Design and construction of a nanovaccine consist of EsxV and HspX and Ppe44 fusion proteins encapsulated with Mannosylated PLGA nanospheres and evaluation of its immunogenicity after subcutaneous immunization in mice

Background: Among the various vaccine candidates, subunit vaccines play an important role in immune protection against tuberculosis (TB). Owing to its great potential to increase immune responses, Mannan decorated PLGA nanoparticles can be considered as a great strategy to improve antigen uptake by antigen presenting cells (APCs). The purpose of this study was to evaluate the specific immune responses following the administration of mannan decorated nanoparticles containing EsxV-Ppe44-HspX antigen and CpG Adjuvant. Methods: After optimizing of proteins expression, proteins were purified by Ni-NTA chromatography. Purification of proteins was confirmed by SDS-PAGE and Western-blot techniques. The different antigen loaded nanoparticles were prepared by W/O/W method. Mannan decorated nanoparticles were achieved by chemical method. The physiochemical properties of the nanoparticles, including morphology, particle size, and zeta potential, and encapsulation rate and antigen release profile were measured in vitro. The subcutaneous immunization with or whitout BCG was performed three times on days 0, 14 and 28. Two weeks after the last administration, concentration of IFN-γ, IL-4, IL-17and TGF-beta in supernatant of splenocytes, serum IgG2aweremeasured by ELISA. Results: The results of SDS-PAGE and Western-blot confirmed that proteins were specifically detected. The HspX-Ppe44-EsxV protein and CpG were encapsulated in PLGA and mannan decorated PLGA nanoparticles. The mean sizes of resultant nanoparticles contain antigen and CpG was 271.3 ± 28.5 and 327.2 ± 12.3 for PLGA and mannan decorated PLGA nanoparticle, respectively. The mannan decorated PLGA nanoparticles loaded with CpG and antigen had stronger ability to induce IFN-γ, IL-4, IL-17, TGF-beta, IgG2a, IgG1 levels. Conclusion: Mannan decorated nanoparticles created strong immune responses against M. tuberculosis antigens. Therefore, these nanoparticles could be considered as an excellent adjuvant/delivery system to design new vaccine candidate against TB infection.

Keywords:

PPE44 , EsxV , HspX , PLGA , Mannan , Vaccine , Tuberculosis