Recent updates and perspectives on approaches for the development of vaccines against visceral leishmaniasis

Visceral leishmaniasis (VL) is one of the most important tropical diseases worldwide. Although chemotherapy has been widely used to treat this disease, problems related to the development of parasite resistance and side effects associated with the compounds used have been noted. Hence, alternative approaches for VL control are desirable. Some methods, such as vector control and culling of infected dogs, are insuffi ciently effective, with the latter not ethically recommended. The development of vaccines to prevent VL is a feasible and desirable measure for disease control; for example, some vaccines designed to protect dogs against VL have recently been brought to market. These vaccines are based on the combination of parasite fractions or recombinant proteins with adjuvants that are able to induce cellular immune responses; however, their partial effi cacy and the absence of a vaccine to protect against human leishmaniasis underline the need for characterization of new vaccine candidates. This review presents recent advances in control measures for VL based on vaccine development, describing extensively studied antigens, as well as new antigenic proteins recently identifi ed using immuno-proteomic techniques.


INTRODUCTION
Leishmaniasis is a disease complex caused by different species of protozoan parasites of the genus Leishmania (1) .The disease causes high levels of morbidity and mortality worldwide, where approximately 1-1.5 million cases of tegumentary leishmaniasis (TL) and 0.2-0.5 million cases of visceral leishmaniasis (VL) are registered annually (2) .VL is caused by parasites of the Leishmania donovani complex, including the species L. donovani and Leishmania infantum (3) .In the Americas, VL is a zoonotic disease caused by L. infantum, where dogs are considered the main domestic reservoirs of the parasites (4) (5) .In human VL, the outcomes of infection can vary from an asymptomatic and/or subclinical disease to a form with acute symptoms; the disease carries a high risk of mortality in the absence of an adequate treatment (6) (7) .
Chemotherapy based on the administration of pentavalent antimonials has been used to treat VL; however, these products present problems related to their toxicity (8) (9) (10) .Other drugs, such as pentamidine, miltefosine, and amphotericin B also present issues of toxicity and/or high cost (11) (12) (13) (14) .Early diagnosis of VL could allow for more effective treatment of the disease; however, parasitological diagnosis, based on the direct observation of amastigote forms has low sensitivity and requires invasive collection procedures (15) .The detection of Leishmania deoxyribonucleic acid (DNA) using the polymerase chain reaction (PCR) technique is highly specifi c; however, its sensitivity is variable (16) (17) .Serological tests based on the detection of antileishmanial antibodies in patient serum samples are also employed for the diagnosis of VL; however, these are also associated with issues related to sensitivity and/or specifi city, depending on the antigens targeted (18) (19) .
Evidence of life-long immunity to leishmaniasis has also inspired the development of prophylactic vaccination protocols against the disease, although few have progressed beyond the experimental stage (14) .An ideal vaccine candidate against leishmaniasis should be safe, affordable to the population, and able to induce both cluster of differentiation 4 + (CD4 + ) and cluster of differentiation 8 + (CD8 + ) T cell responses and long-term immunological memory, which could be boosted by natural infections, thus reducing the number of vaccine doses required.In addition, an ideal vaccine should be effective against different Leishmania species and stable at room temperature or at 4°C, to eliminate the need for storage at -20°C or -80°C (20) .However, the induction and maintenance of long-lasting immunity and protection against different Leishmania species are very diffi cult to achieve, since the majority of candidate vaccines are composed of antigens that only offer speciesspecifi c protection (21) (22) (23) (24) (25) (26) .
In recent years, proteomic screening studies have revealed a number of antigenic proteins specifi c to the Leishmania genus and frequently annotated as hypothetical proteins in genome databases.This review explores recent developments and discusses the prospects for vaccine development against VL, focusing on well-known antigens described in the literature, as well as the discovery of new antigens by immuno-proteomic approaches.

SECOND-GENERATION VACCINES AGAINST VISCERAL LEISHMANIASIS
Advances in recombinant DNA technology have led to the extensive study of several species-or stage-specifi c Leishmania molecules as candidate vaccines in the form of recombinant proteins.The major advantages of these candidates are their purity and the production yields achievable.Several proteins have been frequently investigated as candidate vaccines for the cutaneous form of leishmaniasis; however, few of these have been evaluated in mammalian VL models (39) .Recombinant proteins have been evaluated as second-generation vaccines for VL with variable degrees of success, usually depending on the vaccine formulation and associated immune adjuvants, as well as the animal model used for testing.Amastigote and promastigote parasite antigens are the most common vaccine candidates tested.
Among amastigote-specifi c antigens tested for induction of immune protection against VL, the A2 antigen has emerged as an effective candidate.It is encoded by a multigene family that is abundantly expressed in the amastigote forms of some Leishmania species able to cause VL (40) .Studies of the administration of recombinant A2 protein associated with immune adjuvants (41) (42) or as a DNA vaccine (43) , as well as in attenuated non-replicative viruses (44) , non-pathogenic bacteria (45) , or non-virulent Leishmania tarentolae (46) , have provided evidence of its protective effi cacy in mammalian models.In general, anti-A2 protective immunity is associated with the generation of parasite-specifi c IgG2a antibodies, as well as with the production of high levels of antileishmanial IFN-γ and low levels of IL-10 by T cells in recall response to the A2 protein or parasite extracts (40) .Other amastigote-specifi c antigens that have been considered promising candidates for VL prevention include the cysteine proteinases (CP).These enzymes belong to the papain super-family, three classes of which (CPA, CPB, and CPC) have been identifi ed in Leishmania parasites.Studies have shown that recombinant CPB protein, in combination with an immune adjuvant or as a DNA vaccine, induced protection against Leishmania major infection in BALB/c mice (47) .In another study, recombinant CPA/ CPB polyprotein vaccine was administered in association with poloxamer 407 as an adjuvant, and induced a protective response against L. major in BALB/c mice, which was more robust than the response induced by recombinant CPA and CPB proteins administered as separate individual antigens (48) ; however, these antigens were not tested as vaccines against VL.
In an evaluation of antigens expressed in promastigote forms of Leishmania parasites as vaccine candidates against VL, parasite surface antigen-2 (PSA-2), which comprises three polypeptides with molecular weights ranging from 50.0 to 96kDa (49) , showed satisfactory results.This immunogen was able to induce protection against a Leishmania challenge in mice, through the development of a Th1-type response, when administered associated with Corynebacterium parvum as an adjuvant (50) .Kinetoplastid membrane protein-11 (KMP-11), a highly conserved protein expressed in different Leishmania species, was also verifi ed as protective against L. donovani infection in hamsters (51) (52) .In addition, the nucleoside hydrolase 36kDa (NH36) antigen was shown to be protective against Leishmania infantum, Leishmania mexicana, and Leishmania amazonensis species in BALB/c mice, indicating its potential as a heterologous vaccine to protect against different Leishmania species (53) (54) .

It has been postulated that a formulation containing different
Leishmania proteins expressed in both parasite stages should provide better results, in terms of a more effective and protective vaccine against VL (42) (55) .The use of vaccines combining different proteins could provide the benefi ts of increased simplicity and reduced production costs, since it would only be necessary to produce a single vaccine to protect against different Leishmania species (56) .However, few studies have evaluated chimeric  (40) Aldolase L. donovani Hamster Partial protection Gupta et al (67) Cysteine-peptidases L. infantum Beagle dog No protection Poot et al (68) Cysteine-proteinase III L. infantum BALB/c mice Partial protection Khoshgoo et al (69) Cyclophilin 1 L. infantum BALB/c mice High protection Santos-Gomes (70) dp72 L. infantum BALB/c mice Partial protection Jaffe et al (71) eIF2 L. donovani Hamster 65% protection Kushawcha (72) HASPB1 L. donovani BALB/c mice 70%-90% protection Stager et al (26) LCR1 L. infantum BALB/c mice Partial protection Wilson et al (73) LdSir2HP L. donovani Hamster High protection Baharia et al (60) LeishH1 L. infantum BALB/c mice High protection Agallou et al (27) L3/L5 L. infantum BALB/c mice High protection Ramírez et al (74) NH36 L. infantum BALB/c mice 80% protection Aguilar-Be et al (54) ORFF L. donovani BALB/c mice Partial protection Tewary et al (75) 78 kDa protein L. donovani BALB/c mice High protection Joshi & Kaur (59) A2/CPA/CPB * L. infantum BALB/c mice High protection Saljoughian et al (57) KSAC * L. infantum C57BL/6 mice High protection Goto et al (61) Leish-111f * L. infantum Beagle dog No protection Gradoni et al (76) NS protein * L. donovani BALB/c mice High protection Coler et al (77) Q protein * L. infantum Beagle dog 90% protection Molano et al (78) 8E/p21/SMT * L. donovani C57BL/6 mice High protection Duthie et al (79) dp72: 72 kDa L. donovani protein; eIF2: eukaryotic initiation factor-2; HASPB1: hydrophilic acylated surface protein B; LCR1: complete conservation of an immunogenic gene; LdSir2HP: NAD + -dependent silent information regulatory- vaccines aimed at protection against VL (25) (57) , since the majority of reports have been of investigations of single antigens (14) (58) (59) (60) .The development of a multi-antigenic vaccine requires an appropriate choice of the biological targets for use in its composition.In a recent study, a polyprotein vaccine formulated with monophosphoryl lipid A, KSAC, was shown to be immunogenic and effective in inducing protection against L. infantum and L. major in mice.KSAC is a chimeric protein composed of the Leishmania homolog of the receptor for activated C kinase (LACK), glycoprotein 63 kDa (gp63), thiol-specifi c-antioxidant (TSA), hydrophilic acylated surface protein B (HASPB), sterol 24-c-methyltransferase (SMT), KMP-11, A2, and CPB proteins.In models challenged with both Leishmania species, the protective response was associated with the production of high levels of IFN-γ, combined with low levels of IL-4 and a decreased antileishmanial IgG1 response (61) .Another chimeric protein, Leish-111f, which is composed of a combination of TSA, stress inducible protein 1 (LmSTI-1), and the Leishmania homolog of the eukaryotic translation initiation factor (eIF4A), was also able to protect BALB/c mice against Leishmania infection, when administered in association with immune adjuvants (62) .
Another fi eld that could be developed in relation to the discovery of new candidate VL vaccines is based on vector salivary proteins.To date, evidence indicates that salivary molecules able to induce a Th1-type response in immunized animals could create a protective immunological environment at the bite site, which could infl uence when parasites are injected, allowing control of the disease and concomitant promotion of Leishmania-specifi c immunity (63) .The Th1-type immunological environment in response to these antigens at the bite site could promote a protective immune response against the parasite challenge.In this context, PdSP15, a 15-kDa salivary protein, which is a member of the family of small odorant binding proteins from Phlebotomus duboscqi, was evaluated as a candidate antigen against leishmaniasis in non-human primates (64) .In addition, LJM19, an 11-kDa salivary protein of unknown function and LJL143, a 38-kDa salivary protein with anticoagulant activity (65) , both of which are present in the saliva of Lutzomyia longipalpis, were shown to be protective against VL (66) .Table 1 shows a summary of relevant vaccine candidates evaluated as individual recombinant protein or polyprotein vaccines against VL (67) (68) (69) (70) (71) (72) (73) (74) (75) (76) (77) (78) (79) .

THE APPLICATION OF IMMUNO-PROTEOMIC APPROACHES TO THE IDENTIFICATION OF NEW LEISHMANIA ANTIGENS WITH POTENTIAL TO BE USED AS VISCERAL LEISHMANIASIS VACCINES
Immuno-proteomic approaches have been developed to identify new Leishmania proteins with distinct biological functions, such as new diagnostic markers, vaccine candidates, and/or potential drug targets (80) (81) (82) .The use of antileishmanial antibodies obtained from infected mammalian hosts contributed to the refinement of these analyses, by assisting in the identifi cation of antigens recognized by the immune system during active disease (82) (83) .Immuno-proteomic approaches usually involve protein preparation and separation by bidimensional electrophoresis, followed by immunoblotting experiments, and subsequent identifi cation of protein spots by mass spectrometry (Figure 1).In a recent immuno-proteomic study, several antigenic parasite proteins were identifi ed from serum samples of dogs developing VL (83) .These proteins were analyzed in silico for epitope identifi cation, and the best antigenic determinants were employed in enzyme-linked immunosorbent assay (ELISA) assays aiming to identify antigenic peptides of interest for the serodiagnosis of canine disease.The authors speculated about the use of these candidates as vaccines in future assays, owing to the existence of putative-T cell motifs in the antigens.In another immuno-proteomic approach, developed using protein extracts from the stationary promastigote and amastigote-like stages of L. infantum, several specific promastigote (Table 2) and amastigote (Table 3) hypothetical proteins were identifi ed in serum samples from dogs with asymptomatic and/or symptomatic VL (82) .
Some of these proteins have already been validated as candidate VL vaccines (Table 4).These antigens were selected because they are conserved among different Leishmania species, but are not present in other Trypanosomatidae or in mammalian hosts.In addition, the selected antigens contain specifi c CD4 + and CD8 + T cell epitopes.In this context, the protective effi cacy against L. infantum infection of LiHyp1, a Leishmania protein belonging to the super-oxygenase family, was evaluated in BALB/c mice.Immunization using the recombinant LiHyp1 protein

TABLE 3
Hypothetical proteins identifi ed in amastigote-like Leishmania infantum by an immuno-proteomic approach using serum samples from dogs with asymptomatic and symptomatic VL.

TABLE 2
Hypothetical proteins identifi ed in Leishmania infantum stationary promastigotes by an immuno-proteomic approach using serum samples from dogs with asymptomatic and symptomatic VL.

TABLE 4
Leishmania-specifi c hypothetical proteins validated as vaccine candidates for visceral leishmaniasis.plus saponin adjuvant induced a Th1 immune response in the vaccinated animals, which was primed by protein-and parasitespecifi c IFN-γ, IL-12, and GM-CSF production, combined with the presence of low levels of IL-4 and IL-10.In addition, the protected animals displayed signifi cant reductions in the number of parasites in their livers, spleens, bone marrow, and draining lymph nodes, compared with that in control groups.
The protection was correlated with parasite-specific and dependent IFN-γ production, mainly by CD4 + T cells, which were the major source of IFN-γ in these animals (14) .The same immune profi le was found when the hypothetical LiHyD (31) , LiHyT (32) , LiHyp6 (55) , and LiHyV (84) proteins were evaluated as vaccine candidates.In all cases, the antigens were shown to be protective against infection, since vaccinated and challenged animals presented signifi cantly lower parasite levels in evaluated organs compared with control groups.In addition, vaccinated and challenged animals demonstrated predominantly IL-12 driven IFN-γ production (also mediated mainly by CD4 + T cells) against parasite proteins, whereas unprotected controls showed high levels of anti-Leishmania IgG1 antibodies and a parasite mediated IL-4 and IL-10 response.
An aspect that should be considered when evaluating the effi cacy of a vaccine is the use of adjuvants.Although recombinant protein-based vaccines offer considerable advantages in terms of safety, standardization, purity, and production costs, they generally present limited immunogenicity and require the use of immune adjuvants (85) .It is generally accepted that the adjuvants used in leishmaniasis vaccine formulations should be able to induce a Th1 response, and some adjuvants are capable of this, including recombinant IL-12, saponin, BCG, monophosphoryl lipid A (MPL), CpG, recombinant virus, and others (86) (87) .The induction of IL-12 is critical for vaccine effi ciency and many of these adjuvants activate the innate immune response via Toll-like receptors (TLR), also infl uencing acquired immune responses (88) .Signifi cant protection is not usually achieved when animals are immunized with recombinant proteins in the absence of adjuvants.These fi ndings have been corroborated by studies evaluating other well-known protective antigens against leishmaniasis (31) (32) (40) (54) .
The concept of cross-protective vaccines is based on the presence of common antigens among pathogens and on the ability of formulated vaccines to elicit cellular immunity (89) .
Since multiple Leishmania species are distributed in common geographical areas, it would be desirable to develop vaccines capable of inducing protection against more than one parasite species (90) .In this context, LiHyT, which was fi rstly identifi ed, in L. infantum (32) as protective against this species, was also shown to confer protection in BALB/c mice against L. major and L. braziliensis.Mice immunized with LiHyT and saponin as an adjuvant, developed a robust Th1 immune response, which was responsible for the induction of signifi cant reductions of parasite load in the tissues and organs evaluated (84) .This cross-protective immunity was also found when another hypothetical protein, rLiHyD, was used as an antigen with saponin as an adjuvant (91) .
As described, the use of chimeric vaccines containing multiple proteins and/or polypeptides could provide more robust protective effi cacy against various Leishmania species (61) (76) (78) .In this context, three recombinant proteins were combined in a vaccine and tested for their protective effects against L. infantum infection.These proteins are expressed in both the promastigote and amastigote stages of the parasites, and their combination was able to induce pronounced parasite-specifi c IFN-γ, IL-12, and GM-CSF responses in immunized mice, which was maintained after challenge.The infected and vaccinated animals showed signifi cant reductions in parasite burden in the various organs evaluated compared with control mice, the protection being associated with IL-12-dependent IFN-γ production against parasite extracts, and correlated with the induction of antileishmanial nitrite production.More importantly, this polyprotein vaccine was able to induce a more robust Th1 response associated with better control of parasite dissemination in the organs of the vaccinated animals, compared with the use of the individual recombinant proteins (55) .

CONCLUSIONS
Effective prophylactic measures to control VL are imperative.Such measures include the design of vaccines, which is the most economical way to control neglected diseases.An ideal vaccine candidate should be able to induce robust antileishmanial Th1 immunity, be parasite-specifi c (to avoid adverse effects in mammalian hosts), and exhibit a high degree of homology between different Leishmania species.Hypothetical proteins, considered unknown molecules until their recognition by the immune system of infected mammalian hosts, could be considered for this purpose and explored for use in the prevention of VL.In addition, the development and use of new technologies, such as reverse vaccinology, to identify novel candidate VL vaccines should be also considered.

TABLE 1
Summary of vaccines against visceral leishmaniasis based on individual recombinant proteins or polyproteins.