Leishmania (L.) species are protozoan parasites with a complex life cycle consisting of a number of developmental forms that alternate between the sand fly vector and their host. The non-pathogenic species L. tarentolae is not able to induce an active infection in a human host. It has been observed that, in pathogenic species, extracellular vesicles (EVs) could exacerbate the infection. However, so far, there is no report on the identification, isolation, and characterization of L. tarentolae EVs. In this study, we have isolated and characterized EVs from L. tarentolaeGFP+ (tEVs) along with L. majorGFP+ as a reference and positive control. The EVs secreted by these two species demonstrated similar particle size distribution (approximately 200 nm) in scanning electron microscopy and nanoparticle tracking analysis. Moreover, the said EVs showed similar protein content, and GFP and GP63 proteins were detected in both using dot blot analysis. Furthermore, we could detect Leishmania-derived GP63 protein in THP-1 cells treated with tEVs. Interestingly, we observed a significant increase in the production of IFN-γ, TNF-α, and IL-1β, while there were no significant differences in IL-6 levels in THP-1 cells treated with tEVs following an infection with L. major compared with another group of macrophages that were treated with L. major EVs prior to the infection. Another exciting observation of this study was a significant decrease in parasite load in tEV-treated Leishmania-infected macrophages. In addition, in comparison with another group of Leishmania-infected macrophages which was not exposed to any EVs, tEV managed to increase IFN-γ and decrease IL-6 and the parasite burden. In conclusion, we report for the first time that L. tarentolae can release EVs and provide evidence that tEVs are able to control the infection in human macrophages, making them a great potential platform for drug delivery, at least for parasitic infections.
Leishmaniasis is a neglected vector-borne disease caused by Leishmania parasites transmitted through the infected sand flies bite. Current treatments are limited, partly due to their high cost and significant adverse effects, and no human vaccine is yet available. Sand flies saliva has been examined for their potential application as an anti-Leishmania vaccine. The salivary protein, PpSP15, was the first protective vaccine candidate against L. major. Additionally, PsSP9 was already introduced as a highly immunogenic salivary protein against L. tropica. Herein, we aimed to develop an effective multivalent live vaccine to control Cutaneous Leishmaniasis induced by two main species, L. major and L. tropica. Hence, the two above-mentioned salivary proteins using T2A linker were incorporated inside the L. tarentolae genome as a safe live vector. Then, the immunogenicity and protective effects of recombinant L. tarentolae co-expressing PpSP15 and PsSP9 were evaluated in pre-treated BALB/c mice with CpG against L. major and L. tropica. Following the cytokine assays, parasite burden and antibody assessment at different time-points at pre and post-infection, promising protective Th1 immunity was obtained in vaccinated mice with recombinant L. tarentolae co-expressing PpSP15 and PsSP9. This is the first study demonstrating the potency of a safe live vaccine based on the combination of different salivary proteins against the infectious challenge with two different species of Leishmania.
DNA vaccines with their extraordinary properties are the best choice as vectors for subunit vaccines but are not in compliance with safety regulations, mainly because of the antibiotic resistance genes on their backbone. New generations of plasmids with minimum bacterial backbones are now developed as promising alternatives to pass the safety rules and be replaced for conventional plasmids. Here we have compared the nanoplasmid (with RNA-out selection system and professional HTLV-1 containing promoter) and the conventionally used pcDNA plasmid, as regards the transfection efficiency. The EGFP gene was cloned in both pcDNA-3.1+ and NTC9385R-MSC and transfected into COS-7 cells for expression evaluation by flow cytometry. Meanwhile, qPCR was used to analyze the EGFP mRNA copy numbers. It was concluded that the nanoplasmid, with its extraordinary properties, can be a tempting alternative to conventional pcDNA in equal or equimolar concentrations for vaccine design. These promising results can put DNA vaccines back into focus, especially regarding diseases controlled by robust cellular immune responses.
Background Leishmania parasites are deposited in the host through sand fly bites along with sand fly saliva. Therefore, salivary proteins are promising vaccine candidates for controlling leishmaniasis. Herein, two immunogenic salivary proteins, PpSP15 from Phlebotomus papatasi and PsSP9 from Phlebotomus sergenti, were selected as vaccine candidates to be delivered by live Leishmania tarentolae as vector. The stepwise in silico protocol advantaged in this study for multi-protein design in L. tarentolae is then described in detail. Methods All possible combinations of two salivary proteins, PpSP15 and PsSP9, with or without T2A peptide were designed at the mRNA and protein levels. Then, the best combination for the vaccine candidate was selected based on mRNA and protein stability along with peptide analysis. Results At the mRNA level, the most favored secondary structure was PpSP15-T2A-PsSP9. At the protein level, the refined three-dimensional models of all combinations were structurally valid; however, local quality estimation showed that the PpSp15-T2A-PsSP9 fusion had higher stability for each amino acid position, with low root-mean-square deviation (RMSD), compared with the original proteins. In silico evaluation confirmed the PpSP15-T2A-PsSP9 combination as a good Th1-polarizing candidate in terms of high IFN-γ production and low IL-10/TGF-β ratio in response to three consecutive immunizations. Potential protein expression was then confirmed by Western blotting. Conclusions The approach presented herein is among the first studies to have privileged protein homology modeling along with mRNA analysis for logical live vaccine design-coding multi-proteins.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.