**2. Results**

## *2.1. Characterization of PLA NPs*

The nanoparticles were obtained by the nanoprecipitation method without adding surfactant. Optimum experimental conditions were as follows: 0.2 g PLA polymer was dissolved in acetone (10 mL) and added to MilliQ water (1v/3v). A white and milky suspension was obtained after the formation of the nanoparticles. The production yield, based on recovered PLA after solvent evaporation, was in the 70−80% range. According to the DLS results, the obtained nanoparticles had a mean hydrodynamic diameter of 287.4 nm (±10); and a polydispersity index (PDI) of 0.14 (±0.06) (Figure 1). This indicates that NP distribution was in a quite narrow range. Zeta potential is one of the important parameters affecting the stability of nanoparticles. Their zeta potential value was −45 mV (±5), prone to ensure colloidal stability through electrostatic repulsion.

**Figure 1.** Size distribution of PLA-NPs using DLS technique. NPs were prepared by a nanoprecipitation process in one single step without using surfactant.

For stability analysis, nanoparticles (4 mg/mL) were kept at +4 ◦C for several months. No significant variation in particle size was observed until month 18.

#### *2.2. Characterization of a Recombinant H2B Protein*

Recombinant protein H2B was produced in *Escherichia coli* BL21 bacteria (*E. coli* BL21) using the pET prokaryotic expression system. Proteins were then purified by affinity chromatography over Ni-NTA resin, and purity was assessed by SDS-polyacrylamide gel electrophoresis. Staining of the gel with Coomassie blue revealed two bands of 16 kDa and

30 kDa, respectively, Figure 2. As previously described based on Western blot analysis, bands of 16 kDa and 30 kDa correspond to recombinant H2B monomeric and dimeric forms, respectively [31].

**Figure 2.** SDS analysis of *L. major* recombinant H2B. Protein was expressed in BL21 *Escherichia coli,* then purified by affinity chromatography over Ni-NTA resin and analyzed using SDS-PAGE (15%) followed by Coomassie blue staining. (\*) indicates an additional band that corresponds to H2B dimeric form. Mr, Molecular weight markers (kDa) (GE Healthcare).

#### *2.3. Adsorption Efficiency and Zeta Potential of H2B/PLA Particles*

The characteristics of H2B-adsorbed PLA nanoparticles are reported in Figure 3. The NP concentration was fixed at 0.06 w% (i.e., 0.6 mg/mL). The adsorption process was followed by monitoring both the amount of adsorbed protein and the surface charge of H2B/PLA. The maximum adsorption was reached when an introduced protein/particle ratio ranged from 5 to 8.3 w%. Thus, the adsorption capacity was 2.8% *w*/*w* of protein particles (Figure 3a). Zeta potentials were followed as a function of introduced protein amounts under the adsorption process, as its strongly cationic character is expected to induce charge inversion (Figure 3b). Flocculation of the colloid was observed around the neutralization point (~2 w% protein/particle ratio). The colloidal stability was restored at an introduced H2B/particle ranging from 5 to 8.3% with a zeta potential of +30 mV and no longer changes, indicating the saturation of the colloid surface.

**Figure 3.** Adsorption curves of H2B/PLA-NPs. (**a**) Adsorption efficiency of the peptide/particles (0.06% *w*/*v*) at different protein-to-particle ratios. (**b**) Zeta potentials of H2B/PLA as a function of initial protein amount (% *w*/*w*).

In further studies, H2B-coated NPs were prepared in the presence of excess protein to avoid the flocculation process (5 w% initial protein/NP ratio) and washed from unbound protein (centrifugation/redispersion steps). The H2B/PLA exhibited a size and a zeta potential of 341 nm and +30.9 mV, respectively, Table 1.

**Table 1.** Main characteristics of H2B/PLA-NPs selected for animal experiments.


## *2.4. In Vitro Desorption of Adsorbed H2B*

The H2B/PLA-NPs were incubated in PBS buffer (pH 7.4) at 37 ◦C. The amount of protein in each supernatant was quantified at a fixed time. As shown in Figure 4, protein release followed a specific profile over time. No protein was detected in the supernatant collected during the first three days until day 7. Continuous H2B release was quantified from day 7 to day 21 with a cumulative percentage value of 20%. After that, the release continues to slow down until the end of the test and the maximum percentage value of 30% was obtained (Figure 4).

**Figure 4.** Release of adsorbed H2B peptide over time. A known amount of H2B/PLA was suspended in PBS buffer solution (pH~7.4) (GIBCO) at 37 ◦C. Protein desorption was followed at predetermined time intervals. Supernatants were collected at each point, and protein concentration was determined using a micro BCA assay kit (St. Louis, MO, USA, Sigma-Aldrich). Desorption rate was expressed as the cumulative percentage.

## *2.5. Assessment of the Antibody Response*

To demonstrate an antigen-specific adjuvant effect, BALB/c mice were subcutaneously immunized two times on days 1 and 14, with the following vaccine formulations; H2B alone and H2B/PLA-NPs. As a positive control, H2B plus CpG-ODN7909 was also included because it triggers cytotoxic immunity. For non-specific responses, one group was given PLA-NPs, while the other was given CpG-ODN7909 only. For infection control, one group was given PBS alone. Specific IgG titers were assessed by ELISA on day 30 at the end of the immunization schedule. A significant increase in the specific IgG titer was found after one month of primary immunization for groups vaccinated with either H2B/PLA-NPs or H2B + CpG in contrast to groups that were exposed to soluble protein (Figure 5). There was an increase in the average antibody levels. These results indicate that PLA-based vaccine formulation induced humoral immunity.

**Figure 5.** Specific anti-H2B antibody immune response. Pooled sera from each group were reacted in ELISA with recombinant H2B protein. Total IgG titers were calculated for each group. Titers were scored positively at the last dilution of immune sera. Results are represented as mean ± standard deviation of optical density (O.D.). (\*) *p* < 0.05 between PBS control and protein-received groups.

### *2.6. Analysis of the Anti-H2B IgG Isotype*

IgG1 and IgG2 antibody isotypes are markers of the humoral and cellular immune response, respectively. The evaluation of their titers allows them to determine by which pathway, Th2 or Th1, the immune system reacts to H2B/PLA formulation. Both adjuvants (particulate, PLA and molecular, CpG) produced a significant IgG2 isotype level. This suggested that the PLA immune response tends towards a Th1-type response, as confirmed with the CpG-ODN adjuvant (as control) (Figure 6).

**Figure 6.** IgG isotype ratio in pooled sera from immunized mice. Results are represented as mean ± standard deviation (SD) of optical density (O.D.) values obtained from 5 mice per group. (\*\*\*) *p* < 0.001 between PBS control and protein-received groups.

#### *2.7. Protective Potency of H2B/PLA in Mice*

The protective potential of antigen adsorbed onto PLA was compared with that of CpG-ODN7909, which has previously been shown to be a Th1 response enhancer.

The challenge tests were performed using the standard parasite strain, MHOM/TN/ 94/GLC94 (GLC94), isolated from a human lesion of cutaneous leishmaniasis (CL). This isolate belongs to the species of *L. major*, zymodeme MON25 and is the most virulent strain [31,33]. A high dose (2 × 106) of GLC94 parasites was inoculated through subcutaneous injection into the right footpad six weeks after the booster dose (week 8). Then, the course of the infection was recorded weekly for eight weeks (weeks 10 to 17) Figure 7a. The progression of lesions was similar in all groups of mice during the first three weeks post infection. Among the groups that received H2B antigen, PLA NP gave the best results, followed by CpG-ODN, applied as a control adjuvant. Since the soluble protein itself is immunogenic, a moderate protective potential has been observed. For PLA NPs, CpG-ODN, and PBS groups, lesions progressed rapidly to severe necrosis from 3 to 8 weeks post-infection. In contrast, the footpads of mice vaccinated with PLA-H2B were free of necrosis at the end of the experiment (week 8) and were 2-fold thinner than those of the

PBS group (*p* < 0.001). Compared with the CpG-ODN group, the results were significantly similar (Figure 7b).

**Figure 7.** Immune protection of vaccinated BALB/c mice against *Leishmania major* challenge. (**a**) The timeline of the in vivo experiment. Female BALB/c mice (6–8 weeks old) were vaccinated twice on day 1 and day 14 with H2B/PLA (25 μg/protein), H2B (25 μg) + CpG or soluble H2B (25 μg). The control groups received PBS, PLANPs or CpG. Mice were infected with (2 × 106) *L. major* GLC94 promastigotes by subcutaneous injection on the right footpad. (**b**) Footpad swelling. Lesion sizes were monitored weekly for up to eight weeks. The mean lesion size ± SD is shown (*n* = 8). (\*\*\*) *p* < 0.001 between PBS control and protein-received groups.

#### *2.8. Evaluating the Parasite Load in Mice*

Parasites disseminated in infected footpads were quantified using a limiting dilution technique. As expected, mice immunized with PLA (H2B/PLA) and CpG (H2B + CpG) induced good protection against parasites, with 2–3 log fewer parasites than with the PBS group (*p* < 0.001). Unvaccinated mice had the highest parasite load (Figure 8). The results correlated with the lesion thickness observed in respective groups. It suggests that only protein formulated with PLA nanoparticles or CpG-ODN can protect animals against parasite spread.

**Figure 8.** Parasite load in infected footpads. BALB/c mice vaccinated with H2B/PLA and H2B + CPG were challenged with *L. major* promastigotes, respectively. Eight weeks post-infection, footpads were collected. Parasites at the lesion site were counted by limiting dilutions. The results were compared with those obtained from PBS group. (\*\*\*) *p* < 0.001 between PBS and protein-received group.
