Immunogenicity of Mycobacterium tuberculosis Antigens: Ag85B, ESAT-6, Rv2660c, and Ag85B-ESAT-6-Rv2660c Fusion in Mice
Immunogenicity of M. tuberculosis Antigens: Ag85B, ESAT-6, Rv2660c and Ag85B-ESAT-6-Rv2660c Fusion in Mice
Keywords:
Ag85B, ESAT-6, Rv2660c, Immunogenicity in miceAbstract
Tuberculosis (TB) remains a major global public health concern. The Bacillus Calmette–Guérin (BCG) vaccine is currently the only available TB vaccine, offering protection against severe TB in children but showing limited efficacy in adults. Developing new TB vaccines is therefore essential for achieving TB eradication. This study aimed to evaluate the immunogenicity of four recombinant Mycobacterium tuberculosis (Mtb) antigens: Ag85B, ESAT-6, Rv2660c, and Ag85B-ESAT-6-Rv2660c fusion in mice, reported as active and latent infection stages to support the design and development of an mRNA-based TB vaccine. Recombinant proteins were designed and expressed in Escherichia coli ClearColi™ BL21(DE3) and used to immunize C57BL/6 mice. Subsequently, humoral immune responses were assessed using Western blot and ELISA, whereas cellular immune responses were analyzed through cytokine detection (IFN-γ, IL-2, and TNF-α) via a Multiplex assay following ex vivo restimulation. The results showed that Ag85B, ESAT-6, and Ag85B-ESAT-6-Rv2660c fusion proteins induced both humoral and cellular immune responses, while Rv2660c, an antigen associated with latent TB infection, primarily stimulated a cellular immune response. These findings suggest the antigens enrolled are promising candidates for further TB vaccine development.
References
Kiazyk S, Ball TB. Latent tuberculosis infection: an overview. Can Commun Dis Rep 2017; 43(3-4): 62-6.
Setiabudiawan TP, Reurink RK, Hill PC, Netea MG, van Crevel R, Koeken VA. Protection against tuberculosis by Bacillus Calmette-Guérin (BCG) vaccination: a historical perspective. Med 2022; 3(1): 6-24.
Zhou F, Zhang D. Recent advance in the development of tuberculosis vaccines in clinical trials and virus-like particle-based vaccine candidates. Front Immunol 2023; 14: 1238649. (12 pages).
Huygen K. The immunodominant T-cell epitopes of the mycolyl-transferases of the antigen 85 complex of M. tuberculosis. Front Immunol 2014; 5: 321. (11 pages).
Macedo GC, Bozzi A, Weinreich HR, Bafica A, Teixeira HC, Oliveira SC. Human T cell and antibody-mediated responses to the Mycobacterium tuberculosis recombinant 85A, 85B, and ESAT-6 antigens. Clin Dev Immunol 2011; 2011: 351573. (10 pages).
Pathak SK, Basu S, Basu KK, Banerjee A, Pathak S, Bhattacharyya A, et al. Direct extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages. Nat Immunol 2007; 8(6): 610-8.
Boggaram V, Gottipati KR, Wang X, Samten B. Early secreted antigenic target of 6 kDa (ESAT-6) protein of Mycobacterium tuberculosis induces interleukin-8 (IL-8) expression in lung epithelial cells via protein kinase signaling and reactive oxygen species. J Biol Chem 2013; 288(35): 25500-11.
Yihao D, Hongyun H, Maodan T. Latency-associated protein Rv2660c of Mycobacterium tuberculosis augments expression of proinflammatory cytokines in human macrophages by interacting with TLR2. Infect Dis (Lond) 2015; 47(3): 168-77.
Jenum S, Tonby K, Rueegg CS, Rühwald M, Kristiansen MP, Bang P, et al. A Phase I/II randomized trial of H56:IC31 vaccination and adjunctive cyclooxygenase-2-inhibitor treatment in tuberculosis patients. Nat Commun 2021; 12(1): 6774. (13 pages).
Novagen. pET System Manual. 11th ed. [online]. 2005; [cited 2023 Apr 12]; [80 screens]. Available from: URL: https://kirschner.med.harvard.edu/files/protocols/Novagen_petsys-tem.pdf.
Merck. ANTI-FLAG® M2 affinity gel. [online]. 2021; [cited 2023 Mar 10]; [8 screens]. Available from: URL: https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/docu-ments/236/831/a2220bul-mk.pdf.
Novagen. Ni-NTA His•Bind® Resins. [online]. 2023; [cited 2023 Mar 10]; [32 screens]. Available from: URL: https://www.merckmillipore.com/TH/en/product/Ni-NTA-Buffer-Kit,EMD_BIO-70899#anchor_BRO.
Thermo Fisher Scientific. Pierce™ chromogenic endotoxin quant kit user guide. [online]. 2023; [cited 2023 Mar 10]; [6 screens]. Available from: URL: https://assets.thermofisher.com/TFSAssets/LSG/manuals/MAN0017902_ChromogenicEndotoxinQuantKit_UG.pdf.
Zar JH. Biostatistical analysis. London: Pearson Education Limited; 2013.
กรมควบคุมโรค กระทรวงสาธารณสุข. แผนปฏิบัติการระดับชาติ ด้านการต่อต้านวัณโรค ระยะที่ 2 (พ.ศ. 2566 - 2570). นนทบุรี: กองวัณโรค กรมควบคุมโรค กระทรวงสาธารณสุข; 2566.
Brito LA, Singh M. Acceptable levels of endotoxin in vaccine formulations during preclinical research. J Pharm Sci 2011; 100(1): 34-7.
Wang P, Wang L, Zhang W, Bai Y, Kang J, Hao Y, et al. Immunotherapeutic efficacy of recombinant Mycobacterium smegmatis expressing Ag85B-ESAT6 fusion protein against persistent tuberculosis infection in mice. Hum Vaccin Immunother 2014; 10(1): 150-8.
He H, Yang H, Deng Y. Mycobacterium tuberculosis dormancy-associated antigen of Rv2660c induces stronger immune response in latent Mycobacterium tuberculosis infection than that in active tuberculosis in a Chinese population. Eur J Clin Microbiol Infect Dis 2015; 34(6): 1103-9.
Thangamariappan E, Mohan M, Sundar K. Computational prediction of B cell epitopes of Mycobacterium tuberculosis – implications in vaccine design. Mediterr J Infect Microbes Antimicrob 2021; 10: 23. (6 pages).
Kong H, Dong C, Xiong S. A novel vaccine p846 encoding Rv3615c, Mtb10.4, and Rv2660c elicits robust immune response and leviates lung injury induced by Mycobacterium infection. Hum Vaccin Immunother 2014; 10(2): 378-90.
Ahmad F, Zubair S, Gupta P, Gupta UD, Patel R, Owais M. Evaluation of aggregated Ag85B antigen for its biophysical properties, immunogenicity, and vaccination potential in a murine model of tuberculosis infection. Front Immunol 2017; 8: 1608. (19 pages).
Dong H, Jing W, Yingru X, Wenyang W, Ru C, Shengfa N, et al. Enhanced anti-tuberculosis immunity by a TAT-Ag85B protein vaccine in a murine tuberculosis model. Pathog Glob Health 2015; 109(8): 363-8.
Zhou L, Luo L, Luo L, Luo H, Ding Y, Lu Z, et al. Ag85B-induced M1 macrophage polarization via the TLR4/TRAF6/NF-kappaB axis leading to bronchial epithelial cell damage and TH17/Treg imbalance. Curr Mol Med. [online]. 2025; [cited 2025 Feb 1]; [16 screens]. Available from: URL: https://www.eurekaselect.com/article/145790.
Launois P, Drowart A, Bourreau E, Couppie P, Farber CM, Van Vooren JP, et al. T cell reactivity against mycolyl transferase antigen 85 of M. tuberculosis in HIV-TB coinfected subjects and in AIDS patients suffering from tuberculosis and nontuberculous mycobacterial Infections. Clin Dev Immunol 2011; 2011: 640309. (10 pages).
Komine-Aizawa S, Jiang J, Mizuno S, Hayakawa S, Matsuo K, Boyd LF, et al. MHC-restricted Ag85B-specific CD8(+) T cells are enhanced by recombinant BCG prime and DNA boost immunization in mice. Eur J Immunol 2019; 49(9): 1399-414.
Passos BBS, Araujo-Pereira M, Vinhaes CL, Amaral EP, Andrade BB. The role of ESAT-6 in tuberculosis immunopathology. Front Immunol 2024; 15: 1383098. (11 pages).
Ning H, Zhang W, Kang J, Ding T, Liang X, Lu Y, et al. Subunit vaccine ESAT-6:c-di-AMP delivered by intranasal route elicits immune responses and protects against Mycobacterium tuberculosis infection. Front Cell Infect Microbiol 2021; 11: 647220. (14 pages).
Kooijman S, Vrieling H, Verhagen L, de Ridder J, de Haan A, van Riet E, et al. Aluminum hydroxide and aluminum phosphate adjuvants elicit a different innate immune response. J Pharm Sci 2022; 111(4): 982-90.
OZ Biosciences. AlumVax hydroxide. [online]. [cited 2023 Jul 10]. Available from: URL: https://ozbiosciences.com/aluminumgels/79-alumvax-hydroxide.html.
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2025 BULLETIN OF THE DEPARTMENT OF MEDICAL SCIENCES
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
