Fig 1.
Confirmation of cell wall protein expression using several methods.
(A) By Western blot analysis using different cellular compartments by anti-GFP Ab, expression of PpSP15-EGFP and EGFP proteins was shown in membrane and cell wall fraction of two recombinant bacteria, L. lactis-PpSP15-EGFPcwa and L. lactis-EGFPcwa. BI: Before induction, AI: After induction, M: Membrane, CW: Cell wall fraction and Cyto: Cytoplasmic fraction of bacteria. The rEGFP used as a positive control that showed a super band ~27 KDa. (B) By ELISA, measurement of expression of two cell wall PpSP15-EGFP and EGFP proteins in cell wall fractionation, and different status of bacteria using anti-GFP Ab. BI: before induction (as a negative control); intact live bacteria; heat-killed cell; and CW: cell wall. Cut off = mean (BI) + 2SD. The rEGFP used as a positive control and showed the highest absorbance. *Present statistical differences between different status of bacteria. (C) Using TEM, microscopic micrographs (magnification ×50,000) of the L. lactis-PpSP15-EGFPcwa (left) 3 h after induction and L. lactis wild-type as a negative control (right) clearly showed the cell wall expression of the PpSP15-EGFP on the surface of bacteria. The scale bar represents 100 nm. Two-headed arrows show the thickness on the cell wall of L. lactis-PpSP15-EGFPcwa.
Fig 2.
Footpad thickness and parasite burden measurment in BALB/c mice at 2 months after challenge.
Schematic figure shows the time schedule for immunization, challenge and analysis during the short- and long term study. Two weeks (ST) or 6 months (LT) after immunization, all groups of BALB/c mice were infected with L. major+SGH, and development of infectivity and effectivity of L. lactis-based vaccine was evaluated through footpad thickness and parasite burden measuring. Footpad thickness in both experiments, ST (A) and LT (B), was meseaured weekly during 8 weeks after challenge by digital caliper. Two months after challenge, LNs from 4–6 mice were isolated, homogenized and after serially culturing the dilutions into the 96-wells plates monitored through weekly microscopical observation to find last dilution containing at least one parasite (C). *Present the statistical differences between different mice groups. * = p<0.05, **** = p<0.0001, ns = non-significant.
Fig 3.
Determination of cytokine profile in both short-term (ST) and long-term (LT) experiments after infection with L. major+SGH.
Two months after challenge with L. major+SGH, the cellular immune response in BALB/c mice through determination of the level of generated cytokines was compared between different groups in both ST and LT experiments. Individual splenocytes were re-estimulated with L. major F/T as antigen, and supernatants collected and analyzed for production of different cytokines. The results are depicted individually for each cytokine (A (IFN-γ), B (IL-17), C (IL-5) and D (IL-10)) or ratio between two different cytokines (E (IFN-γ /IL-5), F (IFN-γ /IL-10), G (IL-17/IL-5) and H (IL-17/IL-10)). *Present the statistical differences between different mice groups. * = p<0.05, ** = p<0.001, ns = non-significant.