RESUMO
Introduction: Induction of a protective immune response against Leishmania major requires the activation of both TH1 and CD8+ T lymphocytes. Because L. major is an intra-phagosomal parasite, its antigens do not have access to MHC-I. The present study aimed to evaluate the effect of cysteine peptidase A (CPA)/cysteine peptidase B (CPB) conjugated to α-AL2O3 on autophagy induction in L. major infected macrophages and subsequent activation of cytotoxic CD8+ T lymphocytes. Methods: Recombinant CPA and CPB of L. major were produced in expression vectors and purified. Aldehyde functionalized α-AL2O3 were conjugated to hydrazine-modified CPA/CPB by a chemical bond was confirmed by Fourier-transform infrared spectroscopy (FTIR). The High efficient internalization of α-AL2O3 conjugated CPA/CPB to macrophages was confirmed using a fluorescence microscope and flowcytometry. Induction of the acidic autophagosome and LC3 conversion in macrophages was determined by acridine orange (AO) staining and western blot. Autophagy-activated macrophages were used for CD8+ T cell priming. Cytotoxic activity of the primed CD8+ T cell against L. major infected macrophages was measured using apoptosis assay. Results: α-AL2O3 conjugated CPA/CPB enhances macrophages antigen uptake and increases acidic vacuole formation and LC-3I to LC-3II conversion. Co-culture of autophagy-activated macrophages with CD8+ T cells augmented CD8+ T cells priming and proliferation more than in other study groups. These primed CD8+ T cells induce significant apoptotic death of L. major infected macrophages compared with non-primed CD8+ T cells. Conclusion: α-AL2O3 nanoparticles enhance the cross-presentation of L. major antigens to CD8+ T cells by inducing autophagy. This finding supports the positive role of autophagy and encourages the use of α-AL2O3 in vaccine design.
RESUMO
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. α-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 α-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 α-alumina nanoparticles. CONCLUSION: α-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 CD8 lymphocytes.