Immunogenicity and Efficacy of Live L. tarentolae Expressing KMP11-NTGP96-GFP Fusion as a Vaccine Candidate against Experimental Visceral Leishmaniasis Caused by L. infantum.

BACKGROUND: The aim of present study was to evaluate the protective efficacy of live recombinant L. tarentolae expressing KMP11-NTGP96-GFP fusion as candidates for live engineered recombinant vaccine against visceral leishmaniasis in BALB/c mice.
METHODS: KMP-11 and NT-GP96 genes cloned into the pJET1.2/blunt cloning vector and then into pEGFP-N1 expression vector. The KMP-11, NT-GP96 and GFP fused in pEGFP-N1 and subcloned into Leishmanian pLEXSY-neo vector. Finally this construct was transferred to L. tarentolae by electroporation. Tranfection was confirmed by SDS-PAGE, WESTERN blot, flowcytometry and RT-PCR. Protective efficacy of this construct was evaluated as a vaccine candidate against visceral leishmaniasis. Parasite burden, humoral and cellular immune responses were assessed before and at 4 weeks after challenge.
RESULTS: KMP- NT-Gp96-GFP Fusion was cloned successfully into pLEXSY -neo vector and this construct successfully transferred to L. tarentolae. Finding indicated that immunization with L. tarentolae tarentolae-KMP11-NTGP96-GFP provides significant protection against visceral leishmaniasis and was able to induce an increased expression of IFN-γ and IgG2a. Following challenge, a reduced parasite load in the spleen of the KMP11-NTGP96-GFP immunized group was detected.
CONCLUSION: The present study is the first to use a combination of a Leishmania antigen with an immunologic antigen in live recombinant L. tarentolae and results suggest that L. tarentolae-KMP11-NTGP96-GFP could be considered as a potential tool in vaccination against visceral leishmaniasis and this vaccination strategy could provide a potent rout for future vaccine development.

Introduction

Leishmaniases include a wide variety of diseases that range in severity of self-healing cutaneous leishmaniasis to fatal disseminated visceral leishmaniasis that affect about 12 million people worldwide(1). As per WHO report, nearly 200,000 to 400,000 new cases of VL occur annually with 20,000 to 30,000 deaths per year (2). Unlike cutaneous leishmaniasis, which accounts for almost 20,000 new cases per year, visceral leishmaniasis has been reported sporadically in Iran, but the disease is endemic in northwestern and southern areas of the country (3–5)with about 100–300 new cases of VL reported annually(6). Dogs and wild canines are the domestic reservoir for L. infantum in both the old and new worlds including Iran. The only hope at present is to find an effective vaccine for prevention and control of canine and human visceral leishmaniases (7).

Already, there is no any effective vaccine for human leishmaniases and challenge with the disease is restricted only to chemotherapy of some resistance parasites with limited number of toxic drugs (8, 9). Several antigens such as KMP-11, LeIF, GP63, p36/LACK, CP A-B, LD1, PSA-2, TSA/LmSTI1, PFR2, GP46, HASPB1, LCR1, and A2 have been tested as vaccine candidates(2), but they have not given any completely satisfied results.

Among various Leishmania molecules that have been identified as potential candidate antigens for second-generation vaccines, KMP-11 has attracted much attention because of its highly antigenicity for murine, canine and human T cells (10–13). The KMP-11 protein may be involved with mobility in both the parasite and in binding to the host cell. So, it could be considered as a candidate for vaccine production (14).GP96 is a member of the HSP90 family and plays important roles in innate and adaptive immune responses, besides protein folding and assembly(15, 16). Previously, the GP96 N-terminal domain has potent adjuvant activity toward hepatitis B surface antigen (17, 18).

Live attenuated vaccines are the gold standard for protection against intracellular pathogens (19). Since 1986, Leishmania protozoan could be used to express foreign genes (20, 21). Among Trypanosomatidae family, L. tarentolae is a nonpathogenic parasite of the lizard Tarentolae annularis and has been developed as a new potential eukaryotic expression system, as proven in the expression of erythropoietin and tissue plasminogen activator. This parasite can differentiate into amastigote, but is not able to persist long enough within mammalian macrophages and thus can be used as a vaccine vector to deliver specific L. antigens (22–24).

The aim of present study was evaluation of the protective efficacy of live recombinant L. tarentolae expressing KMP11-NTGP96-GFP fusion as candidates for live-engineered recombinant vaccine against visceral leishmaniasis in BALB/c mice.

Materials and Methods

This study has been carried out in Faculty of Medical Sciences of Tarbiat Modares University (Tehran, Iran) in 2014.

DNA and Plasmids constructs

Genomic DNA of Iranian strain of L.infantum (MCAN/IR/07/Moheb-gh.) promastigotes was extracted by commercial DNA Extraction kit (Bioneer, Korea). A pBluescript-GP96 plasmid containing the Xenopus GP96 DNA (accession number AY187545, 2552 bp) was kindly provided by Dr. Jacques Robert (University of Rochester Medical Center, USA). Escherichia coli strains TOP10 (Novagene Co.), pJET1.2/blunt cloning vector (Clone JET™ PCR Cloning Kit, Fermentas), pEGFP-N1 (Invitrogen Co) and pLEXSY-neo(Jena Bioscience, GmbH) expression vectors were used in this study.

Amplification of KMP-11

Oligonucleotide primers were designed based on the KMP-11 gene sequences (accession number KF150697, 279 base pair) as follows:

Forward primer: 5′-AGA TCTACC ATG GCC ACC ACG TAC GAG GAG-3′ that ACC ATG: Kozak sequence and AGA TCT: Bgl II cut site.

Reverse primer: 5′-GAA TTC CTT GGA TGG GTA CTG CGC AGC-3′that GAA TTC: EcoRI cut site and primers without stop codon.

The PCR amplification with Pfu DNA polymerase (Vivantis) and DNA of L. infantum was done according to: 95°c for 3 min as initial denaturation, 35 cycles at 95 °C for 30s, 60 °C for 30s, 72 °C for 30s and then 72 °C for 10 min as final extension.

Amplification of NT-GP96

The forward and reverse primers for amplifying the NT-GP96 of Xenopus GP96 DNA (accession number AY187545, 1014 base pair) were designed as following:

Forward primer: 5′-CGG GAA TTC GAA GAT GAC GTT GAA -3′ that GAA TTC: EcoRI cut site.

Reverse primer: 5′-AT GGT ACC TTT GTA GAA GGC TTT GTA-3′ that GGT ACC: KpnI cut site.

The following program was used for PCR amplification of NT-GP96 with using pBluescript-GP96 plasmid and Pfu DNA polymerase (Vivantis): 95 °C for 5min as initial denaturation, 30 cycles at 95 °C for 1min, 62 °C for 2min and 72 °C for 1.5 min and then 72 °C for 20min as final extension. Correct insertion confirmed by PCR, restriction enzymes digestion and sending to the Gen Fanavaran ® Company (Iran, Tehran) for sequencing.

Cloning of KMP 11 -NTGP96 in pEGFPN1 expression vector

First the KMP-11 and NT-GP96 PCR products were ligated into pJET1.2/blunt cloning vector and transformed into E. coli TOP10 strain. Then, the plasmids pJET- KMP-11 and pEGFP-N1 were digested by Bgl II and EcoRI restriction enzymes. The digested bandsofKMP-11 (279 bp) andpEGFP-N1 was purified by gel purification kit (Vivantis Co.) and KMP-11 fragment ligated into digested pEGFP-N1 expression vector and transformed into E. coli TOP10 strain. The recombinant plasmids pEGFP- KMP 11 and pJET-NT-GP96 were digested by EcoRI and KpnI restriction enzymes and the digested bandsofNT-GP96 fragment (1014 bp) ligated into digested pEGFP-N1- KMP 11 and transformed into E. coli TOP10.PCR amplifications were performed on the recombinant colonies using forward of KMP-11 and reverse of NT-GP96 genes primers. Colonies containing the recombinant plasmid were selected and recombinant plasmids were extracted by Vivantis plasmid extraction kit and digested by Bgl II / EcoRI (for KMP-11), EcoRI / KpnI (for NT-GP96) and NheI / KpnI (for KMP 11-NTGP96 fusion) restriction enzymes (Fermentas Co.) for digestion confirmation.

Subcloning of KMP 11-NTGP96 Fusion in pJET1.2/blunt cloning vector

The forward primer of KMP-11 and reverse primer of NTGP96 for amplifying the KMP 11 -NTGP96Fusion (1293 base pair) were designed as follows:

Forward primer: 5′-GGA TCCACC ATG GCC ACC ACG TAC GAG GAG-3′that ACC ATG: Kozak sequence and GGA TCC: BamHI cut site.

Reverse primer: 5′-AT GGT ACC TTT GTA GAA GGC TTT GTA-3′ that GGT ACC: KpnI cut site.

The PCR amplification with Pfu DNA polymerase (Vivantis) and pEGFP-N1 - KMP 11 -NTGP96 Fusion plasmid and the following amplification program was done: 95 °C for 5 min as initial denaturation, 30 cyclesat 95 °C for 1 min, 60 °C for 2min and 72 °C for 1.5 minandthen 72 °C for 20 min a final extension.

The KMP 11 -NTGP96 Fusion PCR product band was ligated into Pjet1.2 cloning vector and transformed into E. coli TOP10 strain. Correct insertion confirmed by PCR, restriction enzymes digestion and sending to the GenFanavaran ® Company (Iran, Tehran) for sequencing.

Sub cloning of KMP 11 -NTGP96Fusion in pLEXSY-neo L. expression vector

The recombinant plasmid pJET-KMP 11-NTGP96 Fusion was digested by BamHI /KpnI and pLEXSY-neo was digested by BglII/KpnI restriction enzymes and the KMP 11-NTGP96 Fusion band ligated into digested pLEXSY-neo and ligation transformed into E.coli TOP10 strain competent cells and dispersed onto LB agar plates containing 100 μg/ml of ampicillinat 30 °C for night. After overnight incubation at 30 °C, colonies that appeared on the agar plate were detected and for confirmation, PCR amplifications were performed on these colonies using forward of KMP-11 and reverse of NT-GP96 genes primers and also by using both KMP-11 and NT-GP96 genes forward and reverse primers at a single PCR reaction as a multiplex PCR. Correct insertion confirmed by PCR, restriction enzymes digestion and sending to the Gen Fanavaran ® Company (Iran, Tehran) for sequencing.

Sub cloning of Green Florescent Protein (GFP) gene in pLEXSY-KMP 11-NTGP96

The plasmids pEGFP-N1 and pLEXSY-KMP 11-NTGP96 were digested by NotI and KpnI restriction enzymes and the digested band of GFP fragment (741 bp)ligated into digested p LEXSY-KMP 11-NTGP96 and transformed into E.coli TOP10.pLEXSYKMP11-NTGP96-GFP correct insertion confirmed by PCR, restriction enzymes digestion and sending to the GenFanavaran ® Company (Tehran, Iran) for sequencing.

Design the pLEXSY-KMP 11 –GFP construct

The plasmids pEGFP-KMP-11 and pLEXSY were digested by BglII and NotI restriction enzymes and the digested bando-fKMP-11-GFP fragment (1020 bp) ligated into digested pLEXSY and transformed into E. coli TOP10.pLEXSY-KMP11 -GFP correct insertion confirmed by PCR, restriction enzymes digestion and sending to the GenFanavaran ® Company (Tehran, Iran) for sequencing.

Transfection of molecular constructs into L. tarentolae

The L. tarentolae Tar II (ATCC 30143) strain was cultivated in RPMI-1640 medium (Sigma) supplemented with 10% heat-inactivated fetal calf serum (FCS, Gibco), at 26 °C. For transfection, 3.5×10 7 log-phase parasites were washed and re-suspended in 350 μl of electroporation buffer (21 mM HEPES, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM glucose; pH 7.5) and mixed with 50 μl H 2 O containing 10 μg of linearized pLEXSYKMP11-NTGP96-GFP with SwaI restriction enzymes (Fermentas, USA), stored on ice for 10 min, and electroporated (Bio-Rad Gene Pulser Ecell, Germany) at 450 V and 500 mF as described previously (25). Then, the electroporated promastigotes were added to in 5ml of RPMI-20% FCS medium without any selective drug and incubated for 24 h at 26°C.After this period recombinant parasites selected by adding 50 μg/ml of Geniticine (G418)(Sigma, USA) to overnight growth culture of transected parasites. The growth of cells highly resistant to Neomycin was observed after 7–10 days.

Screening of recombinant L. tarentolae colonies and confirmation of the KMP11-NTGP96-GFP Fusion Gene Expression 2.7.1. PCR confirmation

To confirm the integration of the KMP11-NTGP96-GFP Fusion into the ssu locus of Leishmania genome, PCR was performed by using genomic DNA of transgenic strains as a template and ssu forward primer F3001 (5′-GATCTGGTTGATTCTGCCAGTAG-3′) and reverse primer A1715 hybridizing within the 5′UTR of the target gene (5′-TATTCGTTGTCAGATGGCGCAC-3′) with annealing temperature 60 °C according to the LEXSY Kit protocol (Jena bioscience, Germany). One of the primers hybridizes within the expression cassette and other hybridizes to the ssu sequence not present in the plasmid. Integration of the expression cassette into the ssu locus yielded a 1 kb fragment that was not obtained in the control reactions with the genomic DNA of L. tarentolae wild type.

RNA Extraction and Reverse-Transcription PCR

Total RNA extracted from promastigote of L. tarentolae with the RNX Plus kit (Cinnagene ®) according to the manufacturer’s instructions. The RNA concentration and quality was assessed by both UV absorbance and electrophoresis on the 2% agarose gel. The total RNA reverse was transcribed to cDNA (with using RevertAid™ H Minus Reverse Transcriptase, Fermentas®) and this cDNA was used as template DNA for RT-PCR amplification. To detect the KMP11, NT-GP96 and Fusion, PCR reactions were carried out using specific primer pairs to amplify each gene separately. The RT-PCR product was analyzed by electrophoresis on a 1.2% agarose gel.

Flow Cytometry and Fluorescence Microscopy analysis

Analysis promastigote forms of L. tarentolae-KMP11-NTGP96-GFP were examined for GFP expression by Epifluorescence microscopy. Promastigotes were centrifuged in 3000 rpm for 10 min and after washing once with PBS, cells were re-suspended in PBS and mounted on microscope slides. Expression of EGFP protein was evaluated by Epifluorescent microscopy. For flow cytometric measurement, parasites at two different growth phases (logarithmic and stationary phases) were centrifuged at 3000 rpm for 10 min, washed with PBS and then resuspended at 10 6 cell/ml in PBS and stored on ice. Cells were analyzed on a FACS caliber flow cytometer (BD: Becton Dickinson, Franklin Lakes, NJ) equipped with a 15 mV, 488 nm, air-cooled argon ion laser. 50,000 events were recorded and EGFP expression in transgenic L. tarentolae was measured in comparison with wild type (WT) parasites.

SDS–PAGE and Western Blot Analysis

Promastigote forms of the transformed L. tarentolae-KMP11-NTGP96-GFP and the wild type parasites were harvested by centrifugation at 3000 rpm for 15 min and washed in PBS and the pellets were lysed in 5X SDS-PAGE sample and then boiled for 5 min. Samples were then loaded on a 12.5% SDS-PAGE. The gels were transferred onto a nitrocellulose membrane and Western blotting was performed according to the standard procedure (26).

Mice, Immunization Schedules and challenge infection

Female inbred BALB/c mice, 7 week-old were acquired from the Animal Breeding Facility Centre of Razi Vaccine and Serum Research Institute, Karaj, Iran. They were housed in clean cages and fed ad libitum. The immunization experiments were carried out in four groups of mice (n= 15 at each group) and all tests were done in triplicate. The first group received PBS only and Group 2 immunized with 2×107 Wild L. tarentolae as the control; group 3 vaccinated with 2×10 7 recombinant L. tarentolae-KMP11-GFP; group 4 vaccinated with 2×107 recombinant L. tarentolae-KMP11-NTGP96-GFP. All groups were immunized subcutaneously via footpad. Four weeks after the last immunization, all animals were challenged with 107 stationary phase of L. infantum strain JPCM5 (MCAN/ES/98/LLM-877) virulent promastigotes by intra peritoneal injection.

Determination of Parasite Burden

Five mice from each group were sacrificed at 4 weeks after challenge and parasite burden in the spleens was quantitatively determined by serial dilutions method. Briefly, apiece of spleen was excised, weighed and then homogenized with a tissue grinder in 2 ml of RPMI-1640 medium (Sigma, USA) supplemented with 20% heat-inactivated fetal calf serum. Under sterile conditions, serial dilutions ranging from 1 to 10−20 were prepared in wells of 96 well microtitration plates. After 3, 7 and 14 days after incubation at 26°C, plates were examined with an inverted microscope at a magnification of 40×. The presence or absence of mobile promastigotes was recorded in each well. The final titer was the last dilution for which the well contained at least one motile parasite. The number of parasites per gram was calculated in the following way: parasite burden = −log10 (parasite dilution/tissue weight)(27, 28).

Determination of Antibody response

Before challenge and 4 week after challenge, all groups of mice were bled retro-orbitally and the levels of anti-soluble Leishmania antigen(SLA) IgG1 and IgG2a Abs were evaluated using ELISA method according to the manufacturer’s instruction (Mouse IgG2a&1 detection kit, ebioscience, USA).

Cytokine Assays

To determine the levels of IFN-γ and IL-4, in each group of experiment five mice were sacrificed before and 4 weeks after challenge and spleen of them were removed and homogenized in PBS. After erythrocytes lysis using ACK lysis buffer (0.15 M NH 4 Cl, 10 mM KHCO 3 and 0.1 mM Na 2 -EDTA), splenocytes were washed with PBS and resuspended in RPMI-10% FCS. Cells were then seeded at a density of 3.5×10 6 cells/ml in the presence of L. tarentolae-KMP11-NTGP96-GFPFreeze/Thawed (25 mg/ml). Concanavalin A (Con A; 5 mg/ml) and medium alone were used as the positive and the negative control respectively. Plates were incubated for 72h at 37°C in 5% CO 2 humidified atmosphere for IFN-γ and IL-4 measurement. The IFN-γ and IL-4 production in supernatants of splenocytes cultures was measured by ELISA kits (U-CyTech, Netherlands), according to the manufacturer’s instructions. All experiments were run in triplicates.

Ethics Statement

This research was carried out accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Tarbiat Modares university and all animals experiments including maintenance, handling and blood collection were approved by Institutional Animal Care and Research Advisory Committee of Tarbiat Modares university based on the Specific National Ethical Guidelines for Biomedical Research issued by the Research and Technology Deputy of Ministry of Health and Medicinal Education of Iran.

Statistical Analysis

Statistics were performed using SPSS version 18 (Chicago, IL, USA) and one way ANOVA (Multiple-comparison Tukey post Hoc test) and Student’s t-test was employed to assess the significance of the differences between the mean values of control and experimental groups. Differences were considered statistically significant when P< 0.05. Data shown represent the mean values ± standard error of the mean (SEM) of three independent experiments.

Results

Constructing molecular structures

Following PCR amplification, a 279 bp DNA fragment for KMP-11 and 1014 bp for NT-GP96 were identified by agarose gel electrophoresis. The PCR products were successfully ligated into a pJET1.2/blunt cloning vector and subsequently, KMP-11 fragment was ligated into pEGFP-N1 expression vectors and then recombinant pEGFP-N1-KMP 11-NTGP96, pJET-KMP 11-NTGP96, pLEXSY-KMP11-NTGP96 and pLEXSY-KMP11-NTGP96-GFP plasmids were constructed successfully.Following PCR amplifications using pLEXSY-KMP11-NTGP96-GFP plasmids as the template and forward and reverse primers specific for KMP-11 and NT-GP96 genes all three DNA fragment (279 bp for KMP-11, 1014 bp for NT-GP96 and 1293 bp for KMP11-NTGP96 fusion) was identified by agarose gel electrophoresis of the PCR products (Fig. 1). Accurate presence of all constructs confirmed by enzyme digestion, PCR amplification and sequencing.

Fig. 1:

Electrophoresis of the amplified KMP-11, NT-GP96 and KMP11-NTGP96 fusion genes in a single PCR reaction (multiplex PCR) on 1.2 % (w/v) agarose gel.Lane1:100 bp DNA Ladder; Lane 2: expanded bands of KMP-11, NT-GP96 and KMP11-NTGP96 fusion genes (approximately 279 bp, 1014 bp and 1293 bp respectively);Lane 3: single expanded band of KMP11-NTGP96 fusion (approximately 1293 bp). Lane4:1kb DNA ladder

Confirmation of construction of Recombinant L. tarentolae expressing the KMP11-NTGP96-GFP Fusion

Recombinant L. tarentolae stably expressing the KMP11-NTGP96-GFP Fusion gene were generated by introducing the linearized pLEXSY-KMP11-NTGP96-GFP vector into the 18S rRNA ssu locus of L. tarentolae. Specific targeting of the expression cassette into the ssu locus was confirmed both by genomic PCR with gene specific primers (Fig. 2.A) and vector specific primers (Fig. 3).Amplification of all expected genes in a RT-PCR product, confirmed the expression of KMP11-NTGP96-GFP by L. tarentolae at the mRNA level (Fig. 2.B).

Fig. 2:

Electrophoresis of the amplified KMP-11, NT-GP96 and KMP11-NTGP96 fusion genes in PCR reaction with using: A. recombinant L. tarentolae DNA as the template and genes specific primers that Lane1: 1kb DNA ladder; Lanes 2, 3, 4: expanded bands of KMP-11, NT-GP96 and KMP11-NTGP96 fusion genes (approximately 279 bp, 1014 bp and 1293 bp respectively) ;and B. recombinant parasite cDNA as the template and genes specific primers that Lanes 1, 2, 3: expanded bands of KMP-11, NT-GP96 and KMP11-NTGP96 fusion genes (approximately 279 bp, 1014 bp and 1293 bp respectively); lane 4: wild L. tarentolae as the negative control; Lane5: 1kb DNA ladder

Fig. 3:

Electrophoresis of the amplified fragments (1 Kb) of specific targeting of the expression cassette into the ssu locus in PCR reaction with using recombinant L. tarentolae DNA as the template and vector specific primers on 1.2 % (w/v) agarose gel. Lane1: 1kb DNA ladder; Lanes 2: expanded bands of 1Kb

Expression of GFP (marker for expression of KMP11-NTGP96-GFP) in recombinant L. tarentolae parasites was confirmed by fluorescence microscopy (Fig. 4.B) and by fluorescence-activated cell sorting (FACS) analysis (Fig. 4.C). The KMP11-NTGP96-GFP expression was also assessed by western blot analysis. As shown in Figure 4.A, immuno-reactive bands were detected in recombinant L. tarentolae parasites using polyclonal antibody.

Fig. 4:

Expression of the fusion gene by L. tarentolae. (A) Western blot analysis for evaluating expression of the KMP11-NTGP96-GFP fusion protein. (B) Expression of EGFP by recombinant L. tarentolae promastigotes.(C)Percentage of the EGFP positive population in L. tarentolae promastigotes transfected with either pLEXSY-KMP11-NTGP96-GFP (I) or pLEXSY-KMP11-GFP (II) that shown in green lines in comparison to wild type parasite that shown in red lines as determined by flowcytometry

Immunization with recombinant L. tarentolae and determination of Parasite burden

Four groups of mice were considered for immunization with two subsequent repeats as described in Materials and Methods. The results are shown, obtained from these two independent experiments. The degree of protection against infection was determined by measurement of the parasite burden in the spleen at 4 weeks after challenge with L. infantum. As shown in Fig. 5, immunization using a L. tarentolae-KMP11-NTGP96-GFPreduced the infection in the spleen at 4 weeks after challenge in contrast to the control groups (PBS and wild L. tarentolae).

Fig. 5:

spleen parasite burden in all groups following immunization and infectious challenge with L.infantum. The parasite number in spleen was evaluated at 4 weeks after challenge (*P< 0.05 compared to PBS)

Evaluation of IFN- γ /IL-4 ratio after immunization with Live Recombinant parasites

The levels of IFN-γ and IL-4 production were analyzed before and 4 weeks after challenge in the supernatant of the spleen cells culture of all five groups following stimulation with Freeze/Thawed L. tarentolae-KMP11-NTGP96-GFP. As shown in Fig. 6-I-A, stimulation of isolated splenocytes from vaccinated group with L. tarentolae-KMP11-NTGP96-GFP prior and 4 weeks after challenge elicited a significantly higher IFN-γ production than other groups (P< 0.05).

Fig. 6:

Analysis of the specific cellular and humoral response factors in vaccinated and control groups before and after challenge. (I) Cytokine production by splenocytes in vaccinated and control groups. A: IFN-γ production by splenocytes in vaccinated and control groups after stimulation with F/T L. tarentolae-KMP11-NTGP96-GFP (*P< 0.05 compared to PBS);B: IL-4-γ production by splenocytes in vaccinated and control groups after stimulation with F/T L. tarentolae-KMP11-NTGP96-GFP (*P< 0.05 compared to PBS);C: IFN-γ/IL-4 ratio in vaccinated and control groups after stimulation with F/T L. tarentolae-KMP11-NTGP96-GFP (*P< 0.05 compared to PBS). (II)Analysis of the specific IgG isotypes in vaccinated and control groups. A: Specific IgG2a antibody isotypes detected by ELISA in the sera of mice in vaccinated and control groups (P< 0.01 compared to PBS); Bˊ: Specific IgG1 antibody isotypes detected by ELISA in the sera of mice in vaccinated and control groups (*P< 0.01 compared to PBS); Cˊ: IgG2a/IgG1 ratio in vaccinated and control groups before and 4 weeks after challenge (*P < 0.01 compared to PBS).

The production of IL-4 upon antigen stimulation before and 4 weeks after challenge was also higher in this vaccinated group (Fig. 6-I-B).We further calculated the IFN-γ to IL-4 ratio for each vaccinated group as an indicator of potential immunization (Fig. 6-I-C). The Leishmania specific IFN-γ/IL-4 ratio were higher in L. tarentolae-KMP11-NTGP96-GFP vaccinated group compared to the others both at before and 4 weeks after challenge.

IgG antibody isotypes response to Immunization with Live Recombinant L. tarentolae

To compare IgG isotypes in different groups, all sera were assayed by ELISA before and 4 weeks after challenge. As shown in Fig. 6-II-Aˊ and 6-II-Bˊ, IgG1 and IgG2a isotypes before challenge were higher in groups that vaccinated with L. tarentolae-KMP11-NTGP96-GFP and L. tarentolae-KMP11-GFP in comparison to the control groups (P<0.05). In addition, increased amount of IgG2a was seen in these groups at 4 weeks after challenge. Interestingly, in the group vaccinated with Live L. tarentolae-KMP11-NTGP96-GFP, a decreased amount of specific IgG1 was detected in comparison to other vaccinated and control groups. The ratio of IgG2a/IgG1 was significant in L. tarentolae-KMP11-NTGP96-GFP as compared with all other groups (Fig. 6-II-Cˊ) (P<0.05).

Discussion

Leishmania vaccine development has proven to be a difficult and challenging task, which is mostly hampered by inadequate knowledge of parasite pathogenesis and the complexity of immune responses needed for protection (29). If the patient with visceral leishmaniasis left without any treatment, it can lead to death in 90% of cases (30). The drugs which are currently used as treatment for leishmaniasis cannot be handled easily due to a number of problems including high toxicity and various side effects (30). Therefore, efforts to introduce new candidates for vaccine production are currently being considered. Vaccine trials against leishmaniasis pioneered by South American scientists 75 years ago (31). Despite advances in Leishmania genomics and proteomics (32), modern biotechnology for antigen expression, purification and delivery, and the large availability of murine models in the field of experimental immunology, Leishmania vaccinology still suffers from several bottlenecks that limit the progress towards effective and universal vaccines (33). Some studies have been conducted in various parts of the world including Iran to find an effective vaccine against visceral leishmaniasis that would provide some protection (7, 34, 35). Numerous Leishmania antigens from different laboratories from all over the word have been introduced as vaccine candidates (36).

KMP-11 is found in all kinetoplastid protozoa (37) and is highly conserved (> 95% homology) in all Leishmania species, suggesting an essential role for this protein in the biology of the parasite (38). Studies on KMP-11 protein have shown that KMP-11 has clearly three immunological roles: B-cell immunostimulatory, inducer lymphocyte proliferation and response cytotoxic and immunoprotective in animal models (39, 40). The ability of KMP-11 protein to induce proliferation of T lymphocytes was demonstrated (41, 42). Immunization of BALB/c mice with an attenuated strain of Toxoplasma gondii expressing the L. KMP-11 protein, induces a specific immune-response and immunoprotective in such animals (43).

GP96 as one of the most abundant intracellular heat shock proteins possesses multiple functions. Among these functions, its ability to bridge the innate and adaptive immune systems has attracted extensive interest. Immuno-therapies using HSPs to generate specific anti-tumor responses have been evaluated in clinical studies. GP96 and its N-terminal fragment, but not the C-terminal fragment showed adjuvant effects in enhancing the peptide-specific CTL response against HBV infection and HCC, and this response was peptide concentration dependent (44). The immune effect of GP96 is probably dependent on its ability to bind peptide epitopes. Both the N- and C-terminal fragments of GP96 are able to bind peptides, with the N-terminal fragment behaving at a similar capacity to the full-length GP96 (45, 46).

For effective intervention measures to control VL in endemic areas, it is imperative to design a vaccine, which is the most economical way of controlling infectious diseases (2). For many years, many laboratories around the world have worked on the development of an effective vaccine against leishmaniasis (47). Different vaccination studies in experimental murine models have shown that live attenuated forms of parasites gives better protection when compared to other recombinant antigens (48). L. tarentolae is a parasite of the gecko Tarentolae annularis and because of its several unique features, including higher specific growth rate compared to mammalian cells, cultivation in low cost media, safety for humans, possibility to introduce several copies of a foreign gene into the parasite genome and production of recombinant proteins with an animal-like N-glycosylation pattern is a feasible eukaryotic expression system for high level production of active recombinant biopharmaceuticals (23, 49). A couple of recombinant pharmaceutical and non-pharmaceutical glycoproteins such as human erythropoietin, tissue plasminogen activator and laminin- 332 have already been produced in this expression system and in all cases, the expressed proteins were biologically active (23, 24, 50). These advantages, in addition to feasibility for constitutive or regulative protein production, make L. tarentolae an attractive host for high level production of heterologous proteins (23, 24, 51, 52) and as an ideal candidate for vaccine against leishmaniases.

In our research, following immunization, a notable pattern was observed on the profile of cytokines and immunoglobulin expression. A significant production of IFN-γ cytokine was detected following immunization with L. tarentolae-KMP11-NTGP96-GFP.We also detected considerable levels of IL-4 in immunized mice with this group. These results indicated that our vaccine strategy mediated protection was associated with a mixed Th1/Th2 that is typical of other successful experimental vaccines against visceral leishmaniasis (53). However, the important point is the matter that the level of IFN-γ cytokines were increased after challenge, but this pattern was decreasing about IL-4. Interestingly, the expression ratio of IFN-γ/IL-4 of mice immunized with L. tarentolae-KMP11-NTGP96-GFP was significantly higher before and after challenge in comparison to control groups. In addition, a significant difference in the expression ratio of IgG2a/IgG1 was observed in mice immunized with this vaccination group.

The important result is that this group also displayed a significant reduction in the parasite load in spleen four weeks after challenge, that suggesting this modulation in the immune response is capable to control infection. The significant parasite load reduction observed in the L. tarentolae-KMP11-NTGP96-GFP immunized group after challenge could be a result of the increased expression ratio of IFN-γ/IL-4 and IgG2a/IgG1 that indicated to activation of Th1 cell mediated immune response that could have an impact on parasite establishment and disease development pattern. Vaccines based on combination of different antigen candidates have been shown to improve protection. In fact, a recombinant Q protein formed by genetic fusion of five parasite intracellular antigens has been successfully tested in dogs (54–56). The data shown herein indicates that L. tarentolae-KMP11-NTGP96-GFP group was able to confer a significant degree of protection against L. infantum.

Conclusion

Immunization with live recombinant L. tarentolae-KMP11-NTGP96-GFP provides significant protection against visceral leishmaniasis in the BALB/c mice. According to the results and information presented in this study, it seems that using fusion strategy between KMP-11 and GP96 can lead us to making an effective construct for increasing the efficiency of a live recombinant vaccine against visceral leishmaniasis. Designing of effective live recombinant vaccines is attractive in terms of low cost, optimal safety, stability and potency when compared with other vaccination strategy. In addition, further investigation about its efficacy should be done in the susceptible hamster and dog model of visceral leishmaniasis and this strategy could have a broader impact on vaccine development efforts for visceral leishmaniasis worldwide.