*4.3. Mice*

Female BALB/c mice (6–8 weeks old) were obtained from The Institut Pasteur de Tunis "http://www.Pasteur.tn (accessed on 12 June 2013)". The experimental protocol (registry number: 2015/13/I/LR11IPT08 V1) was approved by the Institutional Ethical Bio-Medical Committee of the Institut Pasteur de Tunis IPT, and the US registry number is IRB00005445, FWA00010074).

## *4.4. Preparation of PLA Nanoparticles*

Surfactant-free PLA nanoparticles (PLA-NPs) were prepared by the nanoprecipitation method as previously described [57]. Briefly, 0.2 g of polymer was dissolved in 10 mL of acetone and added dropwise to 30 mL of milliQ under moderate stirring water without using a surfactant. The organic solvent and a part of the water were removed by evaporation under reduced pressure. The final NP concentration after the preparation was determined by measuring the solid content after heating the NP dispersion (known volume) to a constant weight in an oven at 70 ◦C for 24 h. The size distribution was determined by dynamic light scattering technique (DLS) at 25 ◦C using a Zeta-Sizer Nano ZS (Malvern Instruments, Malvern, UK). Zeta potential was measured by a laser Doppler with the same instrument using diluted colloidal dispersions in 1 mM NaCl.

#### *4.5. Expression and Purification of Recombinant Histone H2B*

The BL21 *Escherichia coli* (*E. coli*) strain containing the recombinant plasmid pET-H2B was kindly provided by Dr. Chenik.

Purification and expression of the H2B protein were carried out as previously described [31]. Luria broth (LB) medium supplemented with ampicillin (100 μg/mL) and 0.1% glucose was used to grow *E. coli* BL21 pLysS DE3-pET-H2B in shaking flasks until the absorbance at 600 nm reached a value between 0.6 and 0.9. After that, IPTG (1 mM) was used to stimulate protein expression for a minimum of 4 h at 37 ◦C. Cells were pelleted before the periplasmic proteins were osmotically shocked out and placed into a Ni-NTA column. The histidine-tagged (His-tagged) proteins were then eluted using a linear gradient of imidazole from 0.05 to 0.5 M after being cleaned with PBS. Membrane dialysis was used to desalt fractions. SDS-PAGE was used to evaluate purified proteins, and the BCA assay kit (Pierce, Rockford, IL, USA) instructions were used to calculate protein quantities.

## *4.6. Synthesis of H2B/PLA, a Nanovaccine Formulation*

In this study, a nanovaccine formulation was synthesized by the adsorption of H2B onto PLA nanoparticles, as previously described [57]. PLA-NPs were diluted at a concentration of 1.2 mg/mL in Milli-Q water. Then, H2B solutions were prepared at different concentrations (0–100 μg/mL) in NaCl (10 mM). One volume of NPs was added to one volume of protein solution to obtain the H2B/PLA formulation. The adsorption medium has a fixed concentration of both NPs (0.6 mg/mL) and protein, which ranged from 0 to 50 μg/mL (corresponding to an H2B/PLA weight ratio from 0 to 8.3%). Protein and particle dispersions were incubated for 15 min at room temperature with gentle stirring. The samples were centrifuged for 20 min at 5000× *g*. Separate tubes were used to collect each supernatant. Each H2B/PLA pellet was cleaned further by centrifugation/redispersion (5000× *g* for 20 min at 25 ◦C). The pellets were then resuspended in saline solution (NaCl 10 mM). In accordance with the manufacturer's recommendations, the QuantiPro BCA assay Kit (Sigma, Germany) was used to quantify the concentration of non-adsorbed H2B. A Zeta-sizer Nano ZS-based DLS technique was used to measure parameters such as the particle size, polydispersity indices and zeta potentials of the H2B/PLA formulations (Malvern Instruments, Malvern, UK).

## *4.7. In Vitro Release Study*

To study the desorption profile of H2B, the H2B/PLA pellet (which was used at a 5% *w*/*w* ratio) was resuspended in PBS (pH 7.4) and incubated on a shaker at 37 ◦C. At set times (12 h, 1, 2, 3, 7, 14, 21 and 30 days), samples (in triplicate) were taken and centrifuged at 5000× *g* for 20 min. Using the QuantiPro BCA assay Kit, the concentration of protein released in supernatants was quantified.

## *4.8. In Vivo Assay*

## 4.8.1. Immunization of BALB/c Mice

In vivo studies have been performed in BALB/c mice, which are the mouse models used for preclinical evaluation of *Leishmania* vaccine candidates [33]. Forty-eight females, between 6 to 8 weeks old, were used in the experiments. The animals were divided into 6 groups, including 8 mice each. They were then administered subcutaneously twice on days 1 and 14 with the following vaccine formulations: H2B alone (25 μg/injection), H2B (25 μg) plus CpG (20 μg), H2B/PLA-NPs (25 μg/0.9 mg), CpG (20 μg), PLA-NPs (0.9 mg) and PBS. In each vaccination, 200 μl of the preparation diluted in PBS was injected into the animals. On day 30, blood samples were taken to measure serum antibody levels.

## 4.8.2. Identifying the IGg1 and IgG2 Isotypes

To evaluate the antibody pattern generated based on the H2B/PLA formulation, the IGg1/IgG2 subtype was examined. Enzyme immunoassay (ELISA) was used to detect specific antibodies generated against H2B formulations (H2B/PLA and H2B + CpG7909) in mouse serum. Briefly, 5 μg/mL of H2B was coated in 100 μL of carbonate-bicarbonate buffer (0.1 M) and incubated overnight at 4 ◦C in 96-well high-binding plates (Nunc). PBS-T20 buffer was used three times to wash the plates. PBS-T20 solution, supplemented with low-fat milk (2%), was used to block the plates at 37 ◦C for 1 h. After the wash steps, sera were added at a dilution of 1/1000 and incubated for 2 h at 37 ◦C.

## 4.8.3. Parasite Infection and Development of Lesions

The purpose of the treatment was to demonstrate an adjuvant effect of H2B/PLA against an experimental *L. major* infection. A minimum of 6 weeks was required between vaccination and infection. Six weeks after receiving the booster dose, animals in each group were challenged with infectious *L. major* promastigotes (GLC94) in order to observe and evaluate the efficacies of both the H2B + CpG and H2B/PLA-NPs vaccine formulations. For this, mice were subcutaneously injected with an infective parasite load (2 × 106) of *L. major* promastigotes suspended in 50 μL of PBS in the right footpad. Within eight weeks, the swelling's growth was tracked once a week using a dial-gauge caliper (Mitutoyo, Japan). Lesion values (in mm) were calculated by subtracting the thickness of the infected footpad from the thickness of the contralateral footpad that was not infected.

## 4.8.4. Determining the Parasite Load

Parasite load was quantified by a limiting-dilution technique adapted from the work of Laskay et al. [58]. The infected pad was cut (3 groups, *n* = 8) and homogenized before serial 10-fold dilutions were plated in triplicate in 96-well flat-bottom microtiter plates (Nunc, Roskilde, Denmark) containing Schneider's Drosophila medium (Gibco-BRL, Paisley, Scotland) with the addition of 100 U of penicillin/mL, 100 μg of streptomycin/mL, 2 mM L-glutamine, and 10% heat-inactivated fetal calf serum. Plates were incubated at 26 ◦C. Live parasites were attested under an inverted microscope. Parasite load is expressed as the average of the log negative of the last dilution in which mobile parasites were detected.

## **5. Statistical Analysis**

Statistical analysis was performed using GraphPad Prism software 5.1 (GraphPad Software 2365 Northside Dr. Suite 560 San Diego, CA 92108). The data are expressed as mean ± standard deviation. Statistical significance *p*-values of less than 0.05 are considered statistically significant.
