Introduction

Leishmaniasis is an important zoonotic disease caused by facultative intracellular protozoa belonging to Leishmania genus, a Kinetoplastida that infects the macrophages of vertebrates and has more than 30 species, more than 20 of which are pathogenic for human (Alvar et al. 2012). According to the World Health Organization (WHO), nearly 350 million individuals are susceptible to the infection, and nearly 12 million are infected annually, among which 1.5–2 million and 500,000 suffer from cutaneous and visceral Leishmaniasis (Vlach et al.), respectively (Conter et al. 2017).

The disease has been observed sporadically in rural parts of Palestine, Jordan, Senegal, Mali, Sudan, northern Nigeria, and Angola (Akilov et al. 2007). In Iran, the disease is endemic in north and northeast of Isfahan, Torkaman Sahra, Gonbad, Sarakhs, and Esferaien. The most important endemic region, however, is northern Isfahan (Talari et al. 2006; Hashemi et al. 2016).

The drugs of choice in Leishmaniasis treatment include antimony sodium gluconate, penthostam, antimony-containing liposomes, and liposomal amphotericin B (Khamesipour 2014). Nonetheless, treatment with some of these drugs has been limited because of toxicity, not to mention they are expensive (Sadeghian and Nilforoushzadeh 2006).

More than 30 different protein antigens have been considered for vaccine development, such as Lipophosphoglycan (LPG), Thiol-specific antioxidant antigen (Memariani et al.), Glycoprotein 63 (gp63), Leishmania homolog of receptors for activated C-kinase (LACK), Leishmania major stress-inducible protein 1 (LmST1), and combinations of them. These antigens have been used as either second-generation vaccines or third-generation vaccines, i.e., DNA vaccines. DNA vaccines have several advantages over the first- and second-generation vaccines. For example, they can induce both CD4+ and CD8+ T cells, several DNA sequences of antigenic proteins can be incorporated into a single DNA vaccine, and their production is more convenient and cheaper than other types of vaccines (Rivas-Santiago and Cervantes-Villagrana 2014; Engwerda and Matlashewski 2015).

Hydrophilic acylated surface protein B (HASPB) is a membrane protein of Leishmania, which is a proper candidate for vaccine development. It is highly immunogenic, and antibodies against it rise rapidly in the serum of CL and VL patients (Mohapatra et al. 2010). In canine models, HASPB DNA vaccine induced long-lasting immunity against L. donovani (Zackay et al. 2013).

Kinetoplastida membrane protein-11 (KMP11) is another immunogenic protein of Leishmania, which was introduced by Jardim and colleagues, for the first time, as a T cell-interacting protein (Jardim et al. 1991). KMP11 is a Kinetoplastida-specific protein, which is capable of inducing both innate and acquired immunity against Leishmania. In addition, it has a low homology with human proteins, which makes it a suitable candidate for vaccine development (Jardim et al. 1995). Therefore, it has been evaluated extensively in immune protection studies against Leishmania, and it was indicated that KMP11 has a strong potential to induce immune protection in mouse (Stebeck et al. 1995; Guha et al. 2013). Moreover, in 2012, Maroof et al. found out that the adenoviral DNA vaccine harboring KMP11 and HASPB resulted in a protective immune response against L. donovani in mice (Maroof et al. 2012). The latest study, first in the human trial, showed that ChAd63-KH, an adenovirus expressing a novel synthetic gene (KH) encoding two Leishmania proteins KMP-11 and HASPB, is safe and induces cytokine-producing CD8+ T cells.

Our results in the past study indicated that lentiviral vaccine expressing the KMP11-HASPB fusion protein of L. infantum is capable of inducing the immune system to produce cytokines and specific antibodies against L. infantum and resulted in decreased parasite burden (Mortazavidehkordi et al. 2016). Since these two proteins have been detected in different species of Leishmania and have a high degree of similarity among species, therefore, for the first time, we developed a lentiviral DNA vaccine containing KMP11-HASPB coding sequence of L. major and evaluated its potentials in a mouse model of L. major.

Materials and methods

Cell cultures, parasite culture, and animals

HEK293T cells (human embryonic kidney, ATCC CRL-3216), purchased from Mede Bioeconomy Company, Iran, were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA). The medium was supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and penicillin (100 units/ml)/streptomycin (100 mg/ml) (Invitrogen, Carlsbad, CA, USA). The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2.

L. major (MHRO/IR/75/ER) was obtained from Pasteur Institute of Iran, and the promastigote form of the parasite was cultured in RPMI 1640 (Gibco, UK), supplemented with 20% FBS and penicillin (100 units/ml)/streptomycin (100 μg/ml) (Invitrogen, Carlsbad, CA, USA) to obtain sufficient number of promastigotes, as described elsewhere (Hashemi et al. 2018).

To evaluate the recombinant vaccine, 30 inbred, 8–10-week-old BALB/c mice, which were purchased from Pasteur Institute of Iran, were maintained in the laboratory. Animal maintenance and sample collections were performed according to the National Animal Care approved by the Institutional animal care of Isfahan University of medical sciences based on the National Ethical Guidelines for Biomedical Research issued by the Research and Technology Deputy of Ministry of Health and Medicinal Education (MOHME) of Iran (2005).

Design, synthesis, and confirmation of the construct

The coding sequences (CDS) of HASPB and KMP11 (Accession numbers AJ237587.1 and M_003722567, respectively) of L.major (Friedlin) were retrieved from GeneBank, NCBI. After codon optimization, the sequences were synthesized by Genscript, USA, and incorporated into pUC57 plasmid. As shown in Fig. 1, the KMP11-HASPB fusion protein and GFP are expressed under pCMV and eEF promoters, respectively. The transfer vector expresses three proteins, KMP11-HASPB fusion protein from one transcript, and GFP and puromycin, separated from another one by T2A (Fig. 1). The plasmid was used to transform competent E. coli DH5α cells. The transformed bacteria were then cultured on ampicillin-containing LB agar at 37 °C for 24 h. Colonies appeared on the plates were used for plasmid extraction. Plasmid was extracted using Miniprep plasmid extraction kit (Qiagen, Germany) according to the manufacturer҆s instructions. The plasmid was then digested with HindIII and EcoRI, electrophoresed on 1% agarose gel. Finally, it was sequenced to further confirm the cloning procedure.

Fig. 1
figure 1

Schematic presentation of the KMP11-HASPB construct. The sequences of KMP11 (Accession#: XM_00372256 and HASPB (Accession#: AJ237587.1) were retrieved from GeneBank, NCBI. The sequences were synthesized by Genscript, USA, and were incorporated into pUC57 plasmid. In this construct, the KMP11-HASPB is transcribed under CMV (Cytomegalovirus) promoter and GFP is expressed under eEF (eukaryotic elongation factor) promoter. Transfer vector expresses three proteins (KMP11-HASPB, GFP, and puromycin resistance factor) from two mRNAs

Full size image

Construction of KMP11-HASPB-containing recombinant lentiviral vector

Previously cloned in pUC57, KMP11-HASPB fragment was sub-cloned in pCDH513B (pCDH-KH hereafter) using XbaI and BamHI. The recombinant pCDH-KH was digested using XbaI and BamHI restriction enzymes and then sequenced in order to confirm the cloning procedure.

Virus production (rLentivirus-KH and lentivirus-empty)

According to Trono protocol, HEK293T cells were transfected using transfer lentiviral vector (pCDH-KH or pCDH-empty) in conjunction with three plasmids (pLP1, pLP2, and VSV-G) using calcium phosphate transfection method (Barde et al. 2010). On the first day, 5 × 106 HEK293T cells were cultured in a 10-cm plate containing DMEM supplemented with 10% FBS. On the second day, 21 μg of the transfer vector (pCDH-KH or pCDH-empty), 15 μg the pLP1 vector, 6 μg pLP2, and 5 μg of VSV-G vector were mixed with transfection buffer and added to the cells drop-wise. The medium was replaced with fresh medium within 14 h after transfection. GFP expression was assessed after 24 h using a fluorescent microscope. Subsequently, the packaged recombinant lentiviruses (named rLentivirus-KH hereafter) were harvested from the supernatant 24, 48, and 72 h post-transfection by centrifugation at 350g at 4 °C for 5 min. The supernatant was then filtered through a 0.22-μm filter. The recombinant viruses were stored at − 70 °C for subsequent experiments. To determine the titer of packaged viruses, the number of GFP positive cells were counted using flow cytometry based on the equation “TU (Transduction Unite/ml) = [F × C/V] × D” in which F is the frequency of GFP-positive cells, C is the total number of cells in the well at the time of transduction, V is the volume of inoculum in mL, and D is the lentivirus dilution factor. The control vector, not containing the synthetic gene, was named Lentivirus-empty. All steps have been previously described in detail (Kutner et al. 2009).

Western blot

Expression of KMP11-HASPB fusion protein was confirmed by performing Western blot analysis on transduced HEK293T cells as described elsewhere (Khorashadizadeh et al. 2015; Hosseini et al. 2017). Briefly, rLentivirus-KH and Lentivirus-empty were used to transduce HEK293T cells. The cells were then treated with 2 μg/mL puromycin (Sigma-Aldrich, USA). Subsequently, the transduced HEK293T cells (r293T-KH) were cultured in 10-cm plates. Non-transduced HEK293T cells (293T-N) were used as the control group. r293T-KH and 293T-N cells were trypsinized after 72 h and centrifuged at 190g for 5 min at 4 °C. The cells were lysed in 200 μL of protease-inhibitor-containing RIPA buffer (Thermo Fisher Scientific, IL). The lysates were centrifuged at 8800g for 12 min at 4 °C. Then, the protein concentration of the supernatants was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, IL). Lysates/lane of 30 μg were loaded onto a 12% SDS–PAGE gel and then transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, CA). Anti-KMP11 mouse monoclonal antibody (Abcam, USA) and mouse anti β-actin antibody (Abcam, USA) at 1:1000 dilution were used for immunoblotting. Five percent non-fat milk was used to block the membranes. The second antibody was Anti-mouse IgG monoclonal rabbit antibody (Abcam, USA) at the dilution of 1:2000. Finally, chemiluminescence (ECL) method was used to visualize the protein band, and the obtained integrated optical density (IOD) of each band was measured and normalized using β-actin internal standard.

Reverse transcription PCR

HEK293T cells pretreated with rLentivirus-KH and Lentivirus-empty for 72 h were subjected to total RNA extraction using RNeasy® Plus Mini Kit (Alameda, CA, United States). cDNA synthesis was performed using QuantiTect Rev. Transcription Kit (Qiagen, Germany). Specific primers for the gene construct were Forward: 5′ATGGCCACCACGTACGA3′ and Reverse: 5′CACGCTGCCGGCAACTAG3′. PCR thermal cycling was 1 cycle of 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 7 min. After electrophoresis on 1% agarose gel, the PCR products, stained with DNA safe stain, were visualized with a UV trans-illuminator (SABZ biomedical, Iran) and digitally photographed.

Immunization of mice

Thirty inbred, 8-week-old BALB/c mice were divided randomly into three groups (n = 10)—group 1 (rLentivirus-KH), group 2 (lentivirus-empty), and group 3 (PBS). The mice in group 1 were subcutaneously injected twice during 1 week (with a 3-day interval) with 1 × 106 TU of recombinant rLentivirus-KH (2 × 106 TU/ml). The control mice in groups 2 and 3 were also injected subcutaneously with 1 × 106 TU of recombinant lentivirus-empty (1 × 107 TU/ml) or 0.1 ml PBS, respectively. Three weeks after the immunization, immunized mice of each group were used for cytokine assay. Then, immunized mice from each group were challenged subcutaneously with 1 × 107 stationary-phase L. major (MHRO/IR/75/ER) promastigotes, in the right hind footpad. The development of infection was followed by measuring footpad swelling using a metric caliper. The lesion size was defined as the mean of thickness of footpad.

Cytokine analysis

Splenocytes of five mice were isolated and analyzed 3 weeks after challenge for the expression of cytokines as described elsewhere (Pirdel et al. 2014). Briefly, 2 × 106 splenocytes/well of mice from all three groups were recovered after homogenization of the spleen. The cells were plated in a 96-well plate in duplicate after three washing steps and stimulated with L. major lysate, SLA (soluble Leishmania antigen), or medium alone. After 72 h, supernatants were collected, and IFN-γ as well as IL-10 were evaluated in triplicate by sandwich ELISA kits (Abcam, USA) according to the manufacturer’s instructions.

Antibody ELISA

Three weeks after challenge with L. major, the humoral immune response was assessed as described elsewhere (Kaur et al. 2015; Seyedeh et al. 2015). The presence of antibodies in the sera of five mice was analyzed in triplicate. 96-well flat-bottom plates were coated with promastigotes of L. major obtained from the stationary phase of the culture. The plates were blocked using 1% (w/v) BSA. The sera were then diluted 1:100 and applied to the plates. Afterwards, the plate was incubated with peroxidase-conjugated goat anti-mouse IgG1 (Abcam, USA) and peroxidase-conjugated goat anti-mouse IgG2a (Abcam, USA) and developed with peroxidase substrate, tetramethylbenzidine (TMB), to detect the attached antibodies. The plate was read at 450 nm using an ELISA microplate reader (Bio-Rad, USA).

Parasite burden

The five mice were subcutaneously injected 3 weeks after the last vaccination with 1 × 107 stationary-phase L. major promastigotes. The parasite burden of the lymph node and spleen of the vaccinated mice were quantified 8 weeks after infection by limiting dilution, as described previously (Soudi et al. 2011). Using a glass grinder, the tissues were homogenized, and the suspensions were serially diluted (two-fold) with RPMI culture medium in 96-well microplates. Ten days after plating, each well was evaluated for the presence of parasites. Calculation of the number of parasites in the original tissues was performed based on the dilution factor of the last positive well using this formula:

$$ \mathrm{Parasite}\ \mathrm{burden}=-\log\ \left(\mathrm{parasite}\ \mathrm{number}/\mathrm{tissue}\ \mathrm{weight}\right) $$

Statistical analysis

Mann-Whitney statistical test was used to analyze the data using SPSS (V.18). A p value < 0.05 was considered as statistically significant.

Results

Construction of the KMP11-HASPB recombinant lentiviral vector

The construct used in this study has been shown in Fig. 1. KMP11-HASPB sequence was sub-cloned in pCDH-cGFP successfully. XbaI and BamHI restriction enzymes were used to digest the construct. Two fragments of 8200 bp length (pCDH) and nearly 800 bp length (KMP11-HASPB) were obtained as shown in Fig. 2. Finally, the cloning of the construct was further confirmed by sequencing.

Fig. 2
figure 2

Construction of recombinant lentiviral vector. Analysis of recombinant pCDH-KH by restriction enzyme digestion using XbaI and BamHI showed two fragments with 8200 bp (pCDH) and nearly 800 bp length (KMP11-HASPB). Lane 1: undigested plasmid, Lane 2: digested plasmid, M: 1 kb DNA marker

Full size image

Packaging and titration of the recombinant lentiviral particles

cGFP expression using fluorescent microscopy was assessed to verify the packaging of the recombinant lentivirus. After 72 h, more than 90% of HEK 293T cells was shown to be successfully transfected using lentivirus containing pCDH-KMP11-HSABP (Fig. 3a). The titer of the recombinant virus (rLentivirus-KH) was 2 × 106 TU/mL based on the cGFP expression. Lentivirus-empty had a titer over 1 × 107 TU/mL.

Fig. 3
figure 3

Transfection of HEK293T by three plasmids (pCDH, psPAX, and pMD2), transduction of HEK293T by lentiviral particles, RT-PCR, and Western blot. a HEK293T 72 h after transfection. b HEK293T cells prior to transduction (left) and transduced HEK293T cells by rLentivirus-KH. Comparison of both pictures showed the high transduction efficiency higher than 90%. c RT-PCR detected the expression of KMP11-HASPB mRNA in HEK293T cells after transduction. Nearly 800 bp band indicates the presence of the mRNA of the fusion protein KMP11-HASPB in transduced cells with recombinant virus (rLentivirus-KH). d Equal amount of the cell extracts (rLentivirus-KH, lentivirus-empty, and mock) were loaded on all lanes. Immunoblotting was performed using mouse monoclonal antibody against KMP11 (Abcam, USA) and mouse anti β-actin antibody (Abcam, USA). A band of KMP11-HASPB confirmed the presence of the fusion protein (KMP11-HASPB) in the HEK293T cell lysate transduced with rLentivirus-KH. However, there was no detectable band in the control cells (transduced with the lentivirus-empty and non-transduced)

Full size image

Transduction and expression of KMP11-HASPB

RT-PCR and Western blot methods were used to evaluate the expression of KMP11-HASPB mRNA and the fusion protein, respectively, in transduced HEK293T cells (Fig. 3). The presence of nearly 800 bp (Fig. 3b) and of KMP11-HASPB fusion protein bands confirmed the expression of the fusion protein at mRNA and protein levels, respectively (Fig. 3c). In controls, transduced with the lentivirus-empty and non-transduced, bands were observed in neither RT-PCR nor Western blot assays.

IgG2a and IgG1 assay

Three weeks post-injection, the titer of IgG1 and IgG2a antibodies against L. major in the sera of the rLentivirus-KH-immunized mice were detected by ELISA method to determine the immune-response-induction ability of the fusion protein. Mice that received PBS or lentivirus-empty were used as controls. According to the results shown in Fig. 4a, mice vaccinated with rLentivirus-KH showed a significant increase (p < 0.05) of IgG1 and IgG2a levels in comparison with control groups. Furthermore, as shown in Fig. 4, IgG2a level was significantly higher than IgG1 level (p < 0.05).

Fig. 4
figure 4

The level of KMP11-HASP IgG antibody in the sera of immunized mice. Three weeks post-injection, the titer of IgG2a (a) and IgG1 (b) antibodies against L. major in the sera of the mice immunized with rLentivirus-KH were detected by ELISA method. IgG1 and IgG2a concentration produced by rLentivirus-KH-immunized mice were significantly higher than that of controls (rLentivirus-empty and PBS). The results showed that the vaccinated mice had a significant increase in IgG2a production in comparison with IgG1. Data are presented as means ± SD. All tests were performed in triplicate. *P < 0.05

Full size image

IL-10 and IFN-γ concentration

The levels of IFN-γ (Th1) and IL-10 (Th2) were measured in the supernatant of splenocytes of mice vaccinated with rLentivirus-KH in presence of SLA to determine whether the rLentivirus-KH vaccine was able to induce Th1/Th2 immune response (Fig. 5). The results indicated that the level of IFN-γ and IL-10 produced by splenocytes of rLentivirus-KH-immunized mice were higher than that of controls significantly (p < 0.05). It should be mentioned that the level of IFN-γ was significantly higher than that of IL-10 (p < 0.05).

Fig. 5
figure 5

Analysis of IFN-γ and IL-10 levels in the supernatants of splenocytes stimulated with lysate of L. major. a IFN-γ produced by splenocytes of rLentivirus-KH-immunized mice was significantly higher than that of controls (lentivirus-empty and PBS). b IL-10 produced by splenocytes of rLentivirus-KH-immunized mice was significantly higher than that of controls (lentivirus-empty and PBS). It is noteworthy that the level of IFN-γ was significantly higher than that of IL-10. Data are presented as means ± SD. All tests were performed in triplicate, *P < 0.05

Full size image

Protection against L. major

We evaluated the efficacy of the rLentivirus-KH vaccine against experimental challenge with L. major. To do so, the immunized mice were challenged by subcutaneous injection of 1 × 107 L. major promastigotes. Compared with those receiving PBS or lentivirus-empty, a significant decrease of parasites was observed in rLentivirus-KH-immunized mice (Fig. 6). The number of parasites in spleen and lymph node of the rLentivirus-KH-immunized mice was significantly lower than that of PBS and lentivirus-empty groups. At 8 weeks post-infection, there are significant differences in footpad swelling between mice immunized in comparison with control groups.

Fig. 6
figure 6

Protection against L. major infection by rLentivirus-KH vaccination. Mice inoculated with PBS, lentivirus-empty, or rLentivirus-KH were challenged with L. major. The number of parasites in the spleen (a) and lymph node (b) was measured 8 weeks after the infection. The immunized mice showed a reduction in the number of parasites in spleen and lymph node compared with PBS and lentivirus-empty groups. c Footpad swelling was measured weekly. According to the results shown in Fig. 6c, mice vaccinated with rLentivirus-KH showed a significant increase of footpad swelling in comparison with control groups. Data are presented as means ± SD. All tests were performed in triplicate, *P < 0.05

Full size image

Discussion

In the present study, for the first time, we evaluated the effects of a lentiviral vaccine, expressing KMP11-HASPB fusion protein, on the induction of the immune system in mice against L. major.

HASPB is an immunogenic protein found in all kinetoplastids. Sera from patients suffering from either VL or CL react with recombinant HASPB with high specificity and sensitivity (Jensen et al. 1996; Jensen et al. 1999) A. Hydrophilic acylated protein recently found in L. braziliensis, the ortholog of HASPB of other Leishmania species, is also detected by sera taken from L. braziliensis-infected patients (Depledge et al. 2010). This indicates that HASPB family of proteins is ubiquitously present in Leishmania species and is a major immunogenic protein of Leishmania. Vaccination with recombinant L. donovani HASPB at a low dose can induce long-term protection when administered in the absence of exogenous antigen in mice (Stager et al. 2000; Stager et al. 2003). It has also been observed that recombinant HASPB is immunogenic in dogs and is capable of inducing a considerable protection against canine Leishmaniasis in canine animal models (Moreno et al. 2007).

KMP11, as mentioned earlier, is a kinetoplastida-specific protein (Jardim et al. 1991). Since it is not similar to human antigens, it is capable of stimulating both innate and acquired immunity. All vaccinations using KMP11, either as a recombinant protein or DNA vaccine, confirmed the potential of this antigen in the induction of immune system. For example, Basu and colleagues showed that KMP11 can protect mice against the parasite by increasing the level of IL-12, IFN-γ, and TNF and decreasing IL-10 level (Basu et al. 2005). In addition, KMP11-expressing DNA vaccine used in hamsters after challenge with L. chagasi resulted in the lowered parasite burden in the lymph node and spleen and increased level of IFN-γ, compared to IL-10 (da Silva et al. 2011). In the majority of the studies, the immunogenicity of this antigen was evaluated in VL including L. infantum and L. donovani. In the present study, we have assessed the protection efficacy of KMP11-HASPB against L. major in a mouse model.

In previous studies, it was demonstrated that suitable protein antigens used for Leishmania vaccination should be capable of inducing the immune system to produce IgG2a. To assess the efficacy of the fusion protein, the level of specific antibodies (IgG1 and IgG2a) was measured in immunized and control mice. The results demonstrated that the level of specific IgG1 and IgG2a significantly increased in vaccinated mice compared with the control group (p < 0.05). In addition, the serum level of IgG2a was more than that of IgG1 (p < 0.05). These findings show the proper stimulation of immune system against Leishmania by rLentivirus-KH vaccine.

Since cytokines play an important role in the type of immunity developed against L. major, we examined the level of IFN-γ and IL-10 produced by mice splenocytes. As expected, splenocytes of vaccinated mice produced a significant level of IFN-γ compared to the control group and that of IL-10 (p < 0.05). Based on previous research, effective vaccination against Leishmania leads to the production of Th1-related cytokines such as IFN-γ which in turn stimulates the differentiation of Th1 cells and activation of TC, natural killer cells (NK), and M1 macrophages. Subsequently, activation of M1 macrophages results in the production of TNF and NO, which play a key role in the activation of macrophages and their abilities to eradicate pathogens (Podinovskaia and Descoteaux 2015). In fact, one of the main reasons why immune system cannot successfully eliminate Leishmania is its ineffective activation which results from the insufficient production of cytokines that activate them. Therefore, an important parameter in evaluating the efficacy of a vaccine is the level of pro-inflammatory cytokines such as IFN-γ, TNF, and IL-12 as well as TGF-β and IL-10 (Latifynia et al. 2013). In a desirable vaccination, the first three cytokines should be produced at high levels, and the last one should be produced at low levels. Nonetheless, the cytokine pattern of the immune system against Leishmania is not perfectly clear, and both inflammatory and anti-inflammatory cytokines have been detected in the sera of Leishmaniasis patients (Kautz-Neu et al. 2012). Maroof et al. and Mortazavidehkordi et al. who evaluated DNA vaccines against L. donovani and L. infantum, respectively, showed that KMP11 and HASPB are highly capable of stimulating mice immune system and can protect mice against the parasite (Maroof et al. 2012; Mortazavidehkordi et al. 2016). In the present study, we showed that KMP11-HASPB fusion protein induced the immune system of mice against L. major in that the parasite load in the lymph node and spleen of the vaccinated mice was significantly lower than that of controls (p < 0.05). Our results are in line with other researchers’, and this indicates that a promising fusion protein has been probably discovered for vaccination against different species of Leishmania. Since these two proteins have been detected in different species of Leishmania and have a high degree of similarity among species, it seems that, by bioinformatics means, we can find common cross-species epitopes that can be used as a single potent vaccine against Leishmania. However, since the course of infection and the immune system of human and mouse are completely different, which prevents researchers to properly evaluate vaccines, more suitable animal models are needed. Moreover, to stimulate the immune system more effectively, adjuvants should also be considered in vaccination and evaluation of vaccines.