Lactococcus lactis expressing salivary protein SP۱۵ of Phlebotomus papatasi as a live vaccine for cutaneous leishmaniasis

Background and Aim : Cutaneous leishmaniasis is a debilitating parasitic disease without effective vaccine. The parasite is transmitted by sand fly blood feeding. Recently, the well-defined role of sand fly proteins has brought them into focus as vaccine candidates. The goal of this study was to evaluate the protective efficacy of a live-vectored vaccine (expressing an immunogenic sand fly protein) against L. major challenge with sand fly saliva homogenate.Methods : Two synthetic optimized sp۱۵-egfp and egfp genes were cloned in pNZ۸۱۲۱ and transferred into E. coli strain MC۱۰۶۱. Both genes were cloned at downstream of PrtP signal peptide to secret proteins on the cell wall. Then, each recombinant plasmids were episomally transferred into L. lactis subsp. Cremoris strain NZ۹۰۰۰ and generating aspNZ۸۱۲۱-sp۱۵-egfp and pNZ۸۱۲۱-egfp recombinants. Confirmation of protein expression on the cell wall was completed by TEM, western blotting and ELISA using anti-GFP antibody. After two times immunization, we assessed the protective effects in BALB/c mice by measuring footpad swelling, parasite burden, nitric oxide, humoral and cellular immune response at one and two months post challenge with L. major plus salivary gland homogenate.Results : Immunization with L. lactis-SP۱۵-EGFPcwa (vaccinated group) caused a significant delay on development of footpad swelling and reduction of lymph node parasite burden in vaccinated group in comparison with control groups (L. lactis and PBS groups). In addition, these results showed that IgG۲a/IgG۱ ratio and nitric oxide production in immunized group were higher than control groups (L. lactis- EGFPcwa, P< ۰.۰۵). After stimulation of lymphocytes with L. major antigens, nitric oxide, IFN-γ and IL-۱۷ production significantly were highest in vaccinated group comparing with control groups in both time points (P< ۰.۰۵). Furthermore, IL-۱۰ and IL-۵ were equal or significantly less than control groups.Conclusion : In our study, the lower level of parasite burden and higher IFN-γ /IL-۵, IFN-γ /IL-۱۰ and also in IL-۱۷/IL-۵ and IL-۱۷/IL-۱۰ ratios in immunized BALB/c mice with L. lactis-PpSP۱۵-EGFPcwa indicated that membrane associated PpSP۱۵ on the L. lactis can induce strong Th۱-immune responses and could act as live-vectored vaccine for human studies

Keywords:

Vaccination , L. major , L. lactis , P. papatasi , SP۱۵ , cutaneous leishmaniasis