Leishmaniasis constitutes a globally widespread group of neglected tropical diseases (NTDs) caused by intracellular parasites of the genus Leishmania, transmitted to mammalian hosts through the bite of phlebotomine sandflies [1]. Cutaneous leishmaniasis (CL) is the most prevalent clinical form of the disease and can be caused by L. (L.) tropica, L. (L.) major, L. (L.) infantum, L. (L.) aethiopica, L. (M.) martiniquensis, and L. (M.) orientalis in the Old World, and by L. (L.) mexicana, L. (L.) amazonensis, L. (L.) venezuelensis, L. (V.) colombiensis, L. (V.) lainsoni, L. (V.) naiffi, L. (V.) lindenbergi, L. (V.) panamensis, L. (V.) peruviana, L. (V.) braziliensis, and L. (V.) guyanensis in the New World [2]. The Global Health Observatory data repository indicates that about 240,000 new autochthonous CL cases had been reported annually between 2016 and 2020, mostly distributed throughout the eastern mediterranean area, America and Africa [3]. However, these numbers are likely to be underestimated, as leishmaniasis occurs in poor remote regions with limited access to diagnostic and treatment services, and many cases go unnoticed by health authorities, since not all affected countries require mandatory reporting [4], [5].

The disease evolution in CL generally shows a painless ulcerative skin lesion at the site of sand fly bites. Ulcers are frequently observed in exposed body areas. Although this clinical form is not life-threatening, as most of the lesions self-heal over 3-18 months, it may cause serious permanent scars which often lead to social stigmatization [6], [1]. Furthermore, up to 10% of CL cases progress to more severe manifestations, such as mucocutaneous leishmaniasis (MCL), disseminated leishmaniasis (DL), or anergic diffuse cutaneous leishmaniasis (ADCL). These clinical forms can result in facial disfigurement with distressing social and psychological consequences or can also lead to physical disabilities that negatively impact on a patient's quality of life [7], [8]. Despite the high morbidity related to CL, no human vaccine is currently available against this disease.

Over the past decade, several efforts have been made to develop a prophylactic vaccine against various species of Leishmania, based on killed or live-attenuated parasites, fractionated antigens, recombinant proteins or DNA vaccines [9]. It is well documented that an effective Leishmania-vaccine should be capable of inducing a predominantly pro-inflammatory response, mainly driven by CD4+ T cells [10], [11], [12], [13], [14], [15]. Thus, Th1-type cytokines, such as IFN-γ and TNF-α, induce parasite killing within macrophages through nitric oxide (NO) production. These cytokines also promote dendritic cells (DCs) maturation. In contrast, Th2-type cytokines, such as IL-4 or IL-10, suppress macrophage activity as well as Th1 CD4+ cells and instead drive polyamine biosynthesis, facilitating intracellular parasite replication [16], [17].

The efficacy of a vaccine depends importantly on an appropriate adjuvant, capable of enhancing the magnitude and durability of the immune response [18]. Vaccine adjuvants represent a heterogeneous group of compounds that can be divided into cytokines, microbial components/products, pattern recognition receptor (PRRs) agonists, mineral salts, emulsions, saponins, and delivery systems [19]. These agents exert their adjuvanticity through different mechanisms, including the formation of antigen depot at the inoculation site, activation of inflammasomes, enhancement of antigen uptake by antigen presenting cells (APCs), activation and maturation of APCs, induction of cellular recruitment at the site of the injection, modulation of immune mediators like cytokines and chemokines, and production of specific antibody isotypes [19], [20]. Despite the potential benefits that adjuvants offer in vaccine performance, only a few have been licensed for clinical use [18].

The adjuvant α-Galactosylceramide (αGalCer) is a synthetic α-galactosylated sphingolipid composed of a α-linked sugar and lipid moieties derived from an extract of the marine sponge Agelas mauritianas [21]. Since its discovery in the early 1990s, several studies have highlighted its potential effect against solid tumors, as it can trigger the early production of IFN-γ by CD1d-restricted invariant Natural Killer T cells (iNKT) [22]. Furthermore, αGalCer has been used as an adjuvant to enhance the efficacy of various experimental vaccine platforms against infectious diseases [23]. It has been shown that co-administration of this glycolipid with irradiated Plasmodium yoelii sporozoites (γ-spz) enhances the protective anti-malaria immunity mediated by both CD1d molecules and Vα14 iNKT cells [24], [25]. In the case of Mycobacterium tuberculosis, the addition of αGalCer or their C-glycoside analogue (α-C-GalCer) to live attenuated Bacille Calmette-Guérin (BCG) vaccine, leads to a strong protective immunity mediated by CD8+ T-cell and Th1-biased iNKT cell responses [26]. In addition, the co-administration of αGalCer with a dual-promoter DNA vaccine, that encodes human immunodeficiency virus (HIV) env and gag antigens (pADVAX-e/g), has shown to enhance and prolong virus-specific CD4+ and CD8+ T-cell responses, which are mediated by CD1d molecules and the IFN-γ receptor [27]. Thus, the glycolipid αGalCer is possibly a promising candidate to be used as adjuvant in vaccines against intracellular pathogens.

In the present study, we evaluated the protective immunity against L. mexicana infection induced by a single-dose injection of αGalCer combined with an amastigote lysate as an antigen source. We found that prophylactic vaccination with αGalCer induced a Th1-biased protective immune response against L. mexicana infection, leading to significant reduction of the parasite load in footpad lesions of infected mice. Furthermore, a single intraperitoneal immunization with αGalCer enhanced Ly6G and MHCII expression in peritoneal cells and also stimulated the maturation of splenic DCs. Serum IFN-γ levels were also markedly enhanced. These results provide evidence that αGalCer improves protection against CL, strengthening its potential use as an adjuvant for Leishmania-vaccines.