparticles Displaying ESAT-6 Antigen

Sihang Dong,1 Shuang Wu,1 Ke Liang,1 Hui Zhang,2 Fangzheng Guo,1 Yamin Song,1 Yanmei Hao,1 Zhongqing Qian,1 Xiaojing Wang,3 Tao Xu,1,2 Hongtao Wang1,2 1Anhui Province Key Laboratory of Immunology in Chronic Diseases, Medicine Experimental Center, Laboratory Medicine College, Bengbu Medical University, Bengbu, 233030, People’s Republic of China; 2Department of Neurosurgery, the First Afliated Hospital of Bengbu Medical University, Bengbu, 233000, People’s Republic of China; 3Anhui Province Key Laboratory of Respiratory Tumor and Infectious Disease, Molecular Diagnosis Center, First Affiliated Hospital of Bengbu Medical University, Bengbu, 233004, People’s Republic of ChinaCorrespondence: Tao Xu, Email taoxu@bbmu.edu.cn Hongtao Wang, Email hongtaowang@bbmu.edu.cnPurpose: The only World Health Organization (WHO)-approved vaccine for tuberculosis (TB), Bacillus Calmette–Guérin (BCG), shows limited efficacy, particularly against adult pulmonary TB. Moreover, traditional subunit vaccines often suffer from poor immunogenicity. To address these challenges, we aimed to enhance the immune activation potential of the early secreted antigenic target of 6 kDa (ESAT-6) by developing a novel nanoparticle-based vaccine platform.Methods: We engineered a nanovaccine using mi3 nanoparticles to display the ESAT-6 antigen on their surface. These nanoparticles self-assembled into uniform dodecahedral structures, enabling enhanced antigen presentation. To evaluate its immunogenicity, mice (n= 6 per group) were primed with BCG and subsequently boosted with the ESAT-6–mi3 nanovaccine. Control groups included mice receiving BCG alone or unconjugated nanoparticles.Results: Compared with mice that received BCG alone or unconjugated nanoparticles, those boosted with the ESAT-6–mi3 nanoparticle vaccine exhibited significantly stronger immune responses. Antigen-specific antibody titers increased by more than 10-fold compared to the BCG-only group. Additionally, elevated levels of pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL-2 were observed, indicating a robust Th1/Th17-biased cellular immune response. Our findings indicate that the vaccine candidate can elicit effective immune responses in vitro. However, it should be noted that this study has limitations, primarily including the lack of an in vivo pathogen challenge to assess actual protective efficacy and the evaluation of immune responses against only a single antigen. Therefore, these results require further validation in future studies involving challenge experiments and multi-antigen strategies.Conclusion: Our findings demonstrate that the ESAT-6–mi3 nanoparticle vaccine markedly enhances both humoral and cellular immune responses, outperforming traditional subunit vaccine approaches. This strategy shows strong potential as a next-generation booster vaccine to complement BCG and improve TB immunization outcomes. Keywords: Mycobacterium tuberculosis, mi3, self-assembled, nanoparticle, vaccine