**1. Introduction**

Leishmaniasis, a neglected tropical disease, is the third most challenging vector-borne disease after malaria and lymphatic filariasis. According to the World Health Organization's (WHO) published data, it has been reported that leishmaniasis is endemic to approximately 100 countries of the world [1]. Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis. The annual registered cases are around 0.7–1.2 million across the globe. The disease is caused by the protozoan *Leishmania* parasite [2]. The principal vector responsible

**Citation:** Ayari-Riabi, S.; Ben khalaf, N.; Bouhaouala-Zahar, B.; Verrier, B.; Trimaille, T.; Benlasfar, Z.; Chenik, M.;Elayeb, M. Polylactide Nanoparticles as a Biodegradable Vaccine Adjuvant:A Study on Safety, Protective Immunity and Efficacy against Human Leishmaniasis Caused by Leishmania Major. *Molecules* **2022**, *27*,8677. https://doi.org/10.3390/ molecules27248677

 Academic Editors: Marek Brzezi ´nski and Małgorzata Ba´sko

Received: 29 October 2022 Accepted: 29 November 2022 Published: 8 December 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

for mammal-to-human transmission is the Phlebotomine sandfly. Two distinct morphological forms of *Leishmania* have been identified in its life cycle: the promastigote is present in the vector, and the amastigote moves into the monocytes/macrophages of the human or mammalian host [3]. CL is a skin infection that reduces patients' quality of life and imparts psychological problems and social stigmatism [4].

When BALB/c mice, an inbred strain of mice, are infected with the *L. major* parasite, the maturation of a Th2 immune response is triggered by the interleukin-4 (IL-4) produced during the first two days. This IL-4 rapidly renders parasite-specific CD4+ T cell precursors unresponsive to interleukin-12 (IL-12) [5].

Effective regulation requires macrophage activation and nitric oxide (NO) in response to the Th1-produced cytokine IFN-γ. Disease prognosis can be improved by overcoming problems such as low efficacy, systemic toxicity, insufficient drug within macrophages, poor antigen presentation to cells and expensive care. Vaccination may be the most appropriate strategy in this context.

Adjuvants for *Leishmania* vaccines to date are categorized into two types: (i) immunostimulatory molecules and (ii) nanoparticulate and/or delivery systems. The first ones include Bacille Calmette–Guerin (BCG) emulsified with antigens, e.g., *Leishmania amazonenzis* isolate IFLA/BR/1967/pH 8 [6], autoclaved *Leishmania major* ALM or GP63 [7,8], the monophosphoryl lipid (MPL) with LEISH-F1 or LEISH-F2 peptides [9,10], the interleukin-12 (IL-12) with pSP Leish-tec peptide [11], saponin with A2 Leish-tec [12], GLA with SMT Leish-tec [13] and CpG-ODN with KMP-11 Leish-tec [14]. However, immunostimulatory adjuvants suffer from rapid clearance and safety issues [15]. Nano-based delivery systems (NDS) applied as potential adjuvants in anti-leishmanial vaccines may be a useful alternative to conventional bacterial adjuvants and virus vectors [16]. An NDS could potentially deliver target vaccines to the site of action within the host's body, enhance immune reactions by facilitating antigens' absorption and uptake by antigen-presenting cells (APCs) [17], prevent its degradation such as peptides, proteins, or oligonucleotides [18], promote their controlled release and modulate the type of immune responses [19,20]. The particulate adjuvants, for example, liposomes, polymeric microspheres, and emulsions, have been utilized effectively to deliver *Leishmania* antigens in preclinical models of leishmaniasis, as well as other infectious diseases [21]. Liposomes (LPS) and derivatives have been widely studied. Despite the relevance of LPS, some shortcomings are also associated with these lipid-based nanocarriers, such as leakage of the entrapped moiety, oxidation, hydrolysis, and inadequate stability [22]. The polymeric particles, when used as adjuvants, could develop more potent immunogenicity against *Leishmania* antigens [23]. Several polymers, such as polylactide (PLA), polyglycolide (PGA), poly-lactide-co-glycolide (PLGA), poly-caprolactone (PCL), poly-cyanoacrylate (PCA) and natural protein polymers, such as albumin and gelatin, and polysaccharides, have been investigated as vaccine carriers. The polymer that has been explored most extensively is polylactide (PLA). The FDA has approved the use of this compound in human applications due to its biocompatibility and lack of toxicity [23,24]. Currently, PLA is commercialized as part of several nanomedicine tools. For vaccine purpose, antigens formulated with PLA-NPs induce broad and potent humoral and cellular immunity in mice, rabbit and macaque models [25,26]. Other researchers have shown that a mycobacterial antigen adsorbed on lamellar particles of lactide polymers have induced cellular immunity [27].

Another strategy to control leishmaniasis is antigen-target-specific vaccines. These are categorized into three types: (i) live *Leishmania* parasites; (ii) killed *Leishmania* or parasite fractions (first generation), and (iii) *Leishmania* recombinant proteins (second generation) or DNA molecules (third generation) [16]. DNA vaccines are of particular interest because they can effectively induce both CD8+ and CD4+ T cells and produce long-lived antigens and properly folded polypeptides [28]. However, these are still in the early phases of clinical trials. Some trials of developing first-generation vaccines have failed to provide convincing results in phase III due to standardization and safety issues, whereas others are still in earlier phases. Second-generation vaccines, e.g., leish-111f, suffer from a lack

of an appropriate adjuvant [9,21]. Among them, the histone H2B protein was described as a potential candidate [7,10]. The histone protein H2B forms with H2A, H3 and H4, the major constituents of the nucleosome in the nucleus of eukaryotic cells [29]. This protein is conserved among various species of *Leishmania*, i.e., *L. major*, *L. infantum*, *L. donovani* and *L. tropica*. Previous studies investigated the immunogenicity and the protective role of the recombinant H2B protein from *L. major* [30]. H2B, in combination with CpG-ODN, confers effective protection to sensitive BALB/c mice infected with the virulent strain of the *L. major* parasite [31]. Later, Meddeb-Garnaoui et al. showed that this recombinant protein H2B induced a specific Th1-type cellular response in individuals who recovered from cutaneous leishmaniasis infection [32].

The purpose of this research was, on the one hand, to develop a particulate vaccine system based on PLA, a polymer that has received FDA approval, and on the other hand, to protect mice against *L. major* parasite infection. To do this, nanoparticle dispersion was developed using PLA polymer (PLA-NPs). Then, BL21 *Escherichia coli* strain cells were used to express the histone H2B protein, a sub-unit vaccine candidate for leishmaniasis. When animals were experimentally infected with the parasite *L. major* promastigotes, the immune protection of immunized mice was investigated and compared to CpG-ODN, a TLR agonist compound.
