Identification of BALB/c Immune Markers Correlated with a Partial Protection to Leishmania infantum after Vaccination with a Rationally Designed Multi-epitope Cysteine Protease A Peptide-Based Nanovaccine

Background Through their increased potential to be engaged and processed by dendritic cells (DCs), nanovaccines consisting of Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with both antigenic moieties and adjuvants are attractive candidates for triggering specific defense mechanisms against intracellular pathogens. The aim of the present study was to evaluate the immunogenicity and prophylactic potential of a rationally designed multi-epitope peptide of Leishmania Cysteine Protease A (CPA160-189) co-encapsulated with Monophosphoryl lipid A (MPLA) in PLGA NPs against L. infantum in BALB/c mice and identify immune markers correlated with protective responses. Methodology/Principal Findings The DCs phenotypic and functional features exposed to soluble (CPA160-189, CPA160-189+MPLA) or encapsulated in PLGA NPs forms of peptide and adjuvant (PLGA-MPLA, PLGA-CPA160-189, PLGA-CPA160-189+MPLA) was firstly determined using BALB/c bone marrow-derived DCs. The most potent signatures of DCs maturation were obtained with the PLGA-CPA160-189+MPLA NPs. Subcutaneous administration of PLGA-CPA160-189+MPLA NPs in BALB/c mice induced specific anti-CPA160-189 cellular and humoral immune responses characterized by T cells producing high amounts of IL-2, IFN-γ and TNFα and IgG1/IgG2a antibodies. When these mice were challenged with 2x107 stationary phase L. infantum promastigotes, they displayed significant reduced hepatic (48%) and splenic (90%) parasite load at 1 month post-challenge. This protective phenotype was accompanied by a strong spleen lymphoproliferative response and high levels of IL-2, IFN-γ and TNFα versus low IL-4 and IL-10 secretion. Although, at 4 months post-challenge, the reduced parasite load was preserved in the liver (61%), an increase was detected in the spleen (30%), indicating a partial vaccine-induced protection. Conclusions/Significance This study provide a basis for the development of peptide-based nanovaccines against leishmaniasis, since it reveals that vaccination with well-defined Leishmania MHC-restricted epitopes extracted from various immunogenic proteins co-encapsulated with the proper adjuvant or/and phlebotomine fly saliva multi-epitope peptides into clinically compatible PLGA NPs could be a promising approach for the induction of a strong and sustainable protective immunity.


Introduction
Leishmaniasis is an infectious diseases complex caused by protozoan parasites of the genus Leishmania. According to epidemiological data, visceral leishmaniasis (VL) is the second most common parasitic cause of death among tropical infections and is prevalent in 47 countries with about 200 million people at risk and an estimated annual incidence of approximately 500,000 cases [1,2]. Despite the fact that vaccination is considered to be the most promising and effective strategy for controlling leishmaniasis, to date no efficacious vaccine exists against human VL. First attempts at developing an anti-leishmanial vaccine were based on the injection of live virulent L. major parasites in healthy people, a process known as "leishmanization". However, this process was discontinued due to safety and ethical reasons and replaced by firstgeneration vaccines composed by attenuated or inactivated pathogens or even pathogen subunits that in many cases showed inconsistent clinical outcomes [3,4,5].
Subsequently, many research efforts are focused on the development of second generation vaccines that are consisted of recombinant proteins or defined peptides. To date many different Leishmania antigens have been found to be potential vaccine candidates delivered by a plethora of immunizations regimens in animal models. However, these promising findings were overshadowed by mostly negative T-cell responses in humans [6,7]. During the last few with the ARRIVE guidelines. The protocol was approved by the institutional Animal Bioethics Committee (Approval Number: 4455/10-07-2014). All efforts were made to minimize animal suffering. Serum samples from domestic dogs (Canis familiaris) were obtained from an already-existing biobank of our laboratory. Also, L. infantum used in the present study was obtained from the already-existing "Leishmania cryobank collection" of the Hellenic Pasteur Institute. All samples were coded and anonymized. No IRB approval was required for using the strain.

Animals, parasites and preparation of soluble antigen
Studies were performed with female 6-8 weeks old BALB/c mice reared in the pathogen-free animal care facility at Hellenic Pasteur Institute. They were housed in a climatically controlled room receiving a diet of commercial food pellets and water ad libitum.
For preparation of soluble Leishmania antigen (SLA), 1x10 9 late-log phase promastigotes were washed thrice in PBS and disrupted by five repeated freezing/thawing cycles (liquid N 2 / 37˚C) followed by 5 min incubation on ice. Partially lysed material was exposed for 30 sec in a sonicator (UP100H, Hielscher Ultrasonics GmbH, Teltow, Germany) and then centrifuged for 30 min at 8,000×g at 4˚C. The supernatant containing SLA was collected and total protein concentration was determined spectrophotometrically using the MicroBCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA) at 570 nm. SLA was stored at -80˚C in aliquots until use.

Canine serum samples and detection of anti-CPA 160-189 antibodies
For the evaluation of CPA 160-189 immunogenicity, peptide-specific IgG antibodies were detected in serum samples of naturally infected with L. infantum asymptomatic (n = 12) or symptomatic (n = 14) and healthy dogs (n = 6) [30]. For the detection of CPA 160-189 -or parasitespecific IgG antibodies, 96-well microtiter plates were coated with 5 μg/ml CPA 160-189 or SLA, respectively, and incubated overnight at 4˚C. After 3 washes with washing buffer (PBS with 0.05% Tween 20), plates were blocked with blocking buffer (2% BSA in PBS) for 2 h at 37˚C and then 100 μl of dog sera were added at 1:400 dilution, and incubated for another 2 h at 37˚C. HRP-conjugated anti-dog IgG were added and incubated for 1 h at 37˚C. The enzymelabeled complexes were detected by reaction with TMB substrate (Thermo Scientific) and the reaction was stopped by adding 2 M sulfuric acid. The absorbance was measured at 450 nm using an ELISA microplate spectrophotometer (MRX, Dynatech Laboratories, Guernsey, UK).

Preparation of PLGA NPs
For NPs synthesis, PLGA 75:25 (Resomer RG752H, MW: 4-15 kDa), polyvinyl alcohol (PVA) (MW: 30-70 kDa, 87-90% hydrolyzed) and MPLA from Salmonella minnesota were purchased from Sigma-Aldrich (Vienna, Austria). CPΑ 160-189 peptide obtained from the sequence of L. infantum CPA protein (GenBank Acc. No.: CAM67356) was synthesized by GeneCust (Labbx, Dudenange, Luxenmbourg) with purity !95%. All other reagents were of analytical grade and commercially available. PLGA NPs containing the peptide CPΑ 160-189 and the adjuvant MPLA were prepared by the double emulsion method, as described previously [31]. Briefly, 2.9 ml of a PLGA chloroform solution (31 mg/ml) were mixed with 0.1 ml of an MPLA solution (10 mg/ml) in methanol:chloroform (1:4 v/v). A water-in-oil (w/o) emulsion was then formed by adding 0.3 ml of the peptide solution in PBS at a final concentration of 6.6 mg/ml into the PLGA/MPLA solution. The emulsification was performed in an ice bath with the aid of a microtip sonicator (Sonicator Sonics Vibra Cell VC-505, Sonics, Newtown, CT, USA) at 40% amplitude for 45 sec. Subsequently, the primary emulsion (w/o) was added into 12 ml of 1% (w/v) aqueous PVA solution. The mixture was then emulsified via sonication at 40% amplitude for 2 min. The resulting double (w/o/w) emulsion was stirred overnight to allow the evaporation of chloroform. The PLGA NPs were then purified by means of four successive centrifugation-redispersion cycles in sterilized water, at 13,860×g for 10 min at 4˚C and were subsequently lyophilized (ScanVac Freezedryers CoolSafe 55-9, Scientific Laboratory Supplies, Yorkshire, UK). For the preparation of the PLGA-CPΑ 160-189 NPs, the same volume of peptide solution as previously was added into 3 ml of a PLGA/chloroform solution at a final concentration of 30 mg/ml. Finally, to prepare blank PLGA NPs for controls, the CPΑ 160-189 aqueous solution was replaced with 0.3 ml of PBS. Lyophilized PLGA NPs were stored at 4˚C.
The surface morphology of the synthesized PLGA NPs was observed by scanning electron microscopy (SEM) (JEOL JSM 6300, Jeol, Peabody, MA, USA). Accordingly, the lyophilized NPs were first double coated with a gold layer under vacuum and then examined by SEM. The average particle diameter of the PLGA NPs was determined by photon correlation spectroscopy and their zeta potential by aqueous electrophoresis measurements (Malvern Nano ZS90, UK). The measurements were performed with aqueous dispersions of NPs prior to lyophilization.

Determination of peptide and adjuvant loadings
The MicroBCA Protein assay kit (Thermo Scientific) was employed to determine CPΑ 160-189 encapsulation (μg/mg) in the PLGA NPs. Accordingly, 2.5 mg of lyophilized PLGA NPs were dissolved in 0.25 ml DMSO for 1 h following a further dissolution in 1.25 ml of 0.05 M NaOH/ 0.5% SDS for 3 h at 25˚C. Blank PLGA NPs were used as negative controls. The absorbance of the samples was measured at 562 nm using a microplate reader (EL808IU-PC, BioTek Instruments, Inc., Winooski VT, USA). Antigen encapsulation efficiency (EE) was calculated by the ratio of the antigen mass in the NPs over the total mass of antigen used. Also, the antigen loading was calculated by the ratio of the encapsulated mass of antigen over the total mass of PLGA NPs.
A Limulus Amebocyte Lysate (LAL) kit (Thermo Scientific) was used for the determination of the MPLA loading (μg/mg) in the PLGA NPs. Standard curve was established using different concentrations of aqueous MPLA solutions ranging from 0.01 to 10 ng/ml, which was found to be linear for the MPLA concentration range used, with a correlation coefficient of R 2 = 0.9994. The encapsulation efficiency of MPLA was calculated by the ratio of the measured MPLA mass in the NPs over the total mass of MPLA used and the MPLA loading was calculated by the ratio of encapsulated MPLA mass over the total mass of the PLGA NPs.
For the determination of in vitro release of CPΑ 160-189 and MPLA, PLGA NPs were dispersed in PBS at a final concentration of 1 mg/ml and were incubated at 37˚C under constant shacking at 120.7×g. At predetermined time points (0, 1, 2, 4, 6, 8, 12, 24, 48 h, 1 and 2 weeks) 1 ml of the dispersion was centrifuged at 13,860×g for 10 min at 4˚C. Then, the supernatants were collected and the amount of CPΑ 160-189 and MPLA were determined using the Micro-BCA and LAL kits, respectively.

Generation of bone marrow-derived DCs
DCs were generated from pluripotent bone marrow stem cells of BALB/c mice in the presence of rmGM-CSF, as reported previously [29]. On day 7, non-adherent and semi-adherent cells were collected and phenotypic analysis was performed by flow cytometry using antibodies against CD11c and CD8a surface markers. According to trypan blue exclusion, cell viability was >95% and the percentage of CD11c + CD8acells was >75%, as assayed by FACS analysis.
Flow cytometry analysis for the detection of DCs maturation and cytokine production DCs maturation induced by PLGA NPs was determined by flow cytometry. All antibodies used were obtained from BD Biosciences (Erembodegem, Belgium). For this purpose, DCs were cultured into a 24-well plate at a density of 1x10 6 cells/ml/well in the presence of PLGA-MPLA, PLGA-CPA 160-189 or PLGA-CPA 160-189 +MPLA NPs, or soluble CPΑ 160-189 in the presence or not of soluble MPLA at various doses for 24 h at 37˚C in a humidified CO 2 incubator. DCs in medium alone and DCs stimulated with LPS (1 μg/ml) were used as negative and positive control, respectively. At the end of incubation, wells were washed to remove free PLGA NPs followed by a wash with FACS buffer (PBS-3% (v/v) FBS). The cells were then labeled with PEconjugated anti-mouse CD40, CD80, CD83, CD86, MHCI (1:100 dilution) or MHCII (1:200 dilution) mAbs for 30 min at 4˚C. For intracellular staining, cells were subjected to brefeldin A (2.5 μg/ml) for the last 4 h of culture and then were fixed with 2% paraformaldehyde and stained with PE-conjugated IL-12p40 mAb (1:100 dilution) in permeabilization buffer (FACS buffer supplemented with 0.1% saponin). After staining, cells were washed with FACS buffer and subjected to flow cytometric analysis using a FACS Calibur system (Becton-Dickinson, San Jose, CA, USA). Data were analyzed using FlowJo software version 10.0 (Tree Star, Inc., Ashland, OR, USA).

In vitro T cell priming assay
DCs (1x10 6 cells/ml) that had been pulsed with medium, PLGA-CPA 160-189 or PLGA-CPA 160-189 +MPLA NPs for 24 h were co-cultured with naive splenocytes of similar origin, at a responder/stimulator ratio of 5:1 in a 96-well round-bottom plate for 96 h. Splenocytes cultured in medium alone or in the presence of Con A (6 μg/ml; Sigma) served as negative or positive control of T cell proliferation, respectively. Proliferation was determined by addition of 0.5 μCi of Real time quantitative PCR for the detection of cytokine expression IFN-γ, IL-4 and IL-10 mRNA expression by DCs-stimulated T cells was determined by real time quantitative PCR (qPCR). Specifically, complementary DNA (cDNA) was synthesized using RT 2 HT First Strand Kit (Qiagen, Maryland, USA) from 1 μg of total RNA isolated from cells from each culture using the RNeasy mini kit (Qiagen) in GeneAmp PCR System 9700 (Applied Biosystems, NY, USA). Determination of IFN-γ, IL-4 and IL-10 mRNA levels was carried out by a SYBR green real time qPCR using a custom RT 2 Profiler PCR Array (CAPM13028, Qiagen) in a Stratagene Mx3005P PCR System (Agilent Technologies, Santa Clara, CA, USA), according to manufacturer's instructions. Mouse Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control to avoid variations between the samples. Briefly, cDNA samples were subjected to an initial denaturation for 10 min at 95˚C, followed by 40 cycles of denaturation at 95˚C for 15 sec, and extension at 60˚C for 30 sec, ending with incubation at 72˚C for 5 min. Each reaction was carried out in triplicate. Non-pulsed DCs-stimulated T cells were used as calibrators and results were expressed as fold change by the ΔΔCt method. Briefly, the normalized to GAPDH gene expression  in cultures of interest were divided by the normalized to GAPDH gene expression (2 -ΔΔCt ) in the calibrators, where value of 1 is the gene expression baseline in non-pulsed DCs-stimulated T cell cultures. A non-template control without genetic material was included to eliminate nonspecific reactions.

Vaccination schedules with peptide-based nanovaccines and challenge infection
Groups of BALB/c mice (n = 25/group) were vaccinated subcutaneously in the upper and dorsal region with (i) PLGA-MPLA NPs, (ii) PLGA-CPA 160-189 NPs or (iii) PLGA-CPA 160-189 +MPLA NPs in a total volume of 100 μl sterile PBS, as described in Table 1. Taken into account the peptide and MPLA loadings, each mouse received 2 μg of peptide with or without 1 μg MPLA or 1 μg MPLA alone. Two booster doses followed at a 2 weeks interval. Mice receiving PBS alone served as control. Two weeks after each injection, mice (n = 5/group) were euthanized and sera samples and spleens were collected to analyze the immune response induced by vaccination. Two weeks after the final boost, the remaining vaccinated and nonvaccinated mice were challenged by injecting freshly transformed 2x10 7 stationary phase L. infantum promastigotes intravenously. Non-vaccinated non-infected mice served as negative control. The prophylactic efficacy of PLGA NPs formulations was assessed in spleen and liver at 1 and 4 months post challenge. The percentage of inhibition of parasite multiplication was calculated in comparison with the non-vaccinated control using the following formula: percentage of inhibition = (number of parasites from non-vaccinated infected control groupnumber of parasites from the vaccinated infected group / number of parasites from non-vaccinated infected control group) x 100.

Evaluation of peptide-based nanovaccines toxicity and induction of inflammation in mice
In order to examine in vivo toxicity of the synthesized PLGA NPs, BALB/c mice (n = 5/group) were injected subcutaneously at the indicated doses in 100 μl PBS. BALB/c mice (n = 5/group) injected with PBS or LPS (1 μg) served as negative or positive control of inflammation, respectively. The viability and the behavior of mice were observed at predetermined intervals over a 3 days post injection period. Furthermore, for the determination of inflammatory mediators, blood was collected from mice (n = 5/group) 4 h post vaccination. Sera were analyzed for IL-1β, IL-6, TNFα and MCP-1 by magnetic bead multiplex array (Millipore, Billerica, MA, USA). Data were acquired on a Luminex 200 (Oosterhoot, The Netherlands) and analyzed using xPONENT software (Luminex).

Evaluation of parasite load by limiting dilution assay
The parasite burden was quantified by a limiting dilution assay as described previously [32]. The wells were examined for viable and motile promastigotes every 3 days, and the reciprocal of the highest dilution that was positive for parasites was considered to be the number of parasites per mg of tissue. The total parasite burden was calculated by reference to the whole organ weight.

Spleen cell lymphoproliferation and determination of cytokine production
Spleens were aseptically excised from mice of all experimental groups two weeks after each vaccination (Table 1)  In parallel, similar spleen cell cultures were performed for cytokine production 2 weeks after the end of vaccinations and 1 month post challenge. Specifically, spleen cells (1x10 6 cells/ ml) from all groups of mice were plated in 24-well plates and incubated with CPA 160-189 (5 μg/ ml) or SLA (12.5 μg/ml) for 72 hours. At the end of incubation period, cell-free supernatants were harvested by centrifugation, aliquoted and stored at -70˚C until assayed for specific cytokines. The levels of IL-2, IFN-γ, TNFα, IL-4, and IL-10 were measured by magnetic bead multiplex array (Millipore).

Flow cytometry analysis for determination of IFN-γ-producing cells
For intracellular analysis of IFN-γ-producing CD4 + and CD8 + T lymphocytes from all groups of mice at the end of vaccinations, flow cytometry analysis was carried out. In brief, two weeks after the end of vaccinations splenocytes were cultured as described above in the presence of 5 μg/ml of CPA 160-189 or medium alone. At the last 4 h of incubation, cells were exposed to brefeldin A, washed in FACS buffer and stained with FITC-conjugated anti-CD4 or anti-CD8 mAbs (1:100 dilution; BD Biosciences) for 30 min. Then, cells were treated with permeabilization buffer and stained for 30 min on ice with PE-conjugated anti-IFN-γ mAb (1:100 dilution; BD Biosciences).
Determination of CPA 160-189 -specific antibodies Sera were collected from mice of all experimental groups 2 weeks after each vaccination ( Table 1) and production of IgG, IgG1 and IgG2a antibodies against CPA 160-189 was determined by ELISA. In brief, 96-well microtiter plates were coated with 5 μg/ml of CPA  and incubated overnight at 4˚C. After 3 washes with washing buffer (PBS with 0.05% Tween-20), plates were coated with PBS-2% (w/v) BSA for 2 h. Then, sera samples were added at a dilution of 1:100 and incubated for 90 min. After that, biotinylated anti-mouse IgG1 (1 μg/ml) and IgG2a (250 ng/ml) (both obtained from AbD Serotec, Oxford, UK), or HRP-conjugated anti-mouse IgG (1:5000) (Thermo Scientific) were added for 1 h at 37˚C. When biotinylated Abs were used, streptavidin-HRP was added (1:5000) and incubated for another 1 h at 37˚C. The enzyme-labeled complexes were detected by reaction with TMB substrate and the reaction was stopped by adding 2 M sulfuric acid. The absorbance was measured at 450 nm using an ELISA microplate spectrophotometer (MRX).

Statistical analysis
All results are expressed as mean±standard deviation (SD). GraphPad Prism version 5.0 software (San Diego, CA, USA) was used for statistical analysis. One-way ANOVA with multiplecomparisons, Tukey-Kramer post test or two-way ANOVA with Bonferroni post test were performed, when required, in order to assess statistical differences among experimental groups. A value of p<0.05 was considered significant for all analyses.

Results
CPA 160-189 peptide is highly immunoreactive in the serum of naturally infected dogs with L. infantum CPA is a highly immunoreactive molecule that is recognized by the sera obtained from either recovered or active cases of CL and VL, as well as by sera from asymptomatic or symptomatic dogs naturally infected with L. infantum. In order to determine whether CPA 160-189 contains epitopes that are recognized by antibodies present in the serum of naturally infected dogs, an ELISA against CPA 160-189 was performed. SLA of L. infantum was used as an internal control and a comparative antigen to CPA 160-189 . According to the results obtained, CPΑ 160-189 peptide was recognized by highly reactive IgG antibodies present in both asymptomatic (OD values: 0.572±0.324, p< 0.05) and symptomatic (OD values: 0.674±0.322, p< 0.01) dog sera with a cut-off value of 0.173 (Fig 1), indicating the presence of antigenic epitopes of the synthetic peptide. As expected, healthy dogs recognized neither SLA nor CPA 160-189 (Fig 1).

Characterization of the synthesized PLGA NPs
Peptide-based vaccines may benefit from particulate delivery systems and the presence of the right adjuvant. For that reason, CPA 160-189 was chosen to be co-encapsulated with MPLA into PLGA NPs serving as antigen vehicle. According to scanning electron microscopy, the synthesized PLGA NPs were characterized by a well-defined spherical shape (Fig 2A and 2B). The average diameter of different type of synthesized PLGA NPs was in the range of 309.9± 15.4 to 316.6±6.6 nm as determined by photon correlation spectroscopy (% intensity) (Fig 2C  and 2D) with a z potential varying from -18.2±7.7 to -19.1±5.7 mV ( Table 2), indicative of the presence of free -COOH groups on the NP surface. The mean encapsulation efficiency (EE) of CPΑ 160-189 ranged from 77.88±0.47 to 83.8±5.6%, and the antigen loading was 17±0.7 μg and 18.2±2.1 μg of CPΑ 160-189 per mg of PLGA-CPA 160-189 and PLGA-CPA 160-189 +MPLA NPs, respectively ( Table 2). MPLA encapsulation in PLGA-CPA 160-189 +MPLA NPs was 80.1±8.2% and in PLGA-MPLA NPs was 54.5±3.1% corresponding to MPLA loading of 8.7±2.7 μg and 6.1±0.3 μg per mg of PLGA NPs, respectively ( Table 2). In vitro release experiments at pH 7.4 revealed an initial burst release of CPA 160-189 in both NPs formulations in the first 1 hour of the study reaching 39.5% and 53.7% in PLGA-CPA 160-189 NPs and PLGA-CPA 160-189 +MPLA NPs, respectively, followed by a gradual increase until 24 hours of incubation (PLGA-CPA 160-189 NPs: 63.1±1.5% and PLGA-CPA 160-189 +MPLA NPs: 76.2±0.6%). After that time point, antigen release profile was characterized by a plateau. Finally, after 2 weeks the cumulative percent of released CPA 160-189 reached 69.6±3.9% and 86.56±3.4% in PLGA-CPA 160-189 NPs and PLGA-CPA 160-189 +MPLA NPs, respectively ( Fig 2E). In contrast, the percent of MPLA released in the same conditions reached only 8% after 24 h of incubation and remained stable until the end of the study (9.9±2%; Fig 2F).
The most potent signatures of DCs maturation were obtained with the PLGA-CPA 160-189 +MPLA NPs vaccine DCs play a central role in linking innate and adaptive immunity by successfully presenting antigens to T cells through MHC class I and/or II molecules. Moreover, MPLA, a synthetic analog of LPS, has the potential to activate DCs without the toxic effects of LPS, leading to activation of CD4 + Th1 and CD8 + T cell populations. In order to evaluate whether the uptake of the synthesized PLGA NPs could induce maturation of DCs, bone marrow-derived DCs were exposed to different PLGA NPs and the surface expression of CD40, CD86, CD80 and CD83 co-stimulatory and MHCI and MHCII molecules was determined by flow cytometry. For that reason, preliminary experiments were conducted in order to determine the optimum dose of PLGA NPs for efficient maturation of DCs assessed by CD40 and CD86 molecules expression. According to the results, PLGA-CPA 160-189 NPs could not induce CD40 and CD86 co-stimulatory molecules expression, as assessed by low levels of MFI compared to DCs treated with medium alone (S1 Fig). In contrast, co-encapsulation of MPLA led to a dose-dependent upregulation of both maturation markers with the optimum dose of PLGA NPs ranging between 1 and 2 μg of CPA 160-189 and 0.5 and 1 μg of MPLA, whereas at higher doses an advert effect on DCs maturation was observed (S1  (Fig 3A-3F). In contrast, the soluble mixture of peptide-adjuvant induced a minimal, not statistically significant increase in most of the maturation markers, except from CD80 and CD83, which was lower compared to that detected when DCs were pulsed with PLGA-CPA 160 (Fig 3A-3F). Furthermore, pulsing with these PLGA NPs having co-encapsulated CPA 160-189 and MPLA resulted to a significant increase of IL-12-producing DCs (7.7±3.1% vs 0.8±0.6%, p<0.05) compared to control (Fig 3G).
Subsequently, the capacity of these DCs to prime naive T cells by presenting CPA 160-189 peptide was evaluated. To this end, DCs pulsed with the synthesized PLGA NPs were co-cultured with naive splenocytes of the same origin and their proliferation was assessed by 3 [H]-thymidine incorporation. According to the results, DCs pulsed with PLGA-CPA 160-189 NPs were capable of presenting the antigen since a 3.6-fold enhanced splenocyte proliferation was detected in comparison to splenocytes primed by DCs cultured in medium alone (SI: 22.4±5.5 vs 6.1±3.7, p>0.05) (Fig 4A). However, co-encapsulation of CPA 160-189 with MPLA into PLGA NPs enhanced by 2-fold spleen cell proliferation compared to PLGA-CPA 160-189 NPs (PLGA-CPA 160-189 + MPLA: 50.5±11.7 vs PLGA-CPA 160-189 : 22.4±5.5, p<0.05) (Fig 4A). In order to unveil whether DCs stimulated with the above PLGA NPs induced the differentiation and proliferation of peptide-specific Th1 or Th2 cell populations, analysis of mRNA expression for IFN-γ, IL-4 and IL-10 cytokines that are indicative of the respective T cell populations, was conducted. According to the results, priming of naive splenocytes with DCs that have been pulsed with the PLGA-CPA 160-89 +MPLA NPs stimulated significant upregulation of both IFN-γ and IL-10 transcripts by 1.5-fold (p<0.05) over those spleen cell cultures treated with immature DCs, whereas splenocytes that proliferated in the presence of PLGA-CPA 160-189 NPs-pulsed DCs did not alter   Peptide-Based Vaccination against Visceral Leishmaniasis their IFN-γ and IL-10 expression compared to the control group (Fig 4B). On the contrary, IL-4 transcripts were not affected compared to the control group with both treatments (Fig 4B).
Taking together, these data indicated the requirement for the peptide and the adjuvant to be present in the same nanoparticulate formulation, since co-encapsulation of CPA 160-189 and MPLA in PLGA NPs led to differentiation of IL-12-producing DCs and in vitro priming of CPA 160-189 -specific T cell populations.

BALB/c mice after vaccination with PLGA NPs did not show any signs of toxicity and inflammation
Before processing to assessment of the synthesized PLGA NPs immunogenicity in vivo, their biocompatibility was investigated. All mice exhibited normal behavior throughout the study period. Moreover, induction of acute inflammation was not detected after the administration of all PLGA NPs, since IL-1β, IL-6, TNFα and MCP-1 production levels in the sera of vaccinated mice were detected at similar levels to the negative control group (Table 3) and were significantly lower to the positive control group injected with LPS.
In vitro analysis of mice sera showed that PLGA-CPA 160-189 +MPLA NPs also generated secondary T-cell dependent sero-responses, since enhanced levels of peptide-specific IgG antibodies were detected after the second vaccination (1 st boost: 6-fold increase, p<0.001) over control groups, followed by PLGA-CPA 160-189 NPs-vaccinated mice (1 st boost: 2.5-fold increase). However, at the end of vaccination both groups of vaccinated mice had comparable levels of CPA 160-189 -specific IgG antibodies (7-fold increase, p<0.001) (Fig 6A). Assessment of IgG1 and IgG2a antibodies showed that both isotypes were produced with a bias towards IgG1 (Fig 6B). Conclusively, the above results suggested that vaccination with PLGA-CPA 160-189 +MPLA NPs stimulated the activation and differentiation of CPA 160-189 -specific CD4 + Th1 and CD8 + T cell effector cells.
Vaccination with PLGA-CPA 160-189 +MPLA NPs induced partial protection against L. infantum In order to elucidate whether the PLGA NPs-induced CPA 160-189 -specific T cells detected could confer protection against Leishmania parasites, the parasite burden was determined in liver and spleen 1 month later by limiting dilution assay. According to the results, vaccination reduced hepatic parasite burden by 34.2% and 48.2% in PLGA-CPA 160-189 NPs-and in PLGA-CPA 160-189 +MPLA NPs-vaccinated mice, compared to the PBS control group, indicating that vaccinations could promote the self-curing response seen in BALB/c livers ( Fig 8A). Moreover, evaluation of splenic parasite burden in mice vaccinated with PLGA-CPA 160-189 NPs showed a 32.2% reduction when compared with non-vaccinated mice, which was further enhanced when mice were vaccinated with PLGA-CPA 160-189 +MPLA NPs resulting in the significant reduction in parasite load of 90.2% (p<0.01) (Fig 8B). Evaluation of parasite burden 4 months post challenge showed that although PLGA-CPA 160-189 +MPLA NPs-vaccinated mice preserved the reduced parasite burden in liver (61.5% reduction), an increase in the splenic parasite burden was detected (30.6% reduction) indicating a partial vaccine-induced protection ( Fig 9A). These results were well correlated with spleen and liver weights as compared with the PBS control group (Fig 9B). In all cases, no effect of the vaccination with PLGA-M-PLA NPs over infection was detected, confirming that the protection seen in liver and spleen through PLGA NPs was CPA 160-189 -specific.

Discussion
Despite the fact that many attempts to design an effective vaccine against leishmaniasis have been made, when these experimental vaccines reached the clinical trials phase they failed to initiate strong T cell responses [3,4,5]. Difficulties in the design of such a vaccine for induction of T cell mediated immunity are originated not only from the high diversity and variability of the parasite, but also the HLA polymorphisms of the human population, thus affecting the specificities of T cell responses in different individuals. Bioinformatic analysis of protein sequences offers a solution towards this problem by designing synthetic peptides consisting of selected conserved epitopes among different parasite strains that are recognized by a number of different HLA molecules [33,34,35]. Accordingly, in the present study, the selected antigen to be used in the experimental vaccine was a 30-mer peptide, namely CPA 160-189 , designed by our research group to contain promiscuous overlapping human and murine MHC class I and II-restricted epitopes. CPA 160-189 peptide's immunogenicity was confirmed when administered in emulsion with Freund's adjuvant in BALB/c mice, since it induced peptide-specific Th1 and CD8 + T cell responses [27].
As DCs play a central role in priming and controlling T cell mediated immune responses, efficient delivery of antigens to this cell population is a critical issue in the design of vaccine formulations for generation of effective cellular immune responses. In this context, nanoparticle delivery systems hold promise for achieving the appropriate type of immune responses. In the present study, the vaccine was designed in order to target effectively DCs, by using PLGA NPs with a relatively small size (~310 nm) to facilitate their uptake by APCs and free drainage to the lymphatic organs. According to previous studies, NPs with size smaller than 10 μm were more efficiently phagocytosed by APCs, including DCs, and trafficked towards the lymph nodes in a DC-dependent manner, which is an important aspect of vaccine delivery [36]. Indeed, in a recently published research of our group, we confirmed that these PLGA NPs were efficiently taken up by DCs in vitro and when were injected subcutaneously in mice they were phagocytosed by APCs in lymphoid organs, such as DCs [37].
Further, in the present study, it was shown that MPLA adjuvant encapsulation in PLGA NPs loaded with CPA 160-189 was pivotal for triggering DCs functional maturation, as expressed by increased surface expression of co-stimulatory, MHC class I and II molecules and IL-12 production. It is known that MPLA-TLR4 interaction on the surface of DCs induces their functional activation characterized by maturation and expression of pro-inflammatory cytokines, such as IL-12, which are crucial for the activation of Th1 immune responses [38,39,40]. Moreover, the detection of enhanced CD83 expression levels on the surface of DCs triggered with PLGA-CPA 160-189 +MPLA NPs indicated the efficiency of these DCs to stimulate peptidespecific CD4 + and CD8 + T cells activation. Respectively, results of this study showed that in vitro priming of naive spleen cells with DCs pulsed with PLGA-CPA 160-189 +MPLA NPs induced the differentiation and activation of CPA 160-189 -specific T cells characterized by upregulated IFN-γ transcripts, further confirming the effectiveness of this delivery system for supporting polarized Th1 and/or CD8 + T cell immune responses through antigen presentation in context of MHC class II and/or MHC class I molecules. In a recent study, Maji et al showed the importance of adding the MPL adjuvant to liposomal rgp63 which led to a significant enhancement of the antigen presentation by DCs through TAP-dependent MHC class I pathway resulting in more efficient antigen-specific CD8 + T cell responses [13]. However, it must be noted that in our study, significant levels of IL-10 transcripts were also detected suggesting the activation of CPA 160-189 -specific T regulatory cell subpopulations.
Evaluation of PLGA NPs effectiveness to target DCs in vivo has been tested indirectly by assessing the development of CPA 160-189 -specific clones after mice vaccination. Despite the fact that mice received an extremely low dose (2 μg) of peptide, PLGA NPs effectively induced T and B cell CPA 160-189 -specific clonal expansion as indicated by recall assays and IgG detection, underlining the efficacy of PLGA NPs as peptide delivery system. Similar results have been observed in other studies using PLGA NPs for the development of experimental vaccines against cancer or various infections, such as malaria, and their efficacy to induce the desirable immune responses was attributed to the encapsulation of the MPLA adjuvant [41,42]. Interestingly, in contrast to in vitro results, vaccination with PLGA-CPA 160-189 NPs led to similar levels of CPA 160-189 -specific T cell clonal expansion compared to that detected after vaccination with PLGA NPs loaded with CPA 160-189 and MPLA. According to flow cytometry results, these proliferating splenocytes obtained from PLGA-CPA 160-189 +MPLA NPs-vaccinated mice contained both IFN-γ-producing CD4 + and CD8 + T cells. The above results confirmed and further extended our previous work, showing CPA 160-189 peptide's immunogenicity by eliciting a mixed Th1/Th2 response followed by almost equal CPA 160-189 -specific IgG2a and IgG1 production, in parallel with CD8 + T cells activation [27]. Possibly, CD8 + T cell activation detected in the present study was catalyzed by the presence of even low amounts of IL-4, as it was shown in the cytokine assay of the present study. The important role of IL-4 in the generation of CD8 + T cell memory against leishmaniasis has been shown in previous studies [43,44]. More specifically, the protective effect of different antigens, such as HASPB1 and histone H1, against VL was attributed to an IL-4-mediated activation of CD8 + T cells [45,46]. Moreover, splenocytes obtained from PLGA-CPA 160-189 +MPLA NPs-vaccinated mice produced also significant amounts of IL-2 and TNFα which along with IFN-γ suggest the existence of vaccineinduced effector T cell populations. These T cell populations are considered significant for vaccine efficacy to induce long-term protection against various pathogens, among them Leishmania [47,48].
In the area of nanoparticles vaccination, previous studies have shown that liposomal delivery of cysteine proteases CPA, CPB and CPC in combinations or alone, soluble leishmanial antigen and recombinant gp63 in the presence of MPLA adjuvant induced short and longterm immunity against VL due to the presence of both antigen-specific CD4 + and CD8 + T cell responses [49,50,51]. Also, it has been shown that when PLGA NPs were used as antigen vehicle in anti-tumor vaccines, a potent activation of antigen-specific CD8 + T cells was detected [52,53,54,55]. Since Leishmania antigen-specific CD4 + and CD8 + T cells are essential for immunity against leishmaniasis [56,57], the protective effect of these populations detected in the present study was assessed by challenging vaccinated mice with a highly virulent strain of L. infantum promastigotes. The immune response elicited by PLGA-CPA 160-189 +MPLA NPs vaccination conferred significant reduction of parasite burden in spleen and liver by 90% and 40%, respectively. This decrease was positively correlated with the enhanced proliferation of splenocytes in response to CPA 160-189 and SLA stimulation in comparison to control infected groups.
Determination of cytokine profile showed high levels of IL-2, IFN-γ and TNFα production contrary to the minimal levels of IL-4 and IL-10 leading to enhanced IFN-γ/IL-4 and IFN-γ/ IL-10 ratios. It is well documented that whereas IL-4 does not play a decisive role in visceral infection establishment, IL-10 production by splenic cells correlates well with disease progression and pathology in human and experimental disease [58,59,60]. IL-10 has been shown to block Th1 activation and consequently cytotoxic response by down-regulating IFN-γ levels. Further, it decreases the ability of macrophages to destroy parasite by deactivating them. On the other hand, protective immunity against VL is dependent on IL-12-driven Th1 immune response and IFN-γ synergizes with TNFα resulting in the induction of parasite killing by macrophages [61]. However, the simultaneous existence of IL-2, IFN-γ and TNFα producers was followed by a partial protection, since determination of parasite load at 4 months post challenge showed that mice had limited vaccine efficacy to restrain uncontrolled parasite expansion in spleen with only 30% reduction, whereas a decrease in hepatic parasite burden was observed (61%). These results, in contrast to previous findings [47,48], indicated that CPA 160-189 -induced cell immune responses were not capable for the maintenance of long-term protective immunity against the parasite.
According to studies exploring CPA's potential as an effective vaccine against leishmaniasis, CPA conferred significant protection in the experimental models of CL and VL, as wells as in the experimental model of canine leishmaniasis when was administered with other immunogenic proteins [62,63,64,65]. However, the fact that a single peptide of CPA (CPA 160-189 ) coencapsulated with MPLA in PLGA NPs, without the presence of peptides extracted from other immunogenic parasitic molecules, proved to confer significant protection further supporting the appropriateness of the current strategy for the peptide design. Furthermore, the proposed vaccine could be improved by encapsulating more than one synthetic peptides obtained from different Leishmania proteins in order to achieve more intense T-cell responses, since previous studies support that vaccines that address a broad range of specificities are capable of inducing polyclonal effector T cells promoting protection [64]. In addition, in the light of recent findings, anti-leishmanial vaccine efficacy could be improved by including vector-derived molecules in combination with parasitic molecules [66].
Taken together, the data of the present study could provide the basis for the development of peptide-based nanovaccines against leishmaniasis, since it reveals that vaccination with nanovaccines that contain rationally designed multi-epitope peptides covering areas that interact both with MHC class I and II molecules in combination with the appropriate adjuvant and biocompatible delivery system could be a promising approach for the induction of desirable protective immune responses.