**3. Discussion**

A significant public health issue is the absence of effective treatments or preventive vaccinations for several diseases brought on by intracellular infections, such as cutaneous leishmaniasis. Strong cellular responses are needed in the vaccine formulations for these illnesses [34]. Preclinical trials are now being conducted on a variety of potential antileishmania vaccine candidates, including live genetically altered parasites, live attenuated parasites, recombinant proteins and DNA vaccines, and vaccinations using antigen combinations [16]. Despite the preclinical effectiveness of some vaccine candidates, their use has been restricted due to the toxicity of a living vaccine on the one hand and the minimal antigenic exposure to the antigen-presenting cells (APCs) for a subunit protein vaccine on the other hand [15,35,36]. However, a satisfactory adjuvant in vaccine formulation has ye<sup>t</sup> to be approved [36]. At present, the WHO still recommends the use of alum in combination with the vaccine antigen; this encourages the investigation of new adjuvants because alum is unable to elicit cell-mediated Th1 response [37].

Some of these limitations can be resolved through the use of nanoparticulate delivery systems. Today, the administration of vaccines to organisms using PLA nanoparticles with surface-adsorbed antigens is a promising approach. The polymer is biocompatible, biodegradable, non-toxic, and non-antigenic [38]. Interestingly, PLA-based vaccines improve efficient cellular immune responses (CTL) to a number of infections [25]. Such an immune response seems crucial for the control of Leishmania, an intracellular pathogen [21].

This research aimed to develop a PLA polymer-based nanoparticle vaccine for the immunization of mice infected with *L. major* parasites. We investigated and compared the immune performance of antigen-coated PLA nanoparticles and CpG 7909, a Th1- activated adjuvant. The recombinant H2B protein, expressed in *E. coli* BL21 strain cells, is a chosen candidate for a leishmanial vaccine [31]. It is interesting to note that this protein does not interact negatively with mammalian histones, is easily expressed in the prokaryotic environment, and is not cytotoxic [39]. The first part of the study was to optimize the physico-chemical conditions of the procedure and the formulation of the vaccine since the size, zeta potential and desorption pattern are parameters that affect the particle uptake by cells. For the development of H2B-coated PLA NPs, we prepared well-dispersed nanoparticles by the nanoprecipitation method. We optimized the protocol so that the particles are obtained in a single step without the use of surface-active agents. This is a benefit in vaccine formulation that overcomes the inherent toxicity of excipients. Many studies demonstrated the emergence of skin toxicity when using surfactants in nano-delivery systems [40]. The PLA-NPs had a mean diameter of 287 nm and had a quite narrow size distribution with a polydispersity index of 0.14. Neither deposits nor agglomerates were noticed in the PLA dispersions. A zeta potential of −45 mV was high enough (in absolute value) to afford colloidal stability through electrostatic repulsion. The carboxyl groups at the particle surface outset the negative global charge. The H2B adsorption on the particle surface resulted in the inversion of the zeta potential from −45 mV to +30 mV. The carboxyl groups were neutralized by protonated amines within the protein backbone. Obviously, histones are classified as proteins with a lightweight structure and a strong basic polycation character [41]. Thus, the H2B adsorption on NPs occurred by electrostatic interactions. The positive global charge of H2B/PLA formulation could optimize cell membrane interactions. This is a crucial parameter of antigen-presenting cells [42]. Several researchers have explored the cell uptake of positively charged particles. Among them, Liu Z. et al. showed that cationic stearylamine lipid-polymer hybrid NPs (LPNPs) of ~200 nm increased the efficacy of AmB through macrophage uptake [23]. As confirmed elsewhere, the sodium alginate-glycol chitosan stearate NPs (AmB-SA-GCS-NP) showed the highest macrophagic uptake in J774.1 macrophages and the rapid in vivo localization tissues of the liver, spleen, lung, and kidney [43]. Many studies have reported that particle size modulates the assimilation of particles into cells. Among them is one concerning PLGA NPs that exhibited low toxicity and efficient uptake by APC in vitro and in vivo when particles were about 300 nm in average size [44].

Finally, the release pattern of H2B/PLA was analyzed at predetermined time intervals for 30 days. The recombinant H2B started desorption on day 7, and the total amount of 30% was reached 3 weeks later (the 30th day).

Weak desorption at pH = 7.4 could be interpreted as a more favorable interaction between protein and polymer than protein and release medium. The progressive H2B release promotes a deposit effect. For vaccination purposes, this can significantly reduce booster doses.

Secondly, we evaluated the vaccine potency of the formulation on the BALB/c mice because they are susceptible to infection with *L. major*, and if allowed to run its course, subcutaneous injection of parasites leads to uncontrolled lesion growth and eventual death [33]. 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 [45]. BALB/c mice were subcutaneously immunized two times with the following vaccine formulations: H2B alone, H2B/PLA-NPs and H2B/CpG-ODN7909. We recorded an increase in the average antibody level against H2B-based adjuvant formulations in contrast to groups that were exposed to free antigens. The humoral response intensity (IgG titers) was substantially similar between both formulations and, therefore, immunogenic in a specific way to adsorbed antigens. These results agree with previous work on PLA NPs/Nod ligand formulations, which induced an increase of up to 100-fold in antibody responses against Gag p24 HIV-1 antigen in comparison to alum [46]. As previously described using PLA as an adjuvant, the p24/PLA vaccine induced high antibody titers with a strong CTL response in mice, rabbits and macaque [25]. As for other infectious diseases, the pattern of the IgG subclass has been shown to play a role in the course of *Leishmania* infection. Mice, similarly to humans, show four different classes of IgGs, named IgG1, IgG2a, IgG2b and IgG3, which functionally correspond to human IgG1, IgG2, IgG4 and IgG3, respectively. In general, it is possible to conclude that in mice and humans, IgG1 (as well as IgG4 in humans) is associated with a Th2 profile, and the other subclasses are mainly associated with a Th1 profile [47]. The evaluation of the IgG2/IgG1 ratio allowed us to predict the type of immune response induced by both adjuvants. The PLA/H2B formulation tends towards a Th1-activated response due to the positive IgG2/IgG1 ratio of antibodies against H2B. Clearly, the CpG7909 preferentially activated cellular immunity, as confirmed by the prevalence of IgG2, by directing the differentiation of LB towards the secretion of specific IgG2a antibodies. Additionally, for LCL-infected individuals, PBMC proliferation and IFNγ levels increased significantly in the presence of a recombinant H2B [32]. The prevalence of the cellular response with H2B/PLA could be explained based on preliminary experiments suggesting that the H2B protein has the ability to induce a specific Th1 response in BALB/c mice [31]. When BALB/c mice were vaccinated with the H2B protein alone or in the presence of CpG adjuvant (two injections), they were able, one month after the last immunization, to produce large amounts of IFNγ when their lymph nodes (injection sites) were re-stimulated in vitro with H2B protein. Furthermore, the amounts of IL-10 and IL-4 were very low or not detectable. Such a Th1-polarized response is curative, while the Th2 response exacerbates or is ineffective in controlling the disease [48–50].

The challenge assay demonstrated that animals do not develop ulcerating lesions until week 8 post infection. Both groups (PLA particles and CpG-ODN) have the lowest lesion thickness. This suggested that H2B/PLA formulation exhibited a protection immune potency. Consequently, the parasite dissemination has been slowed down or even interrupted in vaccinated animals, while control groups lead to uncontrolled lesion growth and eventual death. Despite the effectiveness of CpG-ODN in triggering a cytotoxic immune response, its use is still restricted since the clearance is rapid. A study of its pharmacokinetics and biodistribution characteristics demonstrated its rapid renal clearance when intravenously injected into animals [51].

A previous study from Khatik et al. (2014) showed that PS–coated gelatin NPs with encapsulated amphotericin B reduced parasite burden in Wistar rats (85% vs. 50%) when

compared to standard AmB [52]. For application against CL, AmB nano-encapsulation in PLGA/dimercaptosuccinic acid (DMSA) NPs (nano-DMS-AmB) was developed and tested against C57BL/6 mice infected with *L. amazonensis*. The nano DMS-AmB showed a greater reduction in the number of parasites than standard D-AmB [53]. Nanoliposomes used as the nanocarriers for soluble *Leishmania* antigens (SLA) showed parasite clearance in the footpad and spleen of a mouse model injected with this formulation. Liposomal formulations of ChimeraT (a combination of 3 leishmanial proteins), when delivered subcutaneously, protected mice against *L. infantum* infection by reducing the parasite load in the spleen, liver, bone marrow, and lymph nodes [54].

In the context of leishmanial nano-vaccines, Katebi A. and collaborators (2021) loaded PLGA NPs with SLA and TLR receptor agonists PAM3CSK4 and R848 and incubated them in vitro with immune cell lines. The results showed a marked increment in the macrophages' phagocytic potential accompanied by a significant reduction in pro-inflammatory cytokines against *L. major* parasites. However, this immunogenic candidate should be tested against a *Leishmania*-infected animal model to determine the in vivo parasitic and disease reduction potential [55,56].

## **4. Materials and Methods**

## *4.1. Chemicals and Reagents*

Phosphate-buffered saline tablets and acetone were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). A Low Molecular Weight Calibration Kit for SDS Electrophoresis (Code: 17-0446-01) was purchased from GE Healthcare. An s29 Gx 0.5 syringe (insulin) was purchased from Terumo. The adjuvant CpG-ODN (7698 g/mol) (ODN 7909), a human TLR9 ligand, was purchased from InvivoGen. Poly (D,L-lactide) (PLA Mw = 16,000 g/mol, Mw/Mn = 1.6) was purchased from Corbion Purac Biomaterials (4206 AC, The Netherlands). Imidazole reagen<sup>t</sup> was obtained from GE Healthcare-Application Note 28-4067-41 AA. A QuantiPro BCA Assay Kit was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA)). Schneider's Drosophila medium was obtained from Gibco-BRL, Paisley, Scotland.
