ABSTRACT
Background: autophagy as a cellular pathway facilitates several immune responses against infection. It also eliminates invading pathogens through transferring content between the cytosol and the lysosomal vesicles and contributes to the cross-presentation of exogenous antigens to T lymphocytes via MHC class I pathway. Autophagy induction is one of the main targets for new drugs and future vaccine formulations. Nanoparticles are one of the candidates for autophagy induction. Cysteine Peptidase A [CPA] and Cysteine Peptidase B [CPB] are two members of papain family [Clan CA, family C1] enzyme that have been considered as a virulence factor of Leishmania [L.] major, making them suitable vaccine candidates. In this research, Leishmania major cysteine peptidase A and B [CPA and CPB] conjugation to alpha alumina nanoparticle was the main focus and their entrance efficacy to macrophages was assessed
Methods: for this purpose, CPA and CPB genes were cloned in expression vectors. Related proteins were extracted from transformed Escherichia coli [E. coli] and purified using Ni affinity column. Alpha alumina nanoparticles were conjugated to CPA/CPB proteins using Aldehyde/Hydrazine Reaction. Autophagy induction in macrophages was assessed using acridine orange staining
Results: CPA/CPB protein loading to nanoparticles was confirmed by Fourier Transform Infrared Spectroscopy. alpha-alumina conjugated CPA/CPB antigen uptake by macrophages at different concentrations was confirmed using fluorescence microscope and flowcytometry. Highly efficient CPA/CPB protein loading to alpha-alumina nanoparticles and rapid internalization to macrophages introduced these nanocarriers as a delivery tool. Acridine orange staining demonstrated higher autophagy induction in CPA/CPB protein conjugated with alpha-alumina nanoparticles
Conclusion: alpha-alumina nanoparticles may be a promising adjuvant in the development of therapeutic leishmania vaccines through antigen delivery to intracellular compartments, induction of autophagy and cross presentation to CD[8] lymphocytes