Vaccines | Free Full-Text | New Approaches to the Prevention of Visceral Leishmaniasis: A Review of Recent Patents of Potential Candidates for a Chimeric Protein Vaccine

The chimeras described in Table 1 primarily consist of known proteins with recognition of the major histocompatibility complex (MHC), in addition to previous studies demonstrating their potential to be used in vaccines against leishmaniasis, either individually or in association with other proteins. The proteins and some findings of their protective potential against leishmaniasis have been described below and their localization is shown in the schematic picture (Figure 1).

Cysteine proteases (CPs) are enzymes with important functions in the pathogenesis of several parasites during their interaction with the host. They are associated with cell and tissue invasion, protein hydrolysis, autophagy, and modulation of the host’s immune response [80]. The CPA, CPB, and CPC subtypes have been explored as vaccine candidates, capable of inducing protective immunity against cutaneous and visceral leishmaniasis, with the induction of NO and IFN-γ production in mice [81,82,83]. In addition, CPs were able to induce the production of IFN-γ, TNF-α, and IgG2 and low production of IgG1 antibodies and IL-10 in vaccinated dogs [84].

The heat shock protein HSP70 is a ubiquitous 70kDa molecular chaperone, highly conserved, and important in the folding and remodeling processes of cellular proteins [85]. It can thus be present in mitochondria and the endoplasmic reticulum [86]. Its expression is high during the process of passing from the invertebrate vector to the mammalian host, which is important in differentiating between the forms of the parasite [87]. HSP70 can induce high levels of IgG2a, IFN-γ, and IL-2 in mice immunized against L. donovani and suppress the Th2-type immune response [88], as well as the maturation capacity of splenic dendritic cells in mice [89].

K39 is an immunodominant epitope in kinesin-related proteins, consisting of 39 amino acids, highly conserved in the Leishmania donovani complex, present in amastigote forms, and involved in the intracellular process [90,91]. This protein has been used to detect anti-Leishmania antibodies in several diagnostic platforms with a high degree of accuracy. The Kalazar Detect™ (InBios International, Seattle, WA, USA), the IT LEISH^® (BIO-RAD Laboratories Inc., Marnes-la-Coquette, France), and the OnSite™ Leishmania IgG/IgM Combo test (CTK Biotech, Poway, CA, USA) are immunochromatographic tests that have been used in the diagnosis of human VL and are available in the Brazilian public health system [92]. Furthermore, the Dual-Path Platform (DPP—Bio-Manguinhos/Fiocruz, Rio de Janeiro, Brazil), presents a fused protein of rK26/rK39 used for the diagnosis of CVL, also available in Brazil [93]. Regarding immunoenzymatic assays, there are several studies evaluating the performance of rK39 in the diagnosis of both human and canine VL [94,95,96,97]. Despite its wide use in the diagnostic field, only one patent (BR102021000794) [71] was found that used rK39 as a possible vaccine candidate.

Kinetoplastid membrane protein 11 (KMP11) is mainly expressed on the cell surface of amastigotes and promastigotes, but it is also found in membrane structures, intracellular vesicles, and flagellar pockets [98]. This protein can produce strong antigenicity for T cells in humans and mice, being a candidate for the vaccine [99]. Its expression is increased in metacyclogenesis and with greater expression in the amastigote form [100]. Results of studies with this protein have revealed its ability to produce IFN-γ, IL-10, and IgG1 and IgG2a antibodies. Despite stimulating the production of IL-10 and IgG1, their levels were lower than those of the IgG2a and IFN-γ, indicating a more prominent activation of the Th1-type response, resulting in a parasite load reduction in the spleen and lymph nodes in mice [101].

Leishmania homolog of activated C kinase (LACK) is a highly conserved protein, an immunodominant present in all Leishmania species, and expressed in both amastigote and promastigote forms [102,103]. Studies have demonstrated its ability to induce CD8+ T cells and IFN-γ in L. major-infected mice [104]. In addition, the production of IFN-γ and IL-10 by peripheral blood mononuclear cells (PBMCs) was reported in patients with cutaneous leishmaniasis caused by L. amazonensis, L. guyanensis, and L. braziliensis [105,106,107,108]. Similarly, asymptomatic individuals and VL-cured patients presented the production of IFN-γ and TNF-α [29,109]. LACK induced the production of IL-4 [110], and different approaches are needed to redirect the initial IL-4 responses to Th1, such as the use of cytokines or DNA vectors [111,112]. However, some authors have reported failure to protect mice against L. infantum and L. donovani, even when using a DNA plasmid expressing LACK for immunization [113,114]. In contrast, Fernández et al. [115] reported promising results with the use of LACK together with the attenuated Vaccinia virus, in which the protein was recognized by the T cells of asymptomatic individuals infected with L. infantum and VL-cured patients. These data on the LACK antigen reported contradictory results and did not support its use in vaccine formulations against VL.

LiP2a and LiP0 are acidic ribosomal proteins of L. infantum characterized by being immunodominant antigens recognized by the serum of humans and other animals infected by L. infantum [116,117]. These proteins were able to trigger the immune response and induce protection against infection by L. infantum and L. major in mice, with increased CD4+ and CD8+ T cells, and significant production of antigen-specific IL-12 [118]. These proteins also stimulate IFN-γ production via splenocytes in mice immunized with LiP2a [119].

Hypothetical proteins have been described in the genome of Leishmania spp. but without a defined biological role [120]. Through an immunoproteomic study, LiHyp1 and LiHyp2 were recognized by antibodies in serum from VL patients but not in serum of healthy individuals [121]. The use of hypothetical proteins is still a field to be explored. Some authors have demonstrated that hypothetical proteins are capable of reducing the parasitic load on the liver, spleen, bone marrow, and lymph nodes, with an immunogenic profile related to high levels of IFN-γ, IL-12, GM-CSF, and specific IgG2 production [122,123].

Nucleoside hydrolases (NHs) are vital enzymes in the metabolism of DNA, being essential for the replication of parasites, especially during the initial stages of infection. These enzymes are present in all species of Leishmania spp., justifying their use as phylogenetic markers. Additionally, they share high identification levels among many microorganisms but are absent in mammals [124]. These characteristics make NHs targets for an anti-VL vaccine by inducing high immunogenicity [124]. For instance, the NH36 of L. donovani is a non-specific nucleoside hydrolase that is the main antigen of Leishmune^®, a vaccine previously sold in Brazil from 2004 to 2014, which was discontinued due to noncompliance with the requirements of phase III studies for efficacy (Brazil, Technical Note 038/2014). This vaccine had an efficacy and protection rate greater than 80%, being able to induce high levels of IgG2, a predominantly Th1-type immune response with high production of IFN-γ, TNF-α, and IL-17 [44]. Other studies have affirmed the potential of NHs [124,125].

Nucleosomal histones are important proteins in the DNA packaging process, transcription, and gene regulation. Evidence suggests that histones from Leishmania spp. are relevant immunogens during parasite/host interactions [126]. Therefore, histones H2A, H2B, H3, and H4 have been studied as potential vaccines against leishmaniasis. A profile similar to the Th1 response was detected in murine models immunized with histones against L. major and L. donovani infections with IFN-γ and TNF-α induction and low IL-4 production [126,127,128,129]. Moreover, this immunization was able to stimulate immune responses in the PBMCs of cured individuals and patients infected with Leishmania, as well as reduce the parasitic burden by more than 80% in the spleen, liver, and bone marrow in hamsters [129].

Prohibitins (PHBs) are conserved proteins found in all eukaryotic cells in the inner membrane of the mitochondria. These proteins are important in several functions linked to this organelle and the stabilization of its membrane. In Leishmania spp., prohibitins are involved in cell proliferation, greater infectivity, and maintenance of the parasite’s mitochondrial integrity [130,131]. The presence of anti-PHB antibodies in patients infected with L. donovani demonstrated that these proteins are relevant when the disease is active [130]. A study by Lage et al. [132] verified the induction of a Th1-type cellular response with high levels of IFN-γ, IL-12, and GM-CSF in immunized animals. Moreover, a significant reduction in the parasite load was reported in the spleen, liver, lymph nodes, and bone marrow in mice. High levels of IFN-γ in PBMC from healthy individuals and cured VL patients have also been observed [133].

Promastigote surface antigens (PSAs) are members of a family of membrane-bound or secreted proteins from Leishmania spp., involved in resistance to lysis promoted by the complement system during interactions with host cells [134,135]. PSAs are highly conserved and, despite being recognized by immune response cells, preferably Th1 in humans, they do not share homology with mammalian cells [136]. Studies with PSA subtypes have shown that they are capable of inducing Th1-type responses with IFN-γ production in mice against L. major infection [137] and proliferation of PBMCs in patients with cutaneous leishmaniasis in response to PSA-2 [138]. Chamakh-Ayari et al. [136] demonstrated that the PSA-38S of L. amazonensis induced a mixed Th1/Th2 response in individuals with immunity to L. major and L. infantum, in addition to inducing granzyme B production. Petitdidier et al. [139] demonstrated that PSA can induce IFN-γ, nitric oxide, and IgG2 antibodies in vaccinated dogs.

Small glutamine-rich TPR proteins (SGTs) are co-chaperones that interact with HSP70 and HSP90 chaperones to ensure their proper functions, which are essential to the parasite’s life cycle and survival [140]. Dias et al. [141] identified the potential of L. infantum’s SGTs for vaccine and diagnostic approaches against VL. Mice immunized with SGTs developed a specific Th1-type response by producing IFN-γ, IL-12, and IgG2a, which induced a resistance profile against the infection.

Specific amastigote protein A2 corresponds to a family of specific amastigote genes necessary for the parasite’s survival and results in a virulence factor [142]. A2 was the main protein in the Leish-Tec^® vaccine and has been explored as a possible vaccine candidate as it has been shown to induce a Th1 immune response represented by biomarkers IFN-γ, TNF-α, and IgG2 antibodies that conferred partial protection against CVL [14,47,52,143,144].

Sterol 24-c-methyltransferase (SMT) is an enzyme of the transferase family, important in steroid production, especially ergosterol biosynthesis present in the Leishmania spp. membrane [145]. Mice immunized with SMT showed IFN-γ induction and lower spleen and liver parasite loads [64]. SMT, together with the nucleoside hydrolase NH36 of L. donovani, composed a vaccine developed against human VL that reached phase I clinical trials [146].