Abstract
Cutaneous leishmaniasisis a vector-borne disease transmitted by Leishmania infected sand flies. PpSP15 is an immunogenic salivary protein from the sand fly Phlebotomus papatasi. Immunization with PpSP15 was shown to protect against Leishmania major infection. Lactococcus lactis is a safe non-pathogenic delivery system that can be used to express antigens in situ. Here, the codon-optimized Ppsp15-egfp gene was cloned in pNZ8121 vector downstream of the PrtP signal peptide that is responsible for expression and secretion of the protein on the cell wall. Expression of PpSP15-EGFP recombinant protein was monitored by immunofluorescence, flow cytometry and Western blot. Also, expression of protein in cell wall compartment was verified using whole cell ELISA, Western blot and TEM microscopy. BALB/c mice were immunized three times with recombinant L. lactis-PpSP15-EGFPcwa, and the immune responses were followed up, at short-term (ST, 2 weeks) and long-term (LT, 6 months) periods. BALB/c mice were challenged with L. major plus P. papatasi Salivary Gland Homogenate. Evaluation of footpad thickness and parasite burden showed a delay in the development of the disease and significantly decreased parasite numbers in PpSP15 vaccinated animals as compared to control group. In addition, immunized mice showed Th1 type immune responses. Importantly, immunization with L. lactis-PpSP15-EGFPcwa stimulated the long-term memory in mice which lasted for at least 6 months.
Highlights
Leishmaniasis are high-prevalence parasitic diseases with a long history in the world [1]
Two desired genes were cloned downstream of the PnisA promoter and the PrtP signal peptide for protein attachment on the cell wall in pNZ8121 to produce two constructs Expression of PpSP15-EGFP (~42 kDa) and EGFP (~27 kDa) proteins after induction were confirmed through different methods, namely Western blotting (S1C Fig), fluorescent microscopy (S1D Fig) and flow cytometry (S1E Fig)
The specific bands for both expected proteins, namely EGFP and PpSP15-EGFP, clearly confirmed their expression (S1C Fig). Fluorescent microscopy showed both recombinant bacteria could express the EGFP at least 3 h after the induction (S1D Fig). This verification by flow cytometry at 3 h after the induction confirmed that the least intensity for L. lactisWT was 0.88%, and a range between 31.86% and 42.37% was obtained for L. lactisPpSP15-EGFPcwa and for L. lactis-EGFPcwa as a positive control, the range of intensity was 64.67–72.11% (S1E Fig shows results from one experiment)
Summary
Leishmaniasis are high-prevalence parasitic diseases with a long history in the world [1]. Theses group of diseases are exhibited in different forms including cutaneous (CL), mucocutaneous (MCL) and visceral (VL) leishmaniasis [2]. Metacyclic promastigotes are regurgitated by sand flies during the blood feeding process. During this process the sand fly delivers saliva at the infection site. Sand fly saliva contains bioactive proteins including anticoagulants, inhibitors of platelet aggregation and anti-complement molecules among other biological activities [5]. Some of these bioactive have immunomodulatory effects in the host [6, 7]. Immunization with P. papatasi PpSP15 was shown to be protective against L. major infection [13] by inducing a cellular immune response in a form of a delayed type hypersensitivity (DTH) response [14]
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