In this study, female BALB/c mice (6–8 weeks old) were obtained from the Pasture Institute of Iran and kept under standard conditions of the animal facility in Pasture Institute of Iran (a constant 12h light-dark cycles, relative humidity 50–60%, and free access to normal rodent chow diet). All animal experiments were performed according to the animal care guideline approved by Institutional Animal Care and Research Advisory Committee of Pasteur Institute of Iran (ethical code: IR.RII.REC.1394.0201.6417, dated 2015) in full compliance with the Specific National Ethical Guidelines for Biomedical Research (MOHME-2005).

All needed solutions were prepared by pyrogenic deionized water (Milli-Q System, Millipore, France). Sodium dodecyl sulfate (SDS), Ficoll-400, Tris-base, Tris-HCL, and cell culture reagents including and Schneider′s Insect Medium, DMEM medium, HEPES, L-glutamine, adenosine, hemin were purchased from Sigma (Germany). Fetal Calf Sera (FCS), G418 were provided from Gibco (Life Technologies GmbH, Germany). Bovine Serum Albumin (BSA) and Diaminobenzidine (DAB) powder were supplied by Merck (Germany). All materials for gel electrophoresis, PCR, and enzymatic digestion were prepared by Roche Applied Sciences (Germany). Specific primers for target genes amplification were synthesized by metabion (Germany). Cytokine kits (IFN-γ, IL-4, IL-10 and IL-17) were purchased from DuoSet R & D (USA). Quanti Nova SYBR Green Master Mix, Anti-His Tag Antibody, RNeasy kit, QuantiTect Reverse Transcription Kit, and GF-1 Tissue DNA Extraction Kit were purchased from QIAGEN (Germany) and Vivantis (Vivantis Technologies, Malaysia), respectively. Pierce™ BCA^® (Bicinchoninic Acid) protein assay kit was provided by Thermo Fisher Scientific (USA). Horseradish peroxidase-conjugated goat anti-mouse IgG1 and IgG2 were provided by Southern Biotech (Canada). Peroxidase Substrate for ELISA systems was obtained from KPL (ABTS, USA). CpG oligodeoxynucleotide (ODN), known as CpG ODN-1826 (6326.7 g/mol, MW), with the sequence 5′-TCCATGACGTTCCTGACGTT-3′, was prepared from Microsynth (Balgach, Switzerland). pLEXSY-neo2 vector was obtained from Jena bioscience (Germany).

The PpSP15-T2A-PsSP9 construct was synthesized by Thermo Fisher Scientific Company (USA) and delivered in the pMA-RQ plasmid (PpSP15-T2A-PsSP9 sequence arrangement was selected among different combinations by bioinformatic analysis based on RNA secondary structure and protein stability but data is not shown here). Following sequence confirmation, the PpSP15-T2A-PsSP9 fragment was digested out by BglII and NheI restriction enzymes and sub-cloned into pLEXSY-neo2 vector (typically used as an integrative vector for stable genome transfection in L. tarentolae) pre-digested by the same enzymes. According to preset transfection protocols (28), 4×10⁷ log-phase L. tarentolae (Tar II ATCC 30267) parasites were re-suspended in 300 µl of ice-cold electroporation buffer (0.7mM Na₂HPO₄, 137mM NaCl, 5mM KCl, 21mM HEPES, 6mM glucose; pH 7.5) and mixed with 10 µg of linearized pLEXSY- PpSP15-T2A-PsSP9 by SwaI enzyme (5989 bp). After 10 min of incubation on ice, cells were electroporated at 450 V and 500 mF (Bio-Rad Gene Pulser Ecell device (Germany)). The electroporated parasites were cultured in supplemented antibiotic-free M199 (5% FBS) medium at 26°C for 24 hr. Antibiotic-resistant parasites were then selected on a semi-solid medium containing M199, 50% Noble agar, and 50 µg/ml G418.

Stable recombinant L. tarentolae parasites co-expressing PpSP15 and PsSP9 proteins were further confirmed at the DNA, RNA, and protein levels by diagnostic PCR, reverse-transcription PCR (RT-PCR), and Western blot, respectively. Diagnostic PCR was performed using the transfectants’ genomic DNA and 5’SSU specific forward (F3001 5’-GATCTGGTTGATTCTGCCAGTAG-3’) and reverse (A1715 5-TATTCGTTGTCAGATGGCGCAC-3) primers to generate a 1-kb fragment. F3001 primer targets upstream of the 5’SSU region on L. tarentolae genome and A1715 primer binds to the pLEXSY backbone(downstream of 5'SSU region and upstream of PpSP15-T2A-PsSP9 construct). For RT-PCR, total RNA from 2×10⁸ recombinant parasites was extracted using RNeasy kit and cDNA was synthesized according to the QuantiTect Reverse Transcription Kit instructions. Targets were separately amplified using specific primers for a part of each individual gene (PpSP15- forward: 5’-ACGCCTCCAAAATGGCTT-3’ and reverse: 5’-TTGACCTTTTTGGCGCACTC-3, PsSP9- forward: 5’- CATTGTGAATACCGTGCTTACG-3’ and reverse 5’-GAGTTTGTTCGAGTGCTTGG-3’). At the protein level, the expression of PpSP15 and PsSP9 by recombinant L. tarentolae was confirmed by Western blot analysis. Briefly, the concentrated supernatant of recombinant L. tarentolae at stationary phase was mixed with SDS-PAGE sample buffer (4.5mMTris–HCl, pH 6.8, 2%, w/v SDS, 5%, v/v 2-mercaptoethanol, 10%, v/v glycerol, 0.05%, w/v bromophenol blue) and run on 17.5% SDS-PAGE polyacrylamide gel. In the next step, proteins were transferred to the nitrocellulose membrane and free binding sites were blocked by incubation of the membrane with a blocking solution (TBS with 2.5% BSA and 0.05% Tween 20) overnight. Then, the primary Anti-His Tag Antibody (1/2000) was added to the membrane at room temperature. After 2 hr, HRP-conjugated goat anti-mouse IgG (1/7000) as the secondary antibody was used. Following incubation for 2 hr and three washes, Diaminobenzidine tetrahydrochloride (DAB) was applied as the substrate for visualization.

BALB/c mice already infected with L. major (MRHO/IR/75/ER) and L. tropica (MOHM/IR/09/Khamesipour-Mashhad) were sacrificed for parasite isolation. Isolated lymph nodes were then incubated in Schneider medium supplemented with 10% FCS and 100μg/ml of Gentamicin at 26°C for Leishmania promastigote propagation. The infective-stage metacyclic parasites at stationary-phase were separated on Ficoll 400 gradient and were collected for the subsequent infectious challenge. For in vitro stimulation assays, the stationary-phase promastigotes of L. tarentolae PpSP15-T2A-PsSP9, L. major, and L. tropica parasites were repeatedly freeze-thawed to prepare F/T antigen. Briefly, promastigotes were collected from the cultures and washed with PBS (8 mM Na₂HPO₄, 1.75 mM KH₂PO₄, 0.25 mMKCl, and 137 mM NaCl). 2×10⁸ parasites/ml were then embedded in liquid nitrogen for a few seconds and immediately thawed in 37°C water-bath. This process was repeated until no intact parasite was discriminated under the light microscope. The protein concentration was then quantified by BCA method (29).

Immunization was carried out for three female BALB/c mice groups (n= 28 per group) three times in 3-weeks intervals (Figure S1). Initially, all groups received CpG ODNs (45μg/mice) as an adjuvant intraperitoneally. After 3hr, groups were immunized with 2×10⁷ L. tarentolae PpSP15-T2A-PsSP9 (G1), 2×10⁷ wild-type L. tarentolae (G2) or PBS (G3) accordingly (Table 1). All mice were immunized subcutaneously (s.c.) at the left hind footpad in a total volume of 50 μl. Three weeks after the last immunization, each group was divided into two separate boxes for the infectious challenge either by L. major or L. tropica metacyclic promastigotes along with pertinent salivary gland homogenate (SGH, 0.5 pair/mouse) derived from Ph. papatasi and Ph. sergenti respectively (provided by Dr. Shaden Kamhawi and Dr. Jesus G. Valenzuela, NIH, USA). Immunized BALB/c mice were challenged s.c with either 2×10⁵ L. major plus Ph. papatasi SGH or 10⁷ L. tropica plus Ph. sergenti SGH at the right hind footpad (Table 1).

Following L. major infection, footpad thickness was weekly monitored with a digital caliper from week two post-challenge until week eleven for 10 weeks. For this, the mice were restricted and the thickness was measured as the diameter of the swelling site by fixing the caliper on the back and forth sides of the footpad. The uninoculated collateral footpad was simultaneously monitored as control.

Parasite load in popliteal lymph nodes (pLNs) was estimated at 7^th and 11^th week post challenge (7^thWAC and 11^thWAC) by quantitative Real-Time PCR. Four mice per group were sacrificed at each time point. Genomic DNA (gDNA) from homogenized lymph nodes was extracted using GF-1 Tissue DNA Extraction Kit according to the manufacturer’s instruction. DNA purity and concentration were assessed by Nanodrop (ND-1000). According to preset protocols (19, 22), two specific primer sets were used to target a part of kinetoplastid minicircle regions of parasites (L. tropica: KDNAF, 5’-GGGTAGGGGCGTTCTGC-3’ and KDNAR, 5’-TACACCAACCCCCAGTTTGC-3, L. major: RV1F, 5’-CTTTTCTGGTCCCGCGGGTAGG-3’and RV2R, 5’-CCACCTGGCCTATTTTACACCA-3’). The absolute copy number of parasites was measured using the StepOnePlus Real-Time PCR device and software (Applied Biosystems). In parallel, mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a reference gene was quantified with GAPDHF, 5’-CGTCCCGTAGACAAAATGGT-3’ forward and GAPDHR, 5’-TTGATGGCAACAATCTTCAC-3’ reverse primers to normalize the parasite load (30). Parasite load was then determined using the standard curve generated by 10 fold dilutions of gDNA extracted from each parasite in a range of 10⁸−10¹ parasite per reaction (extracted gDNA from L. major and L. tropica was used to measure the load of L. major and L. tropica, respectively). The amount of used gDNA per each PCR reaction was 20 and 80 nanograms for L. major and L. tropica, respectively. Likewise, the standard curve for the reference gene was obtained using serial dilutions of naive mouse gDNA (isolated from LN) in the range of 2.7 ×10⁴ to 1 ×10² DNA copies per reaction. The total volume of each PCR reaction containing 5 μl of 2x Quanti Nova SYBR Green Master Mix, 500 nM of each forward and reverse primers, and 1 μl carboxyrhodamine or ROX (passive dye) was 10 μL. The PCR amplification was set on the following program: 95°C for 20 s, 40 cycles consisting of 95°C for 3s and 60°C for 30 s. PCR reactions were all in duplicate and the efficiencies of target and reference genes were 100%. The normalized parasite load was calculated using the formula: [(parasite numbers)/(GAPDH copy numbers/2)] and plotted as the number of Leishmania parasites per cell.

Cytokine production by vaccinated and control groups was measured at pre-and two time points post-challenge (7^thWAC and 11^thWAC). To this end, three or four mice per group were sacrificed at each time point. Then, the isolated spleens were homogenized after erythrocyte lysis by ACK lyses buffer (0.15MNH₄Cl, 1mM KHCO₃, and 0.1 mM Na₂EDTA). Splenocytes were washed and then re-suspended in complete phenol red-free DMEM medium containing 10% FCS, 1% L-glutamine, 1% HEPES, 0.1% 2ME, and 0.1% gentamicin. 3.5×10⁶ cells were plated in 48 well plates and were re-stimulated by different antigens including L. tarentolae PpSP15-T2A-PsSP9 F/T (20 μg/ml) for pre-challenge, L. major F/T (20 μg/ml), and L. tropica F/T (20 μg/ml) for post-challenge. In all experiments, Concanavalin A (ConA, 5 μg/ml) and culture media were used as positive and negative controls, respectively. The incubation time (37°C in a humidified atmosphere with 5% CO₂) was 72 hr for IL-4 and IL-17, but 120 hr for the other two cytokines, IFN-γ and IL-10. The cytokines concentrations in the cells culture supernatants were measured by sandwich ELISA kits according to the manufacturer’s protocols. The minimum limit of detection for IFN-γ, IL-4, IL-10, and IL-17 was 2, 3, 4, and 5 pg/ml, respectively.

The Nitric oxide (NO) production was also measured at 7^th and 11^th weeks after the infectious challenge. Following re-stimulation of mice splenocytes with the specific antigen including L. major F/T (20 μg/ml) or L. tropica F/T (20 μg/ml) for 120h, Nitrite concentration was measured in the cells supernatant by Griess reagent containing naphthyl ethylenediamine (NED) dihydrochloride (0.1 N) and 1% sulfanilamide in 5%H₃PO₄ mixed together in equal volumes before use. For Nitrite measurement, 100 μL of each sample was mixed with an equal amount of Griess reagent and incubated for 10 min at RT. The absorbance was determined at 540 nm. Different concentrations (0–300μM) of sodium nitrite (in phenol red-free DMEM) as standards were applied to plot the standard curve (31).

Before and 11 weeks after the infectious challenge, BALB/c mice were bled from retro-orbital sinus to determine antibody responses. The mice sera were analyzed using ELISA for specific IgG1 and IgG2a subclasses levels against F/T lysates of L. tarentolae PpSP15-T2A-PsSP9, L. major, and L. tropica (28). In brief, 96-well ELISA plates were coated with 10μg/mL of the above-mentioned antigens in bicarbonate buffer (Na₂CO₃ 0.02 M, NaHCO₃ 0.45 M, pH 9.6) overnight at 4°C. Following three washes (PBS + 0.05% Tween 20), wells were blocked with 100 μl of 1% BSA in PBS for 2hr at RT to avoid nonspecific bindings. Then, 100 μl of diluted sera (1/150) in PBS containing 0.05% Tween 20 and 1% BSA were added and micro-plates were incubated for 2 hr at 37°C. Next, 100 μL diluted peroxidase-conjugated goat anti-mouse IgG1 (1:5000) and IgG2a (1:3000) antibodies were added to each pertinent well. Following incubation (2 hr-37°C) and washing, peroxidase substrate KPL ABTS (100 μL) was used for dye development in each well. The enzymatic reactions were stopped by 1% SDS and the absorbance was measured at 405 nm along with reference (630 nm).

GraphPad Prism 7.0 (GraphPad, CA, USA) was utilized for statistical analyses. Based on Shapiro-Wilk test and data passing normality, Student’s t-test was used for data analysis. P-values below 0.05 (p < 0.05) were considered statistically significant.

Recombinant L. tarentolae stably expressing two different sand fly salivary proteins (PpSP15 and PsSP9) was generated by introducing the linearized pLEXSY-PpSP15-T2A-PsSP9 (5989 bp) into the 18S rRNA locus of L. tarentolae genome. It is worth mentioning that the T2A peptide is a self-cleavage linker which can lead to producing its upstream and downstream proteins separately. The integration of the expression cassette in transgenic L. tarentolae was initially confirmed at the DNA level by amplification of a 1-kb fragment (Figure 1A). Moreover, the expression of the salivary proteins was confirmed by both RT-PCR and Western blot. Each constituent gene of the PpSP15-T2A-PsSP9 cassette was amplified after cDNA preparation by specific primers (Figure 1B). At the protein level, two separate immunoreactive bands of approximately 15 kDa (PsSP9) and 18 kDa (PpSP15-T2A) in size were detected in Western blot using an anti-His-tag antibody (Figure 1C). Furthermore, non-cleaved 36 kDa protein was not detected; an indicator of full dissociation of the fusion into constituent proteins owing to T2A related self-cleavage. Of note, in this study T2A peptide was applied for the first time in L. tarentolae, which, despite its viral origin, was able to well function.

After the last immunization and prior to infectious challenge with L. major and L. tropica, all mice groups were subjected to cellular and humoral immune response evaluation. Four effector cytokines including IFN-γ, IL-4, IL-10, and IL-17 were measured in the culture supernatant of mice splenocytes stimulated with L. tarentolae PpSP15-T2A-PsSP9 F/T antigen. It is well established that the higher level of IFN-γ (as the Th1 main cytokine) relative to IL-4 (as the Th2 main cytokine), IL-10 (as the anti-inflammatory cytokine), and IL-17 (as a neutrophil chemotactic cytokine) is the key factor to control the Leishmania infection. Therefore these cytokines were further evaluated in each differently vaccinated group. As shown in Figure 2A, higher levels of IFN-γ were detected in L. tarentolae PpSP15-T2A-PsSp9 vaccinated group (G1). Meanwhile, IL-4 was detected at a low amount in all three groups with no significant difference (Figure 2B). Given the importance of the IFN-γ/IL-4 ratio as Th1 response deviation indicator, the highest level was detected in G1 vaccinated group (p= 0.0017) (Figure 2C). On the other hand, the production of IL-10 as an indicator of immunoregulatory cytokine revealed no significant difference between groups (Figure 2D). Therefore, G1 indicated the highest level of IFN-γ/IL-10 ratio compared to G2 (p= 0.1721) and G3 (p= 0.0125) groups (Figure 2E). Furthermore, IL-17 production (Figure 2F) was remarkably induced in the vaccinated group (G1) however the ratio of IFN-γ/IL-17 in G1 group was non-significantly higher than G2 group (Figure 2G). This might indicate that IL -17 production is mainly controlled by CpG adjuvant rather than the SP15 or SP9 antigens (the IFN-γ/IL-17 ratio in PBS group was calculated high since both cytokines were produced in low amounts).

The humoral immune responses through the measurement of IgG subclasses were also determined for L. tarentolae PpSP15-T2A-PsSP9 vaccinated group (G1) and L. tarentolae wild-type control group (G2) in response to L. tarentolae PpSP15-T2A-PsSp9 F/T antigen. As known, the production of IgG2a is associated with Th1 immune responses in mice while IgG1 production is related to the Th2 profile. As indicated in Figure 3, the higher level of IgG2a and the lower level of IgG1 were significantly observed in the L. tarentolae PpSP15-T2A-PsSP9 vaccinated group (G1) compared to the control group (G2). Collectively, the above findings demonstrated a Th1-skewed response in vaccinated mice by L. tarentolae PpSP15-T2A-PsSP9 compared to the control groups (G2 and G3) relying on remarkable IFN-γ and IL-17 production versus negligible IL-4 and IL-10 in G1 vaccinated group. These results further indicated that sand fly protein-expressing L. tarentolae adjuvanted with CpG can manipulate the immune system in favor of Th1 response which is obviously the prerequisite of Leishmania infection control after infectious challenge.

Three weeks following the last immunization, half of the BALB/c mice in each G1, G2, and G3 groups were challenged by L. major metacyclic promastigotes along with SGH of Ph. papatasi and denoted as G1Lm, G2Lm, and G3Lm (Table 1). Typically, the vaccine efficacy against L. major infection is evaluated by footpad thickness monitoring and parasite burden assessment. As indicated in (Figure 4A) weekly measurement of footpad thicknesses up to week 11 displayed a non-significant control on the thickness increase in mice immunized by L. tarentolae PpSP15-T2A-PsSP9 (G1Lm) compared to control groups (G2Lm and G3Lm). Meanwhile, quantitative RT-PCR at week 7 post-challenge revealed a significantly lower level of parasite number per lymph node cell in G1Lm group (mean ± SE= 11.5 ± 1.6) compared to G2Lm (mean ± SE= 129.9 ± 26.7) and G3Lm (mean ± SE= 662.4 ± 121.9) groups (Figure 4B). Later quantification at week 11 however indicated no significant difference between groups although the lowest parasite number per lymph node cell was detected in G1Lm (mean ± SE= 5.9×10⁴ ± 1.7×10⁴) versus G2Lm (mean ± SE= 28×10⁴ ± 1.5×10⁴) and G3Lm (mean ± SE= 13×10⁴ ± 5.1×10⁴). In other words, L. tarentolae PpSP15-T2A-PsSP9 plus CpG was able to reduce the parasite load in G1Lm by nearly 80% and 55% compared to G2Lm and G3Lm, respectively at later weeks (Figure 4B).

In order to determine the potential of L. tarentolae co-expressing PpSP15 and PsSP9 adjuvanted with CpG in providing protection against L. major infection and to characterize the type of induced immune responses, cytokines including IFN-γ, IL-4, IL-10, and IL-17 were also assessed at weeks 7 and 11 post-challenge following in vitro stimulation with parasite F/T. As shown in Figure 5A, the level of IFN-γ was detected significantly higher in the L. tarentolae PpSP15-T2A-PsSp9 vaccinated group (G1Lm) compared to both control groups at 7^th WAC. At the same time point, IL-4 production was almost similar in all three groups and respecting the production of IL-10, no significant differences were obtained between different mice groups (Figures 5B, D). Therefore in vitro stimulated splenocytes from L. tarentolae PpSP15-T2A-PsSP9 vaccinated mice (G1Lm), elicited significantly higher ratios of IFN-γ/IL-4 (Figure 5C) and IFN-γ/IL-10 (Figure 5E) in comparison with control groups (G2Lm and G3Lm). Besides, the production of IL-17 was substantially higher in the G1Lm group than in G3Lm while it was comparable with G2Lm (Figure 5F). As a result, the highest ratio of IFN-γ/IL-17 was determined for L. tarentolae PpSP15-T2A-PsSP9 vaccinated mice group (G1Lm) compared to G2Lm and G3Lm control groups (Figure 5G). This early-time cytokine profile well matched the same pre-challenge cytokine profile. L. major infection was then well tolerated by an increase in IFN-γ and decrease in IL-17, IL-10, and IL-4 production in G1Lm group. Interestingly, this cytokine profile at 7^th WAC explained the parasite load at the same time point.

Four weeks later at 11^th WAC, the amount of IFN-γ in the vaccinated group (G1Lm) dropped to lower levels but it was still higher than G2Lm (p= 0.1101) and G3Lm groups (p= 0.5673) (Figure 5A). Of note, no increase was observed at the level of IL-4 (Figure 5B) and IL-10 (Figure 5D) in G1Lm (significantly lower levels of the mentioned cytokines were detected). A remarkable decline in the level of IL-17 was also observed compared to the earlier time point (7^th WAC) in G1Lm and G2Lm groups (Figure 5F), but instead IL-17 substantially increased in G3Lm. Therefore, the highest ratios of IFN-γ/IL-4 (Figure 5C), IFN-γ/IL-17 (Figure 5G), and IFN-γ/IL-10 (Figure 5E) were detected in G1Lm group. This cytokine profile explained the parasite load of week 11 post-challenge with a lower but not statistically significant parasite number per cell in the G1Lm group. These results indicated that the severe pathogenicity of L. major infection in BALB/c mice cannot be tolerated at late time points post-infection relying on the strength of the vaccine formulation used in this study.

IFN-γ production is functionally correlated with macrophage induction of Nitric oxide (NO), a way to control parasite proliferation. Therefore, NO measurement is a functional estimation of the IFN-γ production. The amount of NO in response to L. major F/T antigen was measured for all mice groups at 7^th and 11^th WAC. The findings indicated that the highest level of IFN-γ in G1Lm group well matched the highest level of NO in this group at both measurement times (Figure 5H). Therefore the NO profile was quite in line with IFN-γ production.

Likewise, the antibody responses against L. major F/T antigen at 11 weeks post-challenge showed the highest level of IgG2a in the vaccinated group (G1Lm) and comparable amount of IgG1 in all mice groups. Therefore as shown in Figure 6, the ratio of IgG2a to IgG1 was comparatively higher in G1Lm than in G2Lm (p= 0.1675), and G3Lm (p= 0.0331) groups.

Collectively, these findings demonstrated a Th1 polarized response detectable up to week 7 post-infectious challenge which slightly faded at later weeks. This was illustrated in the drop down of IFN-γ/IL-4 and IFN-γ/IL-17 ratios but not in the IFN-γ/IL-10 ratio. This high amount of IL-10 could account for a suppression in Th1 potential to control L. major infection in BALB/c mice which was well reflected on the non-significant lower parasite load of L. tarentolae PpSP15-T2A-PsSP9 vaccinated group at the late weeks post-challenge.

The other half of immunized BALB/c mice in all three groups were challenged by L. tropica metacyclic promastigotes along with SGH of Ph. sergenti. The potency of L. tarentolae PpSP15-T2A-PsSP9 plus CpG as a new live vaccine candidate to control L. tropica infection was estimated by parasite burden measurement (in this model the footpad monitoring is not measurable due to a mild footpad inflammation). As shown in Figure 7, at week 7 post-infection, there was no obvious difference in the number of parasites per lymph node cell between G1Lt (mean ± SE= 8.9 ± 2.9) and G2Lt (mean ± SE= 6.1 ± 1.1) groups. However, the propagation of L. tropica in the vaccinated group (G1Lt) versus PBS group (G3Lt) (mean ± SE= 21.1 ± 3) was significantly lower. Further evaluation of disease progression up to the week 11 after infectious challenge, revealed a significant control in the number of parasites per cell for G1Lt (mean ± SE= 130 ± 14) compared to G2Lt (mean ± SE= 274 ± 55) and G3Lt (mean ± SE= 671 ± 163) groups.

Cytokine evaluation at week 7 post-challenge indicated a remarkable increase in IFN-γ production versus pre-challenge which was comparable in G1Lt and G2Lt and significantly differed with G3Lt group (Figure 8A). There was no significant difference in IL-4 (Figure 8B), IL-10 (Figure 8D), and IL-17 (Figure 8F) production among groups. This profile was directly reflected on IFN-γ/IL-4 (Figure 8C), IFN-γ/IL-10 (Figure 8E), and IFN-γ/IL-17 (Figure 8G) ratios which were non-significant among all three groups but slightly higher in G1Lt. Nonetheless, the ratio of IFN-γ/IL-17 was remarkably higher in G1Lt compared to the G3Lt group. Thus, it seems that the cytokine profile at 7^thWAC could explain the parasite load at the same time point. However, at week 11 post-challenge due to the higher level of IFN-γ (Figure 8A) on one hand and lower levels of IL-4 (Figure 8B), IL-10 (Figure 8D), and IL-17 (Figure 8F) cytokines on the other hand in G1Lt versus G2Lt and G3Lt control groups, the ratios of IFN-γ/IL-4, IFN-γ/IL-10, and IFN-γ/IL-17 in G1Lt group were significantly higher (Figures 8C, E, G) well explaining the parasite load profile.

Moreover, as shown in Figure 8H, the NO profile confirmed the higher production of IFN-γ in the L. tarentolae PpSP15-T2A-PsSP9 vaccinated (G1Lt) group compared to control groups (G2Lt and G3Lt) at both time points (7^th WAC and 11^th WAC) following infection. Similarly, the Th1 type skewed response at later weeks was reflected on antibody isotype. G1Lt produced higher IgG2a compared to G2Lt and G3Lt groups while the level of IgG1 was comparable within groups (Figure 9).

Taken together, it was concluded that the Th1 deviating potential of L. tarentolae PpSP15-T2A-PsSP9 formulation potentially controlled the infectious challenge by L. tropica up to 11 weeks post-challenge. Of note, we could point to the elevated level of IFN-γ (as ameliorating inflammatory cytokine) compared to IL-4 (as disease promoting) and IL-10 (as Th1 suppressor) cytokines during the course of infection from week 7 to 11. The IFN-γ to IL-17 ratio also increased during the infection course as a good sign of infection control. This is in contrast to L. major infection where all ratios dropped to lower levels at late time points leading to disease progression as expected in BALB/c model of L. major. A remarkable difference between L. major and L. tropica infection was a 2 fold increase in IFN-γ/IL-10 ratio following L. tropica infectious challenge which might best account for slowed down disease progression in BALB/c model of L. tropica.