Design, Construction and Immunogenicity Assessment of pEGFP-N1-KMP11-GP96 (Fusion) as a DNA Vaccine Candidate against Leishmania major Infection in BALB/c Mice

Background: KMP-11 (Kinetoplastid membrane protein-Π) exists in all species of kinetoplastid family. It is fully conserved and the protein produced by this gene can induce a very high cellular immune response. We aimed to design a suitable construction for a Leishmania major DNA vaccine and evaluate the protective efficacy of it as a candidate for DNA vaccine against cutaneous leishmaniasis in BALB/c mice. Methods: This experimental study was conducted in Tehran City, Iran, between April 20, 2015 and May 30, 2016. KMP-11 gene of L. major (MRHO/IR/75/ER, Iranian strain) and NT-GP96 of Xenopus GP96 DNA from a pBluescript-GP96 plasmid were amplified by PCR and the purified PCR products were cloned into the pJET1.2/blunt plasmid vector, then, subcloned into pEGFP-N1 plasmid as an expression vector. Finally, the KMP-11 gene was fused with GP96 and afterward the combination cloned in pEGFP-N1. All the cloned genes confirmed by enzyme digestions. Then, four groups of mice were immunized with PBS, pEGFP-N1, pEGFP-N1-KMP, and pEGFP-N1-fusion. Four weeks after immunization, all animals were challenged with L. major virulent promastigotes. Results: The constructed fusion potentially showed an ability to elicit Th1 responses that led to cutaneous lesion healing. Interestingly, the group received KMP11-GP96 –GFP showed the highest ratio of IFN- γ /IL-4 and IgG2a/IgG1 compare to other groups. No side effect was observed after using the fusion in the mice. Conclusion: The constructed fusion could well stimulate both the cellular and humoral immune systems that led to cutaneous lesion healing in mice.


Introduction
eishmaniasis is a neglected tropical disease that causes human infections varying from self-healing cutaneous lesions to mucosal diffuse cutaneous and visceral forms. Antimonial resistance is a major problem that we faced in controlling the disease in Iran. Considering the increasing of clinical drug resistance (1), introducing a new effective antigen for producing an antileishmanial vaccine seems crucial for preventing the disease in endemic areas.
Kinetoplastid Membrane Protein 11 (KMP-11) is a complex protein strongly associated with lipophosphoglycan of Leishmania promastigotes (14). The lipophosphoglycan is highly antigenic for human and murine T cells (15). This is due to the presence of 11-kDa protein in the protozoan membrane. Among various Leishmania molecules known as potential candidate antigens for second-generation vaccines, KMP-11 has concerned much attention because of highly antigenicity for murine, canine and human T cells (16).
GP96 is a member of the HSP90 family and plays an important role in innate and adaptive immune responses, protein folding and assembly (17). GP96 can cause pro-inflammatory cytokine secretion, such as IL-12 and GM-CSF, and maturation of APCs (18). Previously, the GP96 N-terminal domain has a potent adjuvant activity toward the surface antigen of hepatitis B (19). Protective DNA vaccination has been performed using proteins fusion to the GP96 peptide against Listeria monocytogenes in the mice.
The main aim of the present study was to design the pEGFP-N1-KMP 11-NTGP96 Fusion construction to use it as a L. major DNA vaccine.

Ethics Statement
This research was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Tarbiat

Design
This experimental study conducted in Tehran City, Iran, between April 20, 2015 and May 30, 2016. The design, construction and immunogenicity assessment of pEGFP-N1-KMP11-GP96 (Fusion) is shown in a flow chart (Fig. 1).

Genomic DNA Extraction
Genomic DNA of L. major promastigotes was extracted by commercial DNA Extraction kit (Bioneer, Korea) according to the manufacturer's protocol. Qualityand purity of extracting DNA were assessed by electrophoresis on the 1% agarose gel and spectrophotometerically.

Primer design and PCR amplification KMP-11
A pair of oligonucleotide primers were designed based on the KMP-11 gene sequences (accession number KF150697, 279 base pair) according to previous researches (20) as follows: Forward primer: 5′-AGA TCT ACC 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. major was done according to: 95 °C for 3 min as initial denaturation, 35 cycles at 95 °C for 30 sec, 60 °C for 30 sec, 72 °C for 30 sec and then 72 °C for 10 min as final extension.

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 follows: Forward primer: 5′-CGG GAA TTC GAA GAT GAC GTT GAA -3′ that GAA TTC: EcoRI 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 5 min as initial denaturation, 30 cycles at 95 °C for 1 min, 62 °C for 2 min and 72 °C for 1.5 min and then 72 °C for 20 min as final extension.
Finally, the PCR products were analyzed by electrophoresis on 1.2% (w/v) agarose gel. The KMP-11and NT-GP96 PCR products bands were cut under long (312) UV wavelength and were purified from gel by using a gel purification kit (Bioneer, Korea). Correct insertion confirmed by PCR, restriction enzymes digestion and sending to the Gen Fanavaran ® Company (Iran, Tehran) for sequencing.

Cloning of KMP-11and NT-GP96 inpJET1.2/bluntcloning vector
The purified gene fragment of KMP-11 and NT-GP96 was ligated into pJET1.2 cloning vector at 22 °C for an hour and then at 4 °C overnight with T4 DNA ligase (Fermentas Co.). The ligation products were transformed into E. coli TOP10 strain competent cells and dispersed onto LB agar plates containing 100 µg/ml ampicillin. After overnight incubation at 37 °C, colonies on the agar plate that contained recombinant plasmids were detected. For confirmation, PCR amplifications were performed in these colonies using primers specific for KMP-11and NT-GP96 genes and colonies containing the recombinant plasmid were selected. Recombinant plasmids were extracted by Vivantis plasmid extraction kit and digested by Bgl II / EcoRI (for KMP-11), EcoRI / KpnI (for NT-GP96 ) restriction enzymes (Fermentas Co. ® ) for digestion confirmation.

Design the pEGFP-N1 -KMP 11 -NTGP96 Fusion construct
The recombinant plasmids pEGFP-N1 -KMP 11 and pJET-NT-GP96 were digested by EcoRI and KpnI restriction enzymes. The products of digestion were analyzed by electrophoresis on 1.2% agarose gel and the digested bands of NT-GP96 fragment (1014 bp) and pEGFP-N1-KMP 11 were purified by gel purification kit (Vivantis Co.). NT-GP96 genes ligated into digested pEGFP-N1-KMP 11 by using of T4 DNA ligase enzyme. Recombinant plasmids pEGFP-N1-KMP11-NTGP96 were transformed into E.coli TOP10 strain competent cells and dispersed onto LB agar plates containing 100 µg/ml of Kanamycin at 37 °C for overnight. Then, colonies on the agar plate that contained recombinant plasmids were detected. For confirmation, PCR amplifications were performed in these colonies using primers specific for KMP-11and NT-GP96 genes and colonies containing the recombinant plasmid were selected. 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.

Protein expression, and verification of protein expression in eukaryotic cells
Previously (40), KMP11-NTGP96 -GFP Fusion were Subclonned in pLEXSY-neo Leishmania expression vector, and recobbinant constructs were transfected into L. tarentolae promastigotes as eukaryotic cell, that production of recombinant protein of KMP11-NTGP96 -GFP gene was confirmed by SDS-PAGE and Western Blotting.

Mice, Immunization Schedules
Female inbred BALB/c mice, 6-week-old were purchased from 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 pEGFP-N1; group 3 vaccinated with pEGFP-N1-KMP11; group 4 vaccinated with pEGFP-N1-KMP11-GP96 (FUSION). All groups were immunized with 100µg of the materials three times (0, 21, 42 d) in the quadriceps muscle. Four weeks after last immunization, all animals were challenged with 2×10 6 stationary phase of Leishmania major (MRHO/IR/75/ER) virulent promastigotes by intradermal inoculation in the tail. Cutaneous lesion size was measured by digital vernier caliper every day until 30 wk after challenge.
After 30 wk, with full respect to the principles of medical ethics, all live mice initially anesthetized with halothane as an inhalant anesthetics euthanasia, in a chemical fume hood. Then the animals sacrificed, and their liver, bone marrow and spleen were studied for the presence of the Leishmania by smear preparation and cultivation in the culture medium.

Cytokine Assays
To determine the levels of IFN-γ and IL-4, in each group of experiment five mice were sacrificed before and 4 wk after challenge and spleen of them were removed and homogenized in PBS. After erythrocytes lysis using ACK lysis buffer (0.15 M NH4Cl, 10 mM KHCO3 and 0.1 mM Na2-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 -GFP Freeze/Thawed (25 mg/ml) that were designed and constructed in previous research (20). Concanavalin A (Con A; 5 mg/ml) and medium alone were used as the positive and the negative control respectively. Plates were incubated for 72 h at 37 ºC in 5% CO2 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.

Determination of Antibody response
Before challenge and 4 wk after that, all groups of mice were bled retro-orbitally and the levels of IgG1 and IgG2a Abs were evaluated using ELISA method according to the manufacturer's instruction (Mouse IgG2a&1 detection kit, eBioscience, USA).

Statistical Analysis
Statistics were performed using SPSS ver. 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.

Fusion construct
Following PCR amplification a 279 bp DNA fragment for KMP-11 and 1014 bp for NT-GP96 was identified by agarose gel electrophoresis of the PCR products (Figs. 2-4). The PCR products were ligated into a pJET1.2/blunt cloning vector and transformed into E. coli TOP10 strain. Bacterial colonies containing the recombinant pJET-KMP-11 and pJET-NT-GP96 plasmids were confirmed by PCR using KMP-11, NT-GP96 and pJET1.2/blunt specific primers. Subsequently, KMP-11 and NT-GP96 genes were successfully subcloned into pEGFP-N1expression vectors and transformed into E. coli TOP10 strain. The presence of the inserted genes was confirmed by PCR, using specific primers for them. The extracted pEGFP-N1-KMP11 plasmids successfully digested with Bgl II and EcoRI enzymes that after analyzing on 1.2 %( w/v) agarose gel showed a 279 bp fragment (Fig. 3).

The IFN-γ /IL-4 ratio
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 four groups following stimulation with Freeze/Thawed L. tarentolae-KMP11-NTGP96 -GFP. Stimulation of isolated splenocytes from the vaccinated group with recombinant plasmid pEGFP-N1-KMP11-NTGP96 prior and 4 wk after chal-lenge elicited a significantly higher IFN-γ production than other groups (P<0.05) (Fig. 6).
The calculation of the IFN-γ to IL-4 ratio for each vaccinated group were used as an indicator of potential immunization. The Leishmania specific IFN-γ /IL-4 ratio was higher in the pEGFP-N1-KMP11-NTGP96 vaccinated group compared to the others both at before and 4 wk after challenge.

IgG antibody isotypes response to Immunization and challenge
To compare IgG isotypes in different groups, all sera were assayed by ELISA before and 4 wk after immunization. The ratio of IgG2a/IgG1 was highest in the pEGFP-N1-KMP11-NTGP96 vaccinated group that the differences were significant as compared with all other groups (P<0.05) (Fig. 7) .

Mortality rate of immunized and control mice after challenge
The mortality rate in the vaccinated group was significantly less than the control groups (P<0.05). In the groups immunized with pEGFP-N1-KMP11-GP96 and pEGFP-N1-KMP11 the survival rates were higher compared to control groups (P< 0.05) (Fig.8).
Only 20% of the mice that received pEGFP-N1-KMP11-GP96 or pEGFP-N1-KMP11 dead during 30 wk after challenge where 100% of control groups dead within 27 wk. After 30 wk, no parasite was isolated from liver, bone marrow and spleen of the live mice. No side effect was observed after using the fusion in the live mice.

Lesion size in immunized and control groups mice
Lesions with a mean size of 1.5 mm were observed at three weeks after challenge in the control groups; while, the mean size of observed lesions in the immunized group with pEGFP-N1-KMP11-GP96 was 0.50 mm three weeks after the challenge. The mean size of the lesions in both vaccinated group was meaningfully smaller than those in the control group (P< 0.05) ( Table 1). In mice vaccinated with pEGFP-N1-KMP11-GP96 and pEGFP-N1-KMP11, the cutaneous lesions were cured 197 and 201 d after challenge, respectively, while no healing was observed in the mice that received pEGFP-N1 and PBS.

Discussion
Drugs currently used to treat Leishmaniasis have a number of problems, including high toxicity and various side effects (21). Therefore, attempts to introduce new candidates for the production of effective drugs and vaccines are still ongoing.
In general, Leishmania vaccines are divided into 3 categories (22). The first-generation vaccines consist of dead parasites, which have gradually replaced leishmanization. The firstgeneration vaccine have been made using complete killed parasites or their extracts. Numerous efforts have been made to develop such vaccines in Brazil, Colombia, Ecuador, Venezuela and the Islamic Republic of Iran against leishmaniasis and in Sudan against visceral leishmaniasis .
Second-generation vaccines include genetically modified residual leishmaniasis, or bacteria or viruses carrying Leishmania antigen genes, or sub-units of synthetic or recombinant species, and native fractions purified from the parasite. Various proteins of Leishmania have been identified as antigens, gp63 (2), Hsp70 (3), TSA (4), PSA-2 (5), Histone H1 (6), LACK (7,8). Many of these antigens have been identified that, when combined with adjuvants, they are able to provide protection in experimental animals, but to date, only one vaccine has been obtained in clinical evaluations (23). The rest have been provided little immunity against more than one species in animal models.
Third-generation vaccines include genes encoding an immunogenic antigen cloned in a eukaryotic promoter vector. Compared to the recombinant vaccine, DNA vaccines are more stable and have advantages such as cheap production, lack of need for a cold chain and flexibility in combining different genes of different species. These vaccines induce both humoral and cellular immune responses, and these responses can be shifted by altering the vector or attaching cytokine genes with adjuvant properties. Therefore, the immune re-sponse to the encoded antigen from the plasmid can be located in a pathway that produces resistance to the parasite (24). These vaccines provide a better immunity against Leishmania compared to dead or live vaccines, as they can induce expression of different Leishmania antigens without any kind of alteration in structure and antigenicity. DNA vaccines are naturally immunogenetic due to the presence of non-methylated CpG patterns in their structure, leading to the expression of Th1 cytokines and increased TCD8 + cell responses (25). In recent years, antigens such as LACK, LeIF, TSA, LmSTI1, H1, CPA + CPB, KMP-11, and NH36 have been used for production of third-generation vaccine against leishmaniasis.
Studies on KMP-11 protein have shown that KMP-11 clearly has three major immunological features: stimulation of B cell, inducing lymphocytic proliferation and cytotoxic response, and producing protective immunity in animal models. A study showed that immunoglobulin subtypes in response to KMP-11 protein include IgG1, IgG3, IgG2 and IgG4 (26). Some researchers have shown the ability of KMP-11 protein to induce proliferation of Tlymphocytes. KMP-11 of L.donovani, T. rhodesiense, T. brucei, T. congolense and T. simiae was a potent stimulator of CD4 +, CD8-in mice immunized with KMP-11 protein (16). Immunization of BALB/c mice with an attenuated strain of Toxoplasma gondii, which expresses the Leishmania KMP-11 protein, provides a specific and protective immune response in such animals (27). The evidence strongly suggests that this gene is an excellent target for immunotherapy and immunization against leishmaniasis.
GP96 as one of the most abundant intracellular heat shock proteins possesses multiple functions and its ability to bridge the innate and adaptive immune systems has attracted extensive interest. The immunogenicity of GP96 is 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 (20).
In our study, the constructed fusion showed a potency to elicit both Th1 &Th2 responses, but it showed the higher ratio of IFN-γ / IL-4 and IgG2a/IgG1 compare to control groups, indicating that the constructed fusion could well stimulate the cellular immune system in mice.
However, the role of Th1 was greater in the immune response against the infection. These results are in accordance with those of other reports that a higher ratio of the IgG2a/IgG1 isotype is associated with protective immunity against L. major infection in Balb/c mice. Nevertheless, of whether the healing resulted from low doses of parasites (28) or the healing resulted from immunization (29,30).

Conclusion
We successfully cloned fusion of two genes in pEGFP-N1 to use it as a DNA vaccine. The constructed fusion could well stimulate both the cellular and humoral immune systems that led to cutaneous lesion healing in mice.