Design and Preliminary Characterization of Ag85B-ESAT-6-Rv2660c mRNA-Lipid Nanoparticles for TB Vaccine Development
Design and Characterization of Ag85B-ESAT-6-Rv2660c mRNA-LNPs
Keywords:
Tuberculosis, mRNA vaccine, Ag85B-ESAT-6-Rv2660c, Lipid nanoparticleAbstract
Tuberculosis (TB) remains a major global public health challenge, and Thailand is one of the High Burden Country Lists due to its incidence rate exceeding the global average. The only TB vaccine available, Bacillus Calmette–Guérin (BCG), effectively protects against severe TB in children but has limited efficacy in preventing pulmonary TB in adults. Therefore, developing new TB vaccines is crucial to achieving Thailand’s goal of TB elimination and sustainable disease control. This study focused on designing and characterizing modified mRNA constructs for a TB vaccine development using three key antigens: Ag85B, ESAT-6, and Rv2660c, which target different stages of Mycobacterium tuberculosis (MTB) infection. Five mRNA constructs encoding TB antigens were designed and synthesized in vitro, including three single-antigen constructs and two fusion proteins composed of all three antigens. Notably, the fusion mRNA constructs Ag85B-G4S-ESAT-6-G4S-Rv2660c mRNA and Ag85B-AAY-ESAT-6-AAY-Rv2660c mRNA exhibited greater secondary structure stability compared to single-antigen constructs. Encapsulation of both fusion mRNA constructs in lipid nanoparticles (mRNA-LNPs) demonstrated high encapsulation efficiency, excellent size uniformity (~140 nm), and reliable stability. Moreover, the delivery of mRNA-LNPs into HEK293 cells confirmed the successful expression of fusion proteins for both mRNA constructs, as indicated by TB antigen-specific antibody staining. Therefore, the modified Ag85B-ESAT-6-Rv2660c mRNAs exhibited suitable characteristics and significant potential for further development as the tuberculosis vaccine.
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