Immunogenicity Evaluation of a Novel Lentiviral Vaccine Expressing Multi- Epitope Against of Leishmania Major in BALB/c Mice

Background: Nowadays, the prevention of parasitic diseases including leishmaniasis is one of the health concerns in the world, and cutaneous leishmaniasis is the most common type of these diseases. So far, no drug or vaccine has been approved for denitive treatment of this disease. Methods: In this study, the recombinant lentiviral vaccine containing a new multi-epitope of KMP11 and HASPB of the Leishmania major (L. major) was synthesized that had previously been designed in-silico. The designed multi-epitope was subcloned into the pCDH513 lentiviral vector, and the recombinant lentiviral multi-epitope vaccine (rLV-multi-epitope) was synthesized in the HEK293T cell by the packaging vectors. Also, the Western Blotting method was used to conrm the gene expression. Then, the rLV-multi-epitope vaccine was injected twice, along with two control groups, PBS, and rLV-empty to immunize the BALB /c mice. Twenty-one days after the second injection, the splenocytes of the mice were is olated and stimulated with the Leishmania lysate. Results: The results of the enzyme-linked immunoassay (ELISA) test not only showed the titer of IFN-γ and IL-4 was increased in the immunized group compared to the controls, but also indicated that the ratio of IFN-γ to IL-4 cytokines in the main group was increased signicantly. As a result, the Th1 response was generated in the main group. Moreover, the humoral immune response was assessed and the results showed that the ratio of IgG2a to IgG1 antibody in the sera of the immunized mice was increased compared to the control groups. Also, the ratio of IgG2a to IgG1 was increased in the main group. Therefore, the humoral immune response was increased, which can also have a positive effect on increasing the Th1 response. Conclusions: Our results showed that immunization by the new rLV-multi-epitope vaccine could stimulate the immune system towards the Th1 through increasing the IFN-γ


Background
The outbreak of leishmaniasis is among the concerning health issues in many parts of the world, and it is estimated that 350 million people are at the risk for affecting with it around the world. Cutaneous leishmaniasis has the highest prevalence among the most common forms of the disease, with Leishmania major (L. major) as one of the causing factors (1).
Thus, there is a need for de nite treatment of the cutaneous leishmaniasis due to the complications of this disease including the skin ulcers and social harms (2). Unfortunately, there is still a challenge for treatment, the drugs used have high costs, and side effects of these drugs may endanger one's health.
Therefore, it is essential to design and develop a vaccine that can stimulate the immune system to provide the defense against the parasites (3).
Although, various types of vaccines including live -attenuated, killed parasites, subunit vaccines, recombinant proteins, and DNA vaccines have been studied in -vitro for treatment of the leishmaniasis in the past decade, but because of the contradictory observed results, the researchers have not yet reached a de nitive conclusion on the use of these vaccines for treatment of the humans, and a few vaccines have entered the clinical trials (1,4). Thus, there is an urgent need for development of a vaccine that can provide more effective immunity than the previous vaccines (5).
Among the types of vaccines studied, the use of DNA vaccines containing the clusters of differentiation (CDs) of whole immunogenic protein has shown promising results. But the use of these antigens in the form of proteins is accompanied with some problems, such as high production cost, short half-life, low immunogenicity, low stimulation of cellular immunity, and cross-allergic reactions (6). Therefore, in the synthesis of vaccines, the use of immunoinformatics tools can be very useful in order to increase the immunogenicity and decrease the adverse reactions (7).
The most appropriate multi-epitope vaccine can be designed to stimulate the immune system using the immunoinformatic servers and performing tests, such as T-cell prediction, docking, and molecular dynamic simulation (8). DNA vaccines expressing the multi-epitope have many advantages over DNA vaccines expressing a whole protein including: (i) they have several epitopes that can stimulate the helper T lymphocyte (HTL), cytotoxic T lymphocyte (CTL), and B cell lymphocytes and thus, can both develop cellular and humoral immune responses, (ii) in their design, several epitopes of different antigens can be used that can greatly stimulate the immune system; and (iii) they will prevent the unwanted reactions due to the allergen tests and other evaluations carried out on them in -silico (9,10).
The most important step in the design of a multi-epitope vaccine is selecting the appropriate antigen to predict the epitopes. If the appropriate antigen is selected, one can hope to produce an effective vaccine (11). A de nite approach to select the strong and effective antigens is predicting the antigenic epitopes proven in the previous studies to have an immunogenic effect, but it does not have the potential to become an effective vaccine due to the mentioned reasons (12).
Also, adding a linker can be very helpful for enhancing the immunogenicity of the vaccines. Among the linkers used, glycine-rich linkers that also contain the hydrophilic amino acids can provide high exibility and bind the epitopes together in a construct without altering the performance of the domains (13).
In our previous studies, we found that the lentiviral vaccine containing the kinetoplastid membrane protein 11 (KMP11) and hydrophilic acylated surface protein B (HASPB) whole proteins are capable of stimulating the immune system in the BALB/c mice (14). Therefore, in this study, the immunogenic effects of the epitopes of these two antigens were investigated.
In the present study, the lentiviral vector with the GDGDG linker and HTL and CTL antigen epitopes selected by the immunoinformatics server was introduced to increase the immunogenicity and e cacy of the vaccine. After producing the recombinant lentiviral multi-epitope, the immunogenicity of this vaccine was evaluated in the BALB/c mice.

Methods
Design of multi-epitope lentiviral vaccine The protein sequences of KMP11 and HASPB of L.major (Friedlin strain) were obtained from GenBank, protein database of the National Center for biotechnology information (NCBI, http://www.ncbi.nih.gov/genbank/). The ID of KMP11 and HASPB was AAR84616.1 and CAB39972.1, respectively. Then, with the help of immunoinformatics software such as T-cell epitope prediction, B-cell epitope prediction, docking studies and molecular dynamics simulation, the epitopes of KMP11 and HASPB proteins were selected in silico space (15). Also, using GDGDG linker, nal multi-epitope vaccine was designed ( Figure.  The cells were supplemented with 10% foetal bovine serum (FBS) (Gibco, USA), penicillin (100 units/ml)/streptomycin (100 mg/ml) (Invitrogen, Carlsbad, CA, USA), nally the cells were incubated in a humidi ed atmosphere with 5% CO2 at 37°C.

Synthesis and con rmation of the pCDH-multi-epitope construct
The designed multi-epitope was synthesized by Genscript, USA, and merged into pUC57 plasmid. Then, the construct was subcloned into pCDH513B lentiviral vector using XbaI and BamHI restriction enzymes. The recombinant pCDH-multi-epitope was digested using XbaI and ApaI and sequenced. In this construct, to express multi-epitope cytomegalovirus (CMV) promoter was used. Also, GFP and puromycine were expressed using eukaryotic elongation factor (eEF) promoter. Moreover, GFP and puromycine were separated by T2A factor. As a result, transfer vector expressed multi-epitope, GFP and puromycine resistance factor from two mRNAs ( Figure. 2).
The pCDH-multi-epitope plasmid was used to transform the competent E. coli DH5α cells. After transformation of bacteria, the LB agar containing ampicillin (100 µg/ml) was used to bacteria culture at 37°C for 24 h. Then, from the colonies appeared on the plate, plasmid extraction was carried out using Miniprep plasmid extraction kit (Qiagen, Germany) according to manufacturer's protocol. Afterward, the restriction enzymes containing XbaI and ApaI were used to digest the extracted plasmid and electrophoresed on 1% agarose gel. Finally, sequencing of plasmid was done to further con rm the cloning. Also, in order to extract of PAX, MD, and pCDH lentiviral plasmids, all procedures were performed.
Virus production (rLV-multi-epitope and rLV-empty) By using the transfer lentiviral vector (pCDH-multi-epitope or pCDH-empty) and two PAX and MD plasmids, transfection of HEK293T cells were done based on Trono protocol (calcium phosphate transfection method) (16). On the rst day, 3×10 6 HEK293T cells were seeded in a 10-cm plate in Dulbecco's modi ed Eagle's medium (DMEM) (Gibco, USA), supplemented with 10% FBS (Gibco, USA). On the second day, a mixture of 21 μg of the transfer vector (pCDH-multi-epitope or pCDH-empty), 21 μg of psPAX2 vector and 10 μg of pMD2.G vector with transfection buffer was added dropwise to the cells. Then, 14 hours' post-transfection, fresh medium was used to replace the previous medium. After 24 hours, with the help of a uorescent microscope, evaluation of GFP expression was performed. Moreover, 24, 48 and 72 hours' post-transfection the supernatant of the packaged lentivirus (named thereafter rLVmulti-epitope or rLV-empty) were harvested and centrifugation at 350 g at 4°C for 5 minute was done. Moreover, the supernatant was ltered through a 0.22-μm lter. Finally, the rLV-multi-epitope or rLV-empty were stored at −70°C for next experiments.

Titarion of virus
Using ow cytometry, the number of GFP-positive cells were counted and, the titre of packaged viruses was de ned. See the virus count formula TU/mL= (1∕4) (F * N * D * 1000) ∕V, Where F = percentage of uorescent cells (EGFP), N = number of cells at the time of transduction (about 1×10 5 cells per well), D = fold dilution of vector sample used for transduction and V = volume (mL) of diluted vector sample added into each well for transduction. All steps have been previously described (17).

Western blot
In order to con rm the expression of rLV-multi-epitope, western blot was carried out (18).
The First, the rLV-multi-epitope and rLV-empty were used to transduce of HEK293T cells. Thereafter, the cells were treated with 2 μg/mL puromycin (Sigma-Aldrich, USA). Subsequently, the transduced HEK293T cells (rHEK-multi-epitope, rHEK-empy) and non-transduced HEK293T cells (HEK-non) were seeded in 6well plates. Then, after 72 h, the cells were trypsinized and centrifuged at 190g for 5 min at 4 °C. Also, the cells were lysed in 200μL of protease inhibitor-containing RIPA buffer (Thermo Fisher Scienti c, 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 Scienti c, 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).
Thereafter, the protein was transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, CA). Then, using 2.5% non-fat milk, the membranes were blocked and using Anti-polyHistidine antibody (anti-His antibody) (Abcam, USA) conjugated with horseradish peroxidase (HRP) at 1:2000 dilution immunoblotting was performed.
Finally, to visualize the protein band, the substrate of the tetramethylbenzidine (TMB) was used. Finally, the optical density (IOD) of the band was obtained and it was integrated.
Immunization of BALB/c mice Experiments were performed on twenty-four female inbred BALB/c mice, 6-week-old which were purchased from Pasteur Institute of Iran and were maintained in the Institutional animal care of the Fasa Three groups of mice were considered and each group was randomly received 8 mice (n= 8), group 1 (rLV-multi-epitope), group 2 (rLV-empty) and group 3 (PBS). The injections were carried out subcutaneously twice during the 1 weeks (with a 3-day interval) with 1×10 6 TU of rLV-multi-epitope (3×10 6 TU/mL) to group 1, and 1×10 6 TU of rLV-empty (1×10 7 TU/ mL) and 0.1 mL PBS to control groups. Finally, three weeks post-immunization of mice, cytokine assay was done.

Parasite culture
In order to obtain a su cient number of L. major (MHRO/IR/75/ER (parasite, promastigote parasites were cultured and enriched in RPMI 1640 (Gibco, UK) supplemented with 20% foetal calf serum (Gibco, Darmstadt, Germany), 100IU/ml of penicillin and 100 μg/mL of streptomycin. 100 IU/mL of penicillin and 100 μg/mL of streptomycin to obtain an adequate number of promastigotes, as described by Dehkordi et al (19).

Antibody analysis
To investigate the humoral immune response, three-week post-injection the sera of vaccinated mice were collected and analyzed in triplicate as previously described. To determine the presence of antibodies, 96well at-bottom plates were coated with L. major promastigotes that there was obtained from stationary phase of culture. To block the plates, 1% (w/v) BSA was used. Also, mice sera were diluted 1:100 and applied to the plate. Then, detection of attached antibodies was done, and the plate was incubated with anti-His antibody (Abcam, USA) and developed with peroxidase substrate tetramethylbenzidine (TMB). Finally, we used an ELISA microplate reader (Bio-Rad) to read the plate at 410 nm.

Cytokine analysis
In order to evaluate the level of cytokine expression, the splenocytes of mice were separated and analyzed as previously complained. Three weeks after injection, in aseptic conditions the spleens of immunized mice and control groups were separated and homogenized. Then, the splenocytes were recovered, washed three times, and seeded in triplicate in a 96-well plate. Moreover, the cells were stimulated using L. major lysate, Soluble Leishmania Antigen (SLA).
Finally, to determine the levels of IL-4 and IFN-γ, after 72 hours Supernatants were harvested and mentioned cytokines were assayed in triplicate by using sandwich ELISA kits (Abcam, USA) based on the manufacturer's protocol.

Statistical analysis
According to Mann-Whitney U-test, data analysis was performed and SPSS (V.18). A P-value < 0.05 was considered as statistically signi cant.

Results
Construction of rLV-multi-epitope vector Figure 1 shows the construct synthesized in this study. pUC57-multi-epitope was sub-cloned in pCDH513 lentiviral vector successfully. Using restriction enzymes ApaI and XbaI two fragments were obtained with Lengths of 1478 and 6355 bp ( Figure. 3). Furthermore, sequencing was performed, and as a result, the cloning of the construct was con rmed.

Packaging And Titration Of The Recombinant Lentiviral Particles
Using the uorescent microscopy, evaluation of cGFP expression was performed and the packaging of the recombinant lentivirus veri ed. Using pCDH-multi-epitope, transfection of more than 90% of HEK 293T cells after 72 hours were successful (Fig. 4a). According to expression of cGFP, the titre of the recombinant virus (rLV-multi-epitope) and empty vector (rLV-empty) was 1 × 10 6 TU/mL, and 1 × 10 7 TU/mL, respectively.

Transduction And Expression Of Rlv-multi-epitope
The expression of rLV-multi-epitope at protein level was assessed and Western blot analyse was done. The expression of the multi-epitope in the trancduced HEK293T cell in the presence of a 10 KD band was con rmed (Figure.3b). Transduced with the rLV-empty was used as a control and no bands was observed.

Igg1 And Igg2a Assay
In order to evaluate the ability of rLV-multi-epitope vaccine in the induction of humoral immune response, three post-injection, the sera from mice immunized with rLV-multi-epitope and the control groups were collected and titre of IgG1 and IgG2a antibodies against L. major was detected. According to ELISA test the rLV-multi-epitope vaccinated mice had a signi cantly higher titre of IgG2a and IgG1 compared to control groups (PBS and rLV-empty) (P < 0.05), and, the level of IgG2a was signi cantly (p < 0.05) higher than IgG1 level ( Figure. 4a, b, and c).

Il-4 And Ifn-γ Concentration
The ability of vaccinated mice splenocytes to induce the Th1 or Th2 immune response was evaluated using Leishmania lysate or SLA. For this purpose, the level of IFN-γ (Th1) and IL-4 (Th2) was measured in the supernatant of splenocytes of vaccinated mice with rLV-multi-epitope and control groups. As a result, the level of IFN-γ and IL-4 produced by splenocytes of immunized mice were signi cantly higher as compared to control groups (P < 0.05). Also, an important nding is that IFN-γ level was signi cantly (P < 0.05) higher than that of IL-4 level in the main group compared to control groups ( Figure. 5a, b, and c).

Discussion
In the recent years, a variety of vaccines have been investigated against different species of Leishmania parasite due to the ineffectiveness of the drugs used for treatment of the leishmaniasis. Although, according to the results of the previous studies, none of the developed vaccines has been approved as a de nitive treatment for this disease (20,21). However, among them, DNA vaccines have been shown to be more suitable due to the activation of major histocompatibility complex I (MHCI) and major histocompatibility complex II (MHCII) and induction of the appropriate response of Th1 and Tc in the host (22,23).
Among the DNA vaccines evaluated in the treatment of various diseases, multi-epitope DNA vaccines designed by the immunoinformatic software in several stages in -silico have been reported to be successful in the in -vivo studies (24). In fact, peptides are better immunogens than the whole proteins, which can prevent the allergic reactions, cross-reactions, as well as the pathological reactions, because these epitopes are designed in -silico before experimental assay. Then, the gene sequence generating these multi-epitope DNA vaccines can be entered into the suitable vector after designing and it can be added to the host (25,26).
Designed epitopes are exactly the same areas of the target immunogens that can activate the Tc and Th1 cells to stimulate the cellular immunity and B-cells to stimulate the humoral immunity. As a result, it can be expected that the designed multi-epitope vaccine can be a good candidate for vaccination (27).
Although, the short sequence of these vaccines can cause them to rapidly degrade within the cell, using viral vectors to transfer them into the cell can lead to the increased stability (28,29). Nowadays, lentiviral vaccines have received a great deal of attention due to the rapid proliferation of the virus in the body and the lack of need for adjuvant because these vaccines can activate the cellular immunity by enhancing the Th1 and Tc responses (30,31). These viral vectors can contain the desired protein or polypeptide for different types of vaccines.
In our previous study, immunogenicity evaluation of the whole protein of KMP11 and HASPB against L. major was successful in the BALB/c mice (14). Therefore, in this study, for the rst time, the immunogenicity of the KMP11 and HASPB as selected epitopes of the L-major was evaluated as a novel lentiviral multi-epitope vaccine.
After designing the vaccine, 4 CTL (or CD8+) and 2 HTL (or CD4+) selected epitopes were linked together using the GDGDG linker. This linker is composed of the glycine (Gly) and aspartic acid (Asp), which are exible and hydrophilic, respectively. This linker can facilitate the expression of the multi-epitope vaccine (32).
Then, the vaccine was subcloned in the PCDH513 lentiviral vector and the immunogenic effect of the vaccine was assessed through determination of interferon gamma (IFN-γ) and interleukin 4(IL-4) cytokines as well as titer of immunoglobulin G 2a (IgG2a) and immunoglobulin G 1 (IgG1) antibodies. In fact, the cytokines and antibodies are studied due to the process occurring after the parasite enters the host and during vaccination where the vaccine attempts to stimulate the defense against the parasite.
After the parasite enters the host, dendritic cells are activated, and the immune system changes with respect to Th1 response and IFN-γ production increases by stimulating the interleukin 12(IL-12) production. Studies have shown that M1 macrophages are stimulated to produce the nitric oxide (NO) by the inducible nitric oxide synthase (iNOS) and kill the parasites by increasing the level of IFN-γ against the parasite (33,34). Also, an increase in the IFN-γ level can lead to activation of Tc, natural killer (NK), and opsonin antibodies, such as IgG2a (35). On the other hand, increased level of cytokines, such as IL-4 and interleukin 23(IL-23) against immunological agents can lead to the altered immune responses to Th2 and IgG1 antibodies (12,36).
In our study, the levels of IFN-γ and IL-4 cytokines were signi cantly increased in the group immunized with the rLV-multi-epitope vaccine compared to the control groups (p < 0.05). Also, due to the importance of the balance between the levels of IFN-γ and IL-4, the ratio of these two cytokines was measured and the results showed a signi cant increase in this ratio in the immunized group compared to the control group (P < 0.05). The increase in this ratio indicated that the splenocytes can induce the memory T-cells and cellular immunity was stimulated in the appropriate direction against the parasite, and Th1 and Tc were dominant to Th2 in the host. In addition, isotype switching occurs and IgG2a is produced with the increase in the IFN-γ level, which can stimulate the humoral immunity in defense against the parasites. However, researchers have not yet reached a de nitive conclusion about the role of antibodies (37).
In the present study, the levels of IgG2a and IgG1 were assessed and according to the results, these antibodies were increased in the group vaccinated with the rLV-multi-epitope (p < 0.05). Moreover, the ratio of IgG2a to IgG1 production in the immunized group was increased signi cantly compared to the control group (P < 0.05). Since, IgG2a production can indicate the progression of the cellular immune pathway to Th1 and IgG1 production can indicate the progression of the cellular immune pathway to Th2, it can be inferred that the Th1 is dominant to the Th2 and possibly it stimulates the host immune system to defend against the parasites.
Hua Cong et al., in a study on the vaccination against the Toxoplasma gondii by a multi-epitope DNA vaccine showed that the vaccine was capable of stimulating the cellular and humoral immunity (36). Also, Li et al., and Escalona et al., in their studies found that the cellular and humoral immunity was stimulated with the increase in the production of IFN-γ cytokine and IgG2a antibodies using the multi-epitope DNA vaccine against the Epstein-Barr virus (EBV) and Brucella Abortus and the vaccine was effective (38,39). Also, Salehi et al., demonstrated that the use of a polyepitope DNA vaccine expressing the KMP11, Leishmania major stress-inducible protein1 (LmsTI1), thiol-speci c antioxidant protein (TSA), and Leishmania homologue of receptors for activated C kinase (LACK) against the leishmaniasis caused an increase in the level of IFN-γ and a Th1 response, although the load of parasite was decreased in the liver after the challenge (40). These studies are consistent with the results of our research, although more research is needed on the design and synthesis of these vaccines.

Conclusion
Results of the present study revealed that the developed recombinant lentiviral vaccine expressing the KMP11 and HASPB multi-epitope was able to create a protective effect in the BALB /c mice. Increased e cacy of the vaccine was con rmed as the initial design of the vaccine was performed in -silico. In addition, less time and cost is required for synthesis of these vaccines compared to the whole protein with the help of immunoinformatics server. Therefore, it is hoped that this vaccine will be economically viable. Our results showed that the rLV-multi-epitope vaccine could stimulate the immune system towards the Th1 well, stimulating the production of Tc and NK, as well as the formation of M1 macrophage by producing the protective cytokines including IFN-γ, consequently leading to the control of the disease. It also contributes to the eradication of the parasites by producing the opsonin antibodies including IgG2a. Although, the results showed that our vaccine can be prophylactic and therapeutic, more tests are needed including the challenge with the parasite, and more cytokine assessments for nal con rmation.

Availability of data and materials
All data analysed during this study are included in this published article.

Con ict of Interest
The authors declare no competing interests