Cloning and expression of EsxV, HspX, and PPE44 tri-fusion proteins as novel antigens for immunization against Mycobacterium tuberculosis and its evaluation as a candidate recombinant antigen for vaccination with adjuvant in the mouse

Abstract

Polymeric nanoparticles have recently been shown to possess significant potential as vaccine delivery systems. In particular, the use of biodegradable polymeric nanoparticles with entrapped antigens represents an exciting approach for controlling the release of vaccine antigens and optimizing the desired immune response via selective targeting of the antigen to antigen presenting cells (APCs).The objective of the present study was to prepare chitosan nanoparticles with resiquimod adjuvant and EsxV، HspX ، PPE44 proteins of Mycobacterium tuberculosis and evaluation of induced protective immunity after intranasal and subcutaneous immunization in mice. Method: After optimizing of proteins expression, proteins were purified by Ni-NTA chromatography. Purification of proteins was confirmed by SDS-PAGE and Western-blot techniques. HPE and resiquimod adjuvant were then encapsulated in chitosan nanoparticles. The physiochemical properties of the nanoparticles, including morphology, particle size, zeta potential, encapsulation rate and antigen release profile were measured in vitro. The subcutaneous and intranasal immunization with or without 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 IgG2a were measured by an indirect enzyme linked immunosorbent assay (ELISA). Results: The results of SDS-PAGE and Western-blot confirmed that proteins were specifically detected. The HspX-Ppe44-EsxV protein and resiquimod were encapsulated in chitosan nanoparticles. The mean sizes of resultant nanoparticles contain antigen and resiquimod was 130 ±12 nm. The nanoparticles had a strong immunostimulatory effects on spelenocyte stimulated culture levels of IFN-γ, IL-17, IL-4 and TGF-β using intranasal and subcutaneous administration in parallel with boosting effects in mice. The highest IFN-γ, IL-17 and IgG2a concentration was detected in BCG-primed mice that were boosted with HPERC. No significant difference in concentration of IL-4 was observed between groups receiving HPERC and BCG-primed and HPERC-boosted group in comparison to group BCG.