Leishmaniasis is a neglected tropical disease complex caused by intracellular protozoan parasites that present an estimated incidence of about one million cases per year in 98 countries worldwide [1]. The clinical manifestations of this disease complex vary from self-limiting cutaneous lesions, characterized as cutaneous leishmaniasis (CL), to the fatal visceral form, visceral leishmaniasis (VL), which can be fatal if acute and untreated [2]. The VL causative agents are the Leishmania donovani and L. infantum species [3]. Treatment against disease is based on the use of pentavalent antimonials, miltefosine, free and liposomal amphotericin B, paramomycin, among others; however, they cause several side effects in the patients, present high cost, require a long time of treatment, and/or there is the emergence of resistant strains [4,5]. In this context, the employ of strategies to reduce the number of disease cases is desirable, and prophylactic vaccination could contribute to solve such questions [6,7]. However, to data, there is no a human vaccine and the few licensed canine vaccines have restricted use, present high cost and variable results are described [8,9].

Regarding the effective vaccine candidates, immunogenic molecules should induce the development of a specific Th1-type cellular response in vaccinated hosts, which will be based on the production of pro-inflammatory cytokines, such as IFN-γ, IL-12, TNF-α, IL-2, as well as by presence of IgG2a isotype antibodies, which contribute to active parasitized cells that kill parasites [10,11]. Otherwise, studies have implicated the presence of immunosuppressive cytokines, such as IL-4, IL-5, IL-10, TGF-β, IL-13, among others, in VL pathogenesis; since they deactivate parasitized cells by hamper the nitric oxide production, contributing then to the development of active disease [12,13].

Distinct Leishmania proteins have been evaluated as vaccine candidates in experimental models to protect against VL [14,15]. However, due to the limited antigenic repertoire found in a single protein; the combination of distinct T-cell epitopes of several parasite proteins could reduce such problem and contribute to better vaccine efficacy [[16], [17], [18]]. In parallel to the selection of immunogenic proteins, the association of immune adjuvants is usually required, since recombinant proteins are poor immunogenic and need of addition of immune system´ activators [19,20]. These compounds contribute to improve the vaccine efficacy, due to their immunogenicity leading to the reduction in the number of doses and long-lasting immunity [21]. One example could be considered the monophosphoryl lipid A (MPLA), which stimulate the development of a Th1-type response by means of the production of IL-2, TNF-α and IFN-γ, as well as by increase the expression of costimulatory molecules in host cells [22]. Poloxamer 407 (Pluronic F127)-based micelles have been also considered for this purpose, mainly by capacity to activate both CD4+ and CD8+ T-cell subtypes and stimulate the development of a Th1-type cellular response [23,24].

In the present study, bioinformatics was used to predict immunogenic T-cell epitopes from peroxidoxin (LINF_230005400) and pyridoxal kinase (LINF_300018100) proteins, aiming to construct the gene codifying a new protein. Peroxidoxins have been showed to be immunogenic in mammalians and to induce protection against Leishmania infection [25,26]. Pyridoxal kinase is also able to induce the development of Th1-type immune response and protect mice against L. infantum infection [27]. In our study, T-cell epitopes of these proteins were predicted and used to construct a new recombinant construct, called LAV, which was then tested in BALB/c mice to protect against VL. The protein was associated with MPLA or Mic as adjuvants, and distinct laboratorial techniques including flow cytometry, capture and indirect ELISA, qPCR, RT-qPCR, limiting dilution assay, among others, were used to evaluate the efficacy of the vaccine candidates to protect against challenge infection.