Two multi-epitope peptides of CPA (CPA_p2: 160-189 aa and CPA_p3: 273-302 aa), two multi-epitope peptides of histone H1 (H1_p1: 1-20 aa and H1_p3: 43-61 aa), and one multi-epitope peptide of KMP-11 (KMP-11_p1: 4-23 aa), derived from a previously described in silico analysis of the three L. infantum proteins (29) and designed in a way to contain HLA-restricted epitopes (Table 1), formed the basis for the design of chimeric peptides (one for each protein) with the use of proper linkers. HLA A*0201-restricted epitopes derived from the L. infantum proteins and included in the chimeric peptides were predicted using the online available algorithms SYFPEITHI (30) and EpiJen (31) with a cutoff score adjusted to ≥18 and <50 nM, respectively (Table 2). SYFPEITHI was also used for the in silico prediction of potent H2-Kb- or H2-Db-restricted epitopes (Table 2). The chimeric peptides (chCPAp, chH1p, and chKMP-11p) were synthesized by GeneCust (Labbx, Dudelange, Luxembourg) with ≥95% purity. Synthetic chimeric peptides were dissolved in DMSO or dH₂O, according to their hydrophobicity, by vigorous pipetting and stored at −80°C in aliquots until use.

Poly(lactic-co-glycolic) acid 75:25 (Resomer RG752H, MW: 4–15 kDa), polyvinyl alcohol (PVA; MW: 30–70 kDa, 87–90% hydrolyzed), MPLA from Salmonella minnesota, phosphate-buffered saline (PBS; 10×, pH 7.4), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide 98% (NHS) were purchased from Sigma-Aldrich (Vienna, Austria). All the other reagents were of analytical grade and commercially available. PLGA NPs containing the chimeric peptide (chCPAp, chH1p, or chKMP-11p) and the adjuvant MPLA were prepared by the double emulsion method, as previously described (32). 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). Water-in-oil (w/o) emulsion was then formed by adding 0.3 ml of the chimeric 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 (Vibra Cell VC-505; Sonics, Newtown, CO, USA) at 40% amplitude for 45 s. Subsequently, the primary emulsion (w/o) was added into 12 ml of 1% (w/v) PVA aqueous 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; LaboGene ApS, Lynge, Denmark). For the preparation of the PLGA NPs loaded with each of the chimeric peptides, 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. Lyophilized PLGA NPs were stored at 4°C.

For specific purposes, PLGA NPs were surface modified with a synthetic octapeptide (p8: CYTYQGKL; JPT, Berlin, Germany) that mimics the TNFα-docking region with the TNFRII. The peptide was conjugated to NPs via a two-step carbodiimide method (33, 34). Accordingly, 1.5 ml of a 7 wt% EDC solution and 1.5 ml of a 0.3 wt% NHS solution both prepared in 20 mM HEPES/NaOH buffer containing 1% (v/v) Pluronic F-68 at pH 7 were added into 1 ml of PLGA NPs (empty or loaded with each of the chimeric peptides) dispersion in the same buffer at a final concentration of 20 mg/ml, in order to activate the PLGA carboxyl groups (35). The mixture was then stirred end-over-end for 2 h at room temperature. The residual reagents were removed by centrifugation at 13,860 × g for 10 min at 25°C. Subsequently, 0.3 ml of 0.1% (w/v) p8 solution in 20 mM HEPES/NaOH buffer containing 1% (v/v) Pluronic F-68 at pH 7 were added to the PLGA NPs dispersion, and the mixture was incubated for 18 h at room temperature. To saturate free-coupling sites, 500 µL of 20% (w/v) glycine in 20 mM HEPES/NaOH buffer containing 1% (v/v) Pluronic F-68 at pH 7.0 was added and incubated end-over-end for 1 h at 25°C. The PLGA NPs were subsequently purified by means of two successive centrifugation–redispersion cycles at 13,860 × g for 10 min at 25°C in the same buffer. The finally collected NPs were subsequently lyophilized and stored at 4°C.

The surface morphology of the PLGA NPs was observed by scanning electron microscopy (SEM) (JEOL JSM 6300). 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 (Nano ZS90; Malvern Instruments Ltd., Malvern, UK). The measurements were performed with aqueous dispersions of NPs prior to their lyophilization.

The MicroBCA Protein assay kit (Thermo Scientific, Rockford, IL, USA) was employed to determine the chimeric peptide load (wt%) in the PLGA NPs according to the manufacturer’s instructions. Briefly, 2.5 mg of lyophilized PLGA NPs was dissolved in 0.25 ml DMSO for 1 h following a further dissolution in 1.25 ml of 0.05 N NaOH/0.5% (v/v) SDS for 3 h at 25°C. PLGA NPs without encapsulated chimeric peptide were used as controls. The absorbance of the samples was measured at 562 nm using a microplate reader (EL808IU-PC, BioTek Instruments Inc., Winooski, VT, USA). Peptide encapsulation efficiency (%) was calculated by the ratio of the peptide mass in the PLGA NPs over the total mass of peptide used. Peptide load (wt%) was calculated by the ratio of the encapsulated mass of peptide over the total mass of PLGA NPs.

A Limulus Amebocyte Lysate (LAL) kit (Lonza, Walkersville, MD, USA) was used for the determination of the MPLA load (wt%) in the PLGA NPs according to the manufacturer’s instructions. Briefly, a 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 R² = 0.9994. The encapsulation efficiency (%) of MPLA was calculated by the ratio of the measured MPLA mass in the PLGA NPs over the total mass of MPLA in the recipe. Similarly, the MPLA load (wt%) was calculated by the ratio of encapsulated MPLA mass over the total mass of the PLGA NPs.

A UV–Vis spectrophotometer (Lambda 35; PerkinElmer, Waltham, MA, USA) was used for the determination of p8 amount (wt%) conjugated on the PLGA NPs surface. Accordingly, 2.5 mg of lyophilized p8-conjugated PLGA NPs was dissolved in 0.25 ml DMSO for 1 h following a further dissolution in 1.25 ml 0.05 N NaOH/0.5% (v/v) SDS for 3 h. The absorbance of the samples was measured at 492 nm. The calibration curve was found to be linear over the p8 concentration range of 0.3125–70 µg/ml with a correlation coefficient of R² = 0.9982. Conjugation efficiency (%) of p8 was calculated by the ratio of the measured p8 mass on the PLGA NPs over the total mass of p8 used. Similarly, p8 load (wt%) was calculated by the ratio of conjugated p8 mass over the total mass of the PLGA NPs.

For the assessment of the in vitro release of the chimeric peptides and MPLA, PLGA NPs were dispersed in PBS at a final concentration of 1 mg/ml and incubated at 37°C in a thermomixer (Eppendorf) under constant stirring at 120.7 × g. At predetermined time points (0, 1, 2, 4, 6, 12, 24, 48 h, and 1, 2 weeks), 1 ml of the dispersion was centrifuged at 13,860 × g for 10 min at 4°C. The supernatants were collected, and the amount of the chimeric peptide or MPLA was determined using the MicroBCA or the LAL kit, respectively.

B6.Cg-Tg(HLA-A/H2-D)2Enge/J humanized transgenic mice, provided by the Jackson Laboratory (Bar Harbor, ME, USA), were used in this study. These transgenic mice express an interspecies hybrid class I MHC gene, aad, which contains the alpha-1 and alpha-2 domains of the human HLA-A2.1 gene and the alpha-3 transmembrane and cytoplasmic domains of the mouse H-2D^d gene, under the direction of the human HLA-A2.1 promoter, while they are created on a C57BL/6 background. BALB/c mice, also used in this study, were obtained from the breeding unit of the Hellenic Pasteur Institute (Athens, Greece). Both strains were reared in institutional facilities under specific pathogen-free conditions at ambient temperature of 25°C, receiving a diet of commercial food pellets and water ad libitum; female mice 6–8 weeks old were used. Animal protocols had been approved by the institutional Animal Bioethics Committee regulating according to the National Law 2013/56 and the EU Directive 2010/63/EU for animal experiments and complied with the ARRIVE guidelines. All efforts were made to minimize animal suffering.

Dendritic cells were generated from pluripotent bone marrow stem cells obtained from HLA A2.1 transgenic mice in the presence of recombinant mouse granulocyte macrophage colony-stimulating factor (rmGM-CSF) as previously described (36). Briefly, 3.5 × 10⁶ bone marrow cells obtained from the tibias and femurs of mice were seeded in a 100 mm Petri dish in 10 ml of RPMI-1640 medium (Biochrom AG, Berlin, Germany) supplemented with 2 mM l-glutamine, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin (complete RPMI-1640 medium), 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco, Paisley, UK), and 20 ng/ml rmGM-CSF (Peprotech, London, UK) and cultured for 7 days at 37°C and 5% CO₂. On days 3 and 6, loosely adherent cells were harvested and resuspended in fresh culture medium supplemented with the same dose of rmGM-CSF. On day 7, non-adherent cells were collected and characterized; cell viability was >95% as determined by trypan blue exclusion, and the percentage of CD11c⁺CD8α⁻ cells was >85% as assessed by R-phycoerythrin (R-PE)-conjugated hamster anti-mouse CD11c (clone HL3; BD Biosciences, Erembodegem, Belgium) and FITC-conjugated rat anti-mouse CD8α (clone Lyt2; BD Biosciences) monoclonal antibodies (mAbs) and flow cytometry. For each sample, 10,000 cells were analyzed on a FACS Calibur (Becton-Dickinson, San Jose, CA, USA), and the data were processed using FlowJo V10 software (Tree Star Inc., Ashland, OR, USA).

To analyze the effect of differentially functionalized PLGA nanoformulations on DCs maturation and functional differentiation, DCs were harvested on day 7, seeded in 24-well plates at a density of 1 × 10⁶ cells/ml, and cultured for 24 h at 37°C and 5% CO₂ in the presence of (i) PLGA, (ii) PLGA-MPLA, (iii) p8-PLGA, (iv) PLGA-chCPAp, (v) PLGA-chH1p, (vi) PLGA-chKMP-11p, (vii) a mix of PLGA-chCPAp, PLGA-chH1p, and PLGA-chKMP-11p (mix A), (viii) PLGA-chCPAp-MPLA, (ix) PLGA-chH1p-MPLA, (x) PLGA-chKMP-11p-MPLA, (xi) a mix of PLGA-chCPAp-MPLA, PLGA-chH1p-MPLA, and PLGA-chKMP-11p-MPLA (mix B), (xii) p8-PLGA-chCPAp, (xiii) p8-PLGA-chH1p, (xiv) p8-PLGA-chKMP-11p, and (xv) a mix of p8-PLGA-chCPAp, p8-PLGA-chH1p, and p8-PLGA-chKMP-11p (mix C). In parallel, DCs were cultured under the same conditions in the presence of each soluble chimeric peptide or a mix of soluble chimeric peptides with or without the adjuvant MPLA (mix D or mix E, respectively). Unstimulated DCs or DCs stimulated with 1 µg/ml LPS (Sigma-Aldrich) were used as negative and positive control, respectively. The optimal dose for each chimeric peptide encapsulated in PLGA NPs was determined at 2 µg according to preliminary experiments (data not shown). At the end of incubation, cells were washed with FACS buffer [3% (v/v) FBS in PBS] and then labeled with R-PE-conjugated 1:100 diluted rat anti-mouse CD40 (clone 3/23; BD Biosciences), hamster anti-mouse CD80 (clone 16-10A1; BD Biosciences), rat anti-mouse CD86 (clone GL-1; BD Biosciences), mouse anti-mouse MHCI H-2 Kb/H-2 Db (clone 5041.16.1; Acris, Herford, Germany), or 1:200 diluted rat anti-mouse MHCII I-Ab chain, H2-Eb1 (clone NIMR; Acris), or 1:10 diluted mouse anti-human HLA-ABC (clone G46-2.6; BD Biosciences) mAbs for 30 min at 4°C in the dark. For intracellular staining, cells were subjected to 2.5 µg/ml brefeldin A (Applichem, Darmstadt, Germany) during the last 4 h of culture and then they were fixed with 2% (w/v) paraformaldeyde (PFA; Sigma-Aldrich) in PBS and stained with PE-conjugated 1:100 diluted anti-mouse IL-12p40/p70 (clone C15.6; BD Biosciences) mAb in permeabilization buffer [0.1% (v/v) saponin in FACS buffer] for 30 min at 4°C in the dark. After a washing step with FACS buffer, cells were analyzed by flow cytometry, and data were processed as previously described.

Spleens from BALB/c mice were aseptically excised and teased into single-cell suspension in complete RPMI-1640 medium supplemented with 10% (v/v) FBS. Red blood cells were removed by treating spleen cells with ammonium chloride lysis solution, pH 7.2 (ACK; 0.15 M NH₄Cl, 1 mM KHCO₃, 0.1 mM Na₂EDTA). Lysis reaction was stopped by adding ice-cold complete RPMI-1640 medium, and spleen cells were washed twice. DCs that have been stimulated as described above for 24 h were washed and co-cultured with 2 × 10⁵ naive spleen cells at different ratios (1:5, 1:10, or 1:20) in 96-well round-bottom plates for 96 h at 37°C and 5% CO₂. Spleen cells cultured in medium alone or in the presence of 6 µg/ml concanavalin A (Con A; Sigma-Aldrich) served as negative and positive control of T cell proliferation, respectively. Cells were pulsed with 1 μCi/ml of ³[H]-thymidine (³[H]-TdR; PerkinElmer, MA, USA) for the last 18 h of the culture period and then were harvested. The ³[H]-TdR incorporation was determined on a microplate scintillation counter (Microbeta Trilux, Wallac, Turku, Finland). All samples were run in triplicate.

In parallel, DCs stimulated for 24 h with (i) the mix of PLGA-chCPAp, PLGA-chH1p, and PLGA-chKMP-11p nanoformulations (mix A), (ii) the mix of PLGA-chCPAp-MPLA, PLGA-chH1p-MPLA, and PLGA-chKMP-11p-MPLA nanoformulations (mix B), (iii) the mix of p8-PLGA-chCPAp, p8-PLGA-chH1p, and p8-PLGA-chKMP-11p nanoformulations (mix C), (iv) the mix of soluble chimeric peptides (mix D), and (v) the mix of the soluble chimeric peptides with the adjuvant MPLA (mix E) were co-cultured with 1 × 10⁶ naïve spleen cells from BALB/c mice at a ratio of 1:5 in 24-well plates for 48 h at 37°C and 5% CO₂. Cells were exposed to 2.5 µg/ml brefeldin A during the last 4 h of culture and then washed with FACS buffer and fixed with 2% (w/v) PFA in PBS. Fixed cells were stained with APC-conjugated hamster anti-mouse CD3e (clone 145-2C11), FITC-conjugated rat anti-mouse CD4 (clone RM4-5) or CD8α (clone 53-6.7) mAbs, and R-PE-conjugated rat anti-mouse IFNγ (clone XMG1.2) or IL-4 (clone BVD4-1D11) mAbs in permeabilization buffer for 30 min at 4°C in the dark. All mAbs used in the protocol were purchased from BD Biosciences. After a washing step with FACS buffer, 20,000 cells from each sample were analyzed by flow cytometry, and data were processed as previously described.

To analyze the effect of differentially functionalized PLGA nanoformulations on DCs gene expression, DCs were stimulated with (i) the mix of PLGA-chCPAp, PLGA-chH1p, and PLGA-chKMP-11p nanoformulations (mix A), (ii) the mix of PLGA-chCPAp-MPLA, PLGA-chH1p-MPLA, and PLGA-chKMP-11p-MPLA nanoformulations (mix B), (iii) the mix of p8-PLGA-chCPAp, p8-PLGA-chH1p, and p8-PLGA-chKMP-11p nanoformulations (mix C), and (iv) the mix of soluble chimeric peptides (mix D) at 37°C and 5% CO₂, for 18 h. DCs cultured in medium alone were used as a reference group. Total RNA was isolated with Trizol Reagent (Invitrogen, Karlruhe, Germany) and purified on a Qiagen RNeasy column (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was quantified with ND-1000 Nanodrop (Thermo Fisher Scientific, Wilmington, DE, USA), and RNA integrity was assessed using the RNA 6000 NanoLabChip kit on Agilent Bioanalyzer 2100 (Agilent Technologies, Inc., Palo Alto, CA, USA). Only samples with intact total RNA profiles (retention of both ribosomal bands and the broad central peak of mRNA) and RIN > 7.0 were utilized in microarray analysis. Approximately 300 ng of total RNA were used to generate biotinylated complementary RNA (cRNA) for each group using the GeneChip^® WT PLUS Reagent Kit protocol for whole transcript (WT) expression array, Rev3. Poly-A RNA control added to the RNA test samples as exogenous positive control, and the RNA was reverse transcribed to double-stranded cDNA, and biotinylated cRNA was synthesized and purified according to the protocol. A second cycle of cDNA synthesis followed along with a second purification step. Then, 6 µg of the single-stranded DNA was fragmented, labeled with the appropriate labeling reagent, and hybridized to GeneChip^® Mouse Gene 2.0 ST arrays (Affymetrix, UK). Hybridization took place for 16 h in an Affymetrix GeneChip^® Hybridization Oven 640. Affymetrix GeneChip^® Fluidics Station 450 was used to wash and stain the arrays with streptavidin–phycoerythrin according to the standard antibody amplification protocol for eukaryotic targets. Arrays were scanned on Affymetrix GeneChip^® Scanner 3000 at 570 nm. The Affymetrix eukaryotic hybridization control and Poly-A RNA control were used to ensure efficiency of hybridization and cRNA amplification. Images and data were acquired and analyzed using the Affymetrix^® GeneChip^® Command Console^® Software (AGCC), where initial quality control of the experiment was performed. AGCC was also used to perform robust multi-array average (RMA) normalization and probe set summarization steps on the raw signal intensity files. Principal component analysis (PCA) and heatmap representation of individual values were conducted utilizing appropriate open-source software. Differential expression analysis was performed for stimulated vs. unstimulated DCs utilizing one-way ANOVA (p < 0.05) and a ±0.585log2 (fold change) threshold on mean gene-expression levels. Probe sets that were unannotated or that mapped to the same gene in a many-to-one fashion were removed from the subsequent analysis. Microarray raw files and RMA-summarized data have been deposited in Gene Expression Omnibus under accession number GSE92869.

Mapping of array-specific probe sets to Entrez Gene IDs was conducted using up-to-date annotation package mogene20sttranscriptcluster.db in R. Significantly deregulated genes [p < 0.05, ±0.585log2 (fold change) threshold] from each comparison (mix A–D vs. reference) were subjected to functional enrichment analysis with the R package clusterProfiler (37), setting parameters “pvalueCutoff” = 0.05 and “pAdjustMethod” = “fdr” for multiple comparisons. Terms from Gene Ontology (Biological Process) database slice (38) were tested for enrichment. Mappings between GO terms and Entrez Gene IDs relied on regularly updated R package org.Mm.eg.db. R package Pathview (39) was utilized to superimpose differentially expressed genes upon KEGG pathways. Red and green colors signify upregulation and downregulation on stimulated DCs, respectively.

HLA A2.1 transgenic mice (n = 5) were immunized subcutaneously in the upper and dorsal region with the mix of PLGA-chCPAp-MPLA, PLGA-chH1p-MPLA, and PLGA-chKMP-11p-MPLA nanoformulations (mix B) in a total volume of 100 µl sterile PBS. Taking into account the chimeric peptide and MPLA loads of the above nanoformulations, each mouse received in total 6 µg of chimeric peptides (2 µg of each chimeric peptide) with 3 µg MPLA. Two booster doses—also subcutaneously injected—followed at a 2-week interval. Control groups (n = 5 mice/group) received PLGA-MPLA nanoformulations in PBS or only PBS.

Two weeks after the last immunization, mice were euthanized, and spleens were removed aseptically. To analyze antigen-specific T cell proliferation, single-cell suspensions were prepared in complete RPMI-1640 medium supplemented with 10% (v/v) FBS, as previously described. Spleen cells were cultured in 96-well round bottom plates at a density of 2 × 10⁵ cells/200 μl/well in the presence of 5 µg/ml soluble chCPAp or chH1p or chKMP-11p for 96 h at 37°C and 5% CO₂. Spleen cells cultured in medium alone or in the presence of 6 µg/ml Con A served as negative and positive control of T cell proliferation, respectively. Cells were pulsed with 1 μCi/ml of ³[H]-TdR for the last 18 h of the culture period and then were harvested. The ³[H]-TdR incorporation was determined on a microplate scintillation counter. All samples were run in triplicate, and the results were expressed as Δcpm (cpm of stimulated spleen cells − cpm of unstimulated spleen cells).

In parallel, intracellular cytokine staining and flow cytometry were performed in the spleen cell suspensions from immunized mice in order to identify antigen-specific IFNγ-producing CD8⁺ T cells. In brief, 1 × 10⁶ spleen cells were cultured in 24-well plates in the presence of 5 µg/ml soluble chCPAp or chH1p or chKMP-11p for 48 h, according to previously described protocols (29, 40), at 37°C and 5% CO₂. Spleen cells cultured in medium alone served as negative control. Spleen cells were exposed to 2.5 µg/ml brefeldin A during the last 4 h of culture and then washed with FACS buffer and fixed with 2% (w/v) PFA in PBS. After fixation, spleen cells were stained with APC-conjugated anti-CD3, PE-conjugated anti-IFNγ, and FITC-conjugated anti-CD8 mAbs in permeabilization buffer and analyzed with flow cytometry, as previously described.

A strain of L. infantum (MHOM/GR/2001/GH8) originally isolated from a Greek patient suffering from VL (41) was cultured in vitro and maintained infective through serial passage in BALB/c mice, as previously described (36). Immunized with the mix B and non-immunized (received only PBS) HLA A2.1 transgenic mice (n = 5/group) were infected by injecting intravenously 2 × 10⁷ stationary phase L. infantum promastigotes in 100 µl PBS, 2 weeks after the final booster dose. Mice immunized with PLGA-MPLA nanoformulations and then infected were used as a control group. The protective effect of immunization with the mix B was assessed in liver and spleen of the infected mice at 1 and 2 months post-infection. The protective effect was determined in comparison with the non-immunized control group according to the formula: percentage of reduction in parasite burden = (number of parasites from the non-immunized infected control group − number of parasites from the immunized infected group)/(number of parasites from the non-immunized infected control group) × 100.

The parasite burden was assessed by a limiting dilution assay following well-established protocols (42) with minor modifications. Briefly, livers and spleens were removed aseptically from the euthanized mice and weighed. Suspensions of liver or spleen tissue were prepared in Schneider’s Drosophila medium (Biosera, Boussens, France) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 20% (v/v) FBS to 1 mg/ml final concentration. Serial twofold dilutions of the tissue suspensions were plated in 96-well culture plates and incubated at 26°C for 7 days. After the incubation period, the presence or absence of viable and motile promastigotes was recorded in each well. The reciprocal of the highest dilution that was positive for parasites was considered to be the number of parasites per milligram of tissue. The total parasite burden was calculated by reference to the whole organ weight.

Results are derived from at least two independent experiments and are expressed as the mean value ± SD. One-way ANOVA and Tukey’s multiple comparisons test or two-way ANOVA and Bonferroni multiple comparisons test were performed, when required, to assess statistical differences using the GraphPad Prism software (version 5.0; San Diego, CA, USA). The probability (p) of <0.05 was considered to indicate statistical significance.

The capacity of the synthesized PLGA-based nanoformulations to directly activate and induce maturation of CD11c⁺ bone marrow-derived DCs after 24 h stimulation was assessed by flow cytometry, measuring the surface expression of the hallmark DCs maturation and T-cell co-stimulation markers MHC class I, MHC class II, CD40, CD80, and CD86. According to preliminary experiments, the optimum dose of each chimeric peptide encapsulated in PLGA NPs for efficient DCs maturation was determined at 2 µg (data not shown). As depicted in Figures 2A,B, the encapsulation of chimeric peptides in PLGA NPs resulted in a significant increase in the number of DCs expressing CD40, CD80, CD86, MHC class I, and MHC class II molecules, in comparison to DCs stimulated with soluble peptides (CD40: 72.40 ± 1.27 vs. 35.53 ± 3.41%, p < 0.001; CD80: 81.20 ± 1.56 vs. 56.20 ± 2.78%, p < 0.001; CD86: 74.33 ± 3.99 vs. 43.20 ± 1.13%, p < 0.001; murine MHC class I: 82.15 ± 4.31 vs. 71.60 ± 2.26%, p < 0.05; murine MHC class II: 82.35 ± 0.35 vs. 62.03 ± 2.97%, p < 0.001; hybrid HLA class I: 39.50 ± 0.30 vs. 22.55 ± 3.82%, p < 0.001). Regarding the mean fluorescence index (MFI) values (Figure 2C), significant increase was observed only for CD80, murine MHC class I, and MHC class II molecules in comparison to DCs stimulated with soluble peptides (CD80: 754 ± 26.87 vs. 503 ± 70.29, p < 0.05; murine MHC class I: 432 ± 50.91 vs. 237 ± 27.58, p < 0.01; MHC class II: 3,990 ± 718.74 vs. 2,370 ± 15.56, p < 0.01). It must be noted that DCs stimulated with each of the PLGA-chCPAp, PLGA-chH1p, and PLGA-chKMP-11p nanoformulations exhibited a similar maturation profile to that of the DCs stimulated with the mix of these nanoformulations (mix A), in comparison to DCs stimulated with the relevant soluble chimeric peptide (Figure S1 in Supplementary Material). The above results confirm that PLGA NPs are promising delivery systems of immunogenic peptides in the development of peptide-based vaccines.

The MPLA incorporation as well as the surface modification of PLGA NPs with p8 further increased DCs maturation. More specifically, as shown in Figures 3A,B, stimulation of DCs with the mix of PLGA-chCPAp-MPLA, PLGA-chH1p-MPLA, and PLGA-chKMP-11p-MPLA nanoformulations (mix B) led to a significant increase in the number of DCs expressing the co-stimulatory molecules CD40 (80.73 ± 3.46 vs. 72.40 ± 1.27%, p < 0.05) and CD86 (86.75 ± 2.33 vs. 74.33 ± 3.99%, p < 0.01), as well as the MHC class I molecules (murine MHC class I: 93.05 ± 0.78 vs. 82.15 ± 4.31%, p < 0.01; hybrid HLA class I: 71.65 ± 4.17 vs. 39.50 ± 0.30%, p < 0.001), in comparison to DCs stimulated with the mix A. This was also accompanied by a significant increase in the MFI values for CD40 (1,338 ± 81.32 vs. 618 ± 72.75, p < 0.0001) and CD80 (1,338 ± 81.32 vs. 618 ± 72.75, p < 0.0001) molecules (Figure 3C). Regarding the surface modification with p8, as depicted in Figures 4A,B, stimulation of DCs with the mix of p8-PLGA-chCPAp, p8-PLGA-chH1p, and p8-PLGA-chKMP-11p nanoformulations (mix C) significantly also enhanced the presence of DCs expressing the co-stimulatory molecules CD40 (84.80 ± 1.57 vs. 72.40 ± 1.27%, p < 0.001) and CD86 (83.60 ± 4.95 vs. 74.33 ± 3.99%, p < 0.01), as well as the MHC class I molecules (murine MHC class I: 90.25 ± 5.02 vs. 82.15 ± 4.31%, p < 0.05; hybrid HLA class I: 73.50 ± 0.05 vs. 39.50 ± 0.30%, p < 0.001), in comparison to DCs stimulated with the mix A. In addition, a significant increase was observed in the MFI values of DCs stimulated with the mix C in comparison to DCs stimulated with the mix A for all the co-stimulatory and MHC class I molecules except from the MHC class II molecule (CD40: 1,084 ± 28.93 vs. 618 ± 72.75, p < 0.0001; CD80: 1,715 ± 223.45 vs. 754 ± 26.87, p < 0.001; CD86: 2,529 ± 201.52 vs. 1,680 ± 347.19, p < 0.01; murine MHC class I: 537 ± 31.82 vs. 432 ± 50.91, p < 0.05; HLA class I: 122 ± 4.24 vs. 96 ± 2.26, p < 0.05). The above results concerning DCs stimulated with the mix B or the mix C were comparable to that observed in the case of DCs cultured in the presence of LPS (positive control of maturation; CD40: 82.80 ± 2.86%; CD80: 89.10 ± 5.61%; CD86: 81.63 ± 3.84%; murine MHC class I: 96.53 ± 1.34%; murine MHC class II: 86.63 ± 2.73%; hybrid HLA class I: 73.30 ± 3.36%) (Figures 2A,B). Moreover, similar results were obtained from DCs stimulated with each of the PLGA-chCPAp-MPLA, PLGA-chH1p-MPLA, and PLGA-chKMP-11p-MPLA nanoformulations (Figure S2 in Supplementary Material) or each of the p8-PLGA-chCPAp, p8-PLGA-chH1p, and p8-PLGA-chKMP-11p nanoformulations (Figure S3 in Supplementary Material), in comparison to DCs stimulated with each of PLGA-chCPAp, PLGA-chH1p, and PLGA-chKMP-11p nanoformulations, respectively. It is noteworthy that the significant (p < 0.001) increase observed in the number of DCs expressing the hybrid HLA-A2.1 molecule confirmed the successful design of the chimeric peptides to harbor T cell epitopes with high-binding affinity to HLA class I molecules.

Furthermore, as demonstrated in Figure 5, stimulation of DCs with the mix B (Figure 5A) or the mix C (Figure 5B) resulted in a significant increase of DCs that intracellularly produced IL-12 (26.94 ± 2.74 and 37.60 ± 1.43, respectively, vs. 12.16 ± 2.20%, p < 0.001), compared to DCs stimulated with the mix A. This observation supports the functional differentiation of DCs toward DC1 type. The high percentage of DCs that produced IL-12 (22.11 ± 0.53%) in response to p8-PLGA NPs indicated a role of the synthetic octapeptide in this biological process.

Since the peptide-based PLGA nanoformulations and especially those with MPLA incorporation or surface modification proved capable to induce DCs maturation and IL-12 production, mixed leukocyte cultures were performed in order to investigate the capacity of these DCs to promote in vitro T cell proliferation. For this purpose, DCs stimulated with the peptide-based PLGA nanoformulations were co-cultured with allogeneic spleen cells at different stimulator/responder ratios and their proliferative potential was determined by ³[H]-TdR incorporation. The results obtained indicated that the optimum stimulator/responder ratio was that of 1:5. As shown in Figure 6, DCs stimulated with the mix of PLGA-chCPAp, PLGA-chH1p, and PLGA-chKMP-11p nanoformulations (mix A) proved able to promote T cell proliferation (cpm: 10,895 ± 953) compared to unstimulated DCs stimulated (cpm: 8,330 ± 1,167). However, the incorporation of the MPLA adjuvant, as well as the surface modification with p8, remarkably enhanced the competency of stimulated DCs to efficiently present the processed antigenic peptides to T cells. In more details, DCs stimulated with the mix of PLGA-chCPAp-MPLA, PLGA-chH1p-MPLA, and PLGA-chKMP-11p-MPLA nanoformulations (mix B) or the mix of p8-PLGA-chCPAp, p8-PLGA-chH1p, and p8-PLGA-chKMP-11p nanoformulations (mix C) triggered significantly increased lymphoproliferative responses in comparison to unstimulated DCs (cpm mix B: 26,014 ± 1,828 vs. 8,330 ± 1,167, p < 0.001; cpm mix C: 23,626 ± 776 vs. 8,330 ± 1,167, p < 0.001). It is noteworthy that DCs stimulated with PLGA-MPLA or p8-PLGA also induced T cell proliferation at significant levels compared to unstimulated DCs (p < 0.001) at the ratio 1:5. This difference was not observed at ratio 1:20 in contrast to DCs stimulated with mix B or mix C. Spleen cells cultured in medium alone and in vitro stimulated with the mitogen ConA were used as positive control of proliferation (cpm: 40,118 ± 1,936, data not shown).

Flow cytometry analysis was conducted to unveil the presence of CD4⁺ T cells producing IL-4 (indicative of T_H2 polarization) or IFNγ (indicative of T_H1 polarization), as well as IFNγ-producing CD8⁺ T cells. Flow cytometry results revealed no increase in the population of IL-4-producing CD4⁺ T cells in comparison to T cells co-cultured with unstimulated DCs (Figures 7A,D,G). On the contrary, a significant increase was observed in the populations of IFNγ-producing CD4⁺ T cells (Figures 7B,E,H) and CD8⁺ T cells (Figures 7C,F,I) upon activation by DCs stimulated with the mix of PLGA-chCPAp, PLGA-chH1p, and PLGA-chKMP-11p (Mix A; CD4⁺ T cells: 12.0 ± 0.14%, p < 0.01 and CD8⁺ T cells: 5.4 ± 0.05%, p < 0.01) compared to unstimulated DCs. A further increase in IFNγ-producing CD4⁺ and CD8⁺ T cells was observed upon activation with DCs stimulated with PLGA-chCPAp-MPLA, PLGA-chH1p-MPLA, and PLGA-chKMP-11p-MPLA nanoformulations (mix B; CD4⁺ T cells: 13.0 ± 0.28%, p < 0.001 and CD8⁺ T cells: 5.88 ± 0.14%, p < 0.001) or the mix of p8-PLGA-chCPAp, p8-PLGA-chH1p, and p8-PLGA-chKMP-11p nanoformulations (mix C; CD4⁺ T cells: 14.85 ± 0.49%, p < 0.001 and CD8⁺ T cells: 7.16 ± 0.86%, p < 0.01).

In an attempt to unveil changes in the gene-expression profile of DCs stimulated with the differentially functionalized PLGA nanoformulations for 18 h, transcriptome analysis was performed using microarrays. PCA and Pearson correlation analysis of samples displayed satisfactory replicate resemblance (Figures 8A,B). Microarray data analysis revealed considerable differences in the gene-expression profiles of DCs stimulated with the different mixes of peptide-based PLGA nanoformulations or the mix of soluble chimeric peptides (Figures 8C,D). The highest number of differentially expressed genes was observed in DCs stimulated with the PLGA-chCPAp-MPLA, PLGA-chH1p-MPLA, and PLGA-chKMP-11p-MPLA nanoformulations (mix B, 2,104 differentially expressed genes; 1,027 up- and 1,077 downregulated), followed by DCs stimulated with the p8-PLGA-chCPAp, p8-PLGA-chH1p, and p8-PLGA-chKMP-11p nanoformulations (mix C, 1,273 differentially expressed genes; 527 up- and 746 downregulated), and then DCs stimulated with the PLGA-chCPAp, PLGA-chH1p, and PLGA-chKMP-11p nanoformulations (mix A, 847 differentially expressed genes; 278 up- and 569 downregulated). Importantly, much milder differences were observed for DCs stimulated with the mix of soluble chimeric peptides (mix D, 191 differentially expressed genes; 69 up- and 122 downregulated) compared to unstimulated DCs. Therefore, the hierarchy of potency in inducing gene-expression changes was mix B > mix C > mix A > mix D, stressing that the overall magnitude of activation was much higher in DCs stimulated with the mix B.

Enrichment analysis of differentially expressed genes for GO terms (Biological Processes) revealed that DCs stimulated with the mix B or the mix C exhibited a higher number of significantly enriched terms compared to DCs stimulated with the mix A, while no significant enrichment was observed in the case of DCs stimulated with the mix D. More specifically, DCs stimulated with the mix B or the mix C shared common genes involved in cytokine production (GO:0001819), inflammatory response (GO:0050727), leukocyte cell–cell adhesion (GO:0007159), and cellular response to cytokine stimulus (GO:0071345), as well as in other biological processes related to antigen processing and presentation or adaptive immunity (Data Sheet S1 in Supplementary Material). Focusing on a number of the most significantly enriched GO terms (Figure 8D; Table 4), genes encoding pro-inflammatory cytokines, such as Il12b, Il6, type-I, and II IFN-response elements (Irf7, Stat5a, Irf8, Gbp5, Ido1, Stat1, Ifi205, etc.) and DCs maturation markers (Cd40) were upregulated in both groups. However, a much higher number of upregulated genes involved in the above biological processes were found to be specifically expressed in DCs stimulated with the mix B. Between these specifically upregulated genes, they were identified IFN-response genes (Ifih1, Ifit1, Ifit2, Iigp1, Irg1, Gbp2, and Gbp4), inflammation-related genes (Il10, Il23a, Il15, and Saa3), and genes encoding chemokines that act to recruit leukocytes (Ccl3, Cx3cl1, Mif, and Cxcl1). Moreover, a remarkable number of genes was identified characteristic for the type 1 DCs phenotype that could induce a subsequent CD8⁺ T cell activation (Psmb10, Hsp90aa1, Hsp90ab1, Igtp, Ccl3, Dhx58, Tnfsf8, Il15, Olr, etc.) and CD4⁺ T_H1 polarization (Cd86, Cish, Dll4, Cxcl9, Cx3cl1, Osm, Il1f6, Tnfsf15, Kit, Clec4e, etc.) (Table 4; Figure 9). Overall, the above findings suggested that DCs exposed to mix B could probably be in a more advanced state of maturity and functional differentiation and might be able to induce specific T cell responses.

The in vitro screening of the differentially functionalized PLGA nanoformulations in DCs gave precedence to the mix of PLGA-chCPAp-MPLA, PLGA-chH1p-MPLA, and PLGA-chKMP-11p-MPLA nanoformulations (mix B) to be evaluated in vivo in terms of immunogenicity, as a promising peptide-based nanovaccine. For this purpose, HLA A2.1 transgenic mice were subcutaneously injected with the mix B and boosted twice in a 2-week interval (Figure 10A). The specific T cell expansion induced by each of the chimeric peptides was initially assessed by ³[H]-TdR incorporation. As shown in Figure 10B, chCPAp, chH1p, and chKMP-11p induced proliferation of spleen cells from mice immunized with mix B compared to spleen cells from non-immunized mice (received only PBS), upon in vitro stimulation. More specifically, chKMP-11p induced the strongest proliferation (Δcpm: 7,908 ± 1,886 vs. 65 ± 20, p < 0.01), followed by chH1p (Δcpm: 3,764 ± 643 vs. 42 ± 6, p < 0.01) and chCPAp (Δcpm: 2,232 ± 112 vs. 66 ± 5, p < 0.05). Mice immunized with PLGA-MPLA nanoformulations did not show specific T cell expansion. Spleen cells cultured in medium alone and in vitro stimulated with the mitogen ConA were used as positive control of proliferation (Δcpm: 39,726 ± 3,061, data not shown).

Flow cytometry analysis was then performed to detect peptide-specific IFNγ-producing CD8⁺ T cell populations (Figures 10C,D). The highest number of peptide-specific IFNγ-producing CD8⁺ T cells was observed upon in vitro stimulation with chKMP-11p of spleen cells from mice immunized with mix B compared to spleen cells from non-immunized mice (1.34 ± 0.35 vs. 0.22 ± 0.03%, p < 0.05). Chimeric peptides chH1p and chCPAp were also able to stimulate specific IFNγ-producing CD8⁺ T cells to a lower level (0.53 ± 0.05 vs. 0.21 ± 0.02%, p < 0.01 and 0.41 ± 0.06 vs. 0.20 ± 0.02%, p < 0.05, respectively). It must be also noted that spleen cells from mice immunized with PLGA-MPLA nanoformulations did not exhibit IFNγ-producing CD8⁺ T cell populations, similar to spleen cells from non-immunized mice.

In order to investigate whether the peptide-specific T cell responses observed in HLA A2.1 transgenic mice immunized with the mix of PLGA-chCPAp-MPLA, PLGA-chH1p-MPLA, and PLGA-chKMP-11p-MPLA nanoformulations (mix B) could confer protection against L. infantum infection, immunized and non-immunized mice were infected intravenously with L. infantum promastigotes and the parasite burden was assessed in liver and spleen by a limiting dilution assay 1 and 2 months post-infection (Figure 11A). According to the infection kinetics in HLA A2.1 transgenic mice, the parasite burden reached a peak at 1 month post-infection in the liver (Figure 11B) and at 2 months post-infection in the spleen (Figure 11D), followed by a decrease in both organs. The results obtained indicated that immunization with the mix B reduced significantly hepatic and splenic parasite burden 1 month post-infection by 72.81% (p < 0.01, Figure 11C) and 61.98% (p < 0.05, Figure 11E), respectively, compared to the non-immunized control group. Assessment of the parasite burden 2 months post-infection revealed a maintenance of the protective effect with 64.4% reduction of parasite burden in the liver (p < 0.01, Figure 11C) and 73.64% reduction of parasite burden in the spleen (p < 0.05, Figure 11E). It also must be noted that immunization with PLGA-MPLA nanoformulations did not confer protection against L. infantum infection, indicating that the protective effect observed in liver and spleen of immunized with the mix B HLA A2.1 transgenic mice was peptide specific.