Leishmaniasis is an important disease mediated by the protozoan parasite Leishmania via the bite of the female sandfly insect vector. Leishmaniasis is endemic in the tropical and subtropical regions. The most common form of the disease is cutaneous leishmaniasis, which affects more than 10 million people worldwide and includes at least 1.5 million new cases every year. So far, treatment of the disease relies on unsatisfactory chemotherapy that can be complicated by the rising appearance of drug-resistant parasites. Furthermore, it is challenging to achieve solid control of the insect vector and animal reservoir. Therefore, the development of a safe and effective vaccine is urgently needed for the treatment and prevention of leishmaniasis. This review focuses on the recent advances in the development of a safe vaccine that could be used for prevention and treatment of cutaneous leishmaniasis. A short outlook for future research efforts is also presented.
Leishmaniasis represents an important global health problem in tropical and subtropical areas, affecting at least 12 million people worldwide. Each year, 2 million new cases arise and 350 million humans are at risk of contracting this disease in more than 88 countries.1 The World Health Organization still considers leishmaniasis as one of the emerging uncontrolled diseases affecting mainly poor regions. Transmission of the disease is achieved through the injection of single-celled parasites by infected female sandflies of the genus Phlebotomus in the Old World and Lutzomyia in the New World. The most common form of leishmaniasis experienced worldwide, as well as in the United States, is the cutaneous form of leishmaniasis (CL), which is caused by approximately 20 species of Leishmania. Cutaneous form of leishmaniasis is considered a zoonotic disease as it is typically passed on from vertebrate animals to humans, who are accidental hosts. Various forms of CL exist. The localized cutaneous form is characterized by a self-healing lesion at the site of the bite and is caused primarily by Leishmania major, Leishmania tropica, and Leishmania aethiopica in the Old World, and Leishmania amazonensis and Leishmania mexicana in the New World. Leishmania braziliensis, Leishmania panamensis, and Leishmania guyanensis account for the more severe form of CL called mucocutaneous leishmaniasis, which is only prevalent in the New World and affects the mouth, nose, and occasionally the ear tissues.2,3 Mucocutaneous leishmaniasis is hard to treat and is often associated with secondary infections that can be lethal. Diffuse cutaneous leishmaniasis is caused primarily by L mexicana and L amazonensis and is characterized by lesions that spread from the site of infection and may cover the whole body. Leishmania donovani and Leishmania infantum are the causative agents of the most severe form of the disease, visceral leishmaniasis, which is lethal if not treated. Occasionally, patients cured of L donovani infection exhibit a syndrome called post kala-azar dermal leishmaniasis.
Leishmania infection predominantly triggers a T-cell–mediated immune response. Shortly after infection, Leishmania parasites are phagocytosed by neutrophils, macrophages, and dendritic cells (DCs). Although neutrophils are among the first cells recruited to the site of infection, their role in disease progression and control is controversial and depends on the strain of the parasite and mice (reviewed in previous works4–7). Dendritic cell functions primarily in antigen processing and presentation for T-cell priming, leading to CD4+ or CD8+ polarization (reviewed in the study by Feijo et al8). Dendritic cell also secretes most of the cytokine interleukin 12 (IL-12), which is necessary for the induction of a protective helper T type 1 (TH1) response characterized by the production of IFN-γ and tumor necrosis factor α (TNF-α).9–12 On activation with IFN-γ and/or TNF-α, macrophages efficiently kill most parasites by inducing the generation of nitric oxide (NO) and reactive oxygen species.13,14 In contrast, a helper T type 2 (TH2)-type response leads to disease progression and is associated with the production of interleukin 10 (IL-10), interleukin 2 (IL-2), and interleukin 4 (IL-4).15,16 Lasting protection against Leishmania is mediated by several subsets of memory T cells (T effector, CD4+) and involves the production of cytokines IL-2, IL-4, and IFN-γ.17,18
The treatment of CL relies primarily on inadequate, expensive chemotherapeutic drugs that display several undesirable side effects and can be sometimes difficult to administer.19 In addition, the rising appearance of drug-resistant parasites complicates the drug treatment of leishmaniasis, and controlling the sandflies’ and/or animal reservoirs represents a real challenge. These compelling facts combined with the rising occurrence of CL make the development of a safe, effective vaccine a necessity for the prevention and treatment of CL.
The development of a CL vaccine has been met by several challenges. Specifically, varying genetic characteristics of individual hosts and parasites and, more importantly, the varying immune responses caused by different Leishmania species make the development of CL vaccine incredibly complex. An effective vaccination geared toward combating the disease must not only be safe and easily accessible but also be capable of efficiently sustaining the prolonged induction of CD4+ and CD8+ memory T cells (reviewed in the study by Glennie and Scott18). This induction is essential and allows the immune system to efficiently respond to a pathogen previously encountered, contributing to a lifelong protection against CL. The various vaccination strategies are described in the following sections and summarized in Table 1.
Summary of CL vaccines.
Vaccination type
Antigens
Live vaccination
Live L major
Whole-killed vaccines
Whole-killed L major, L guyanensis, L braziliensis, or L amazonensis (alone or in combination20)Merthiolate-killed L amazonensis (Leishvacin)21
Histone 1,38 CP A and B,39,40 KSAC,41 ribosomal proteins L3 and L5,42 Leish-111F,43–45LeIF43,44
DNA vaccines
GP63,46–48 LACK,35 A2,49,50 iron superoxide dismutase,51 histone proteins H2A, H2B, H3 and H4,52,53 MKP-11,54 HisAK7054
Sandfly saliva components
Saliva of Phlebotomus papatasi,55,56 PpSP15,57 saliva of L longipalpis,58L intermedia salivary gland extract59
DC-based vaccines
SLA, protein subunits, recombinant proteins, DNA vaccine
Abbreviations: CL, cutaneous form of leishmaniasis; CP, cysteine proteinase; DC, dendritic cell, LACK, Leishmania homolog of receptors for activated C kinase; LeIF, L braziliensis elongation and initiation factor; SLA, soluble Leishmania antigen.
Live Vaccination
Attempts to contrive an effective vaccine date back to the early 20th century when live parasites were first inoculated in healthy individuals through a process known as leishmanization. This procedure led to a lifelong immunity and provided the rational proof that vaccination against leishmaniasis may be possible. Due to safety concerns and problem in standardization, leishmanization was later discontinued in most countries.60 However, the traditional practice of leishmanization is making a strong comeback in certain endemic regions because it mimics a natural infection.61 Due to its potential efficacy, efforts to develop a safer leishmanization process by concomitant stimulation of the immune system to control the growth of the parasite are currently underway.62–65
Whole-Killed Vaccines
Whole-killed parasites of strains L major, L guyanensis, L braziliensis, and L amazonensis (alone or in combination) were tested in human trials but were ineffective in mediating protection. These parasites provided poor antigens and thus did not trigger a robust immune response, even in the presence of adjuvants (summarized in the study by Noazin et al20). The main advantages of using whole-killed parasite vaccines are their safety and easiness in mass production. Unfortunately, intramuscular vaccination of Balb/C mice with merthiolate-killed L amazonensis antigens LaAg (Leishvacin) enhanced susceptibility to cutaneous leishmaniasis due to overproduction of transforming growth factor β (TGF-β).66 Phase 3 trial showed that 3-time intramuscular injection of the Leishvacin formula failed to mediate protection in human subjects.21 However, more recent studies demonstrated that intranasal vaccination using the same antigens provided protection against L amazonensis and L braziliensis in a mouse and hamster model of infection, respectively, proving that the route of administration plays a critical role in the efficacy of a vaccine.67,68
Live Attenuated Parasites
Due to the inefficacy of the whole-killed vaccines, there has been a consequent shift toward “live attenuated” vaccines, which seem to provide a more advantageous substitute. By mimicking the actions of the naturally occurring Leishmania infection, the live attenuated parasites can present a wide variety of antigens to the antigen-presenting cells, leading to a more effective immune response and a better overall defensive result. Nonvirulent microorganisms were generated by knocking out specific virulence genes or alternatively by subjecting the parasites to irradiation69 or long-term in vitro cultivation.
Null mutants as vaccine candidates
The first null mutant strains used as vaccine candidates were developed in the 1990s when genetic manipulation of the parasite became possible.70 Vaccination with a null mutant was originally achieved with dihydrofolate reductase thymidylate synthase (dhfr-ts−/−) knockout parasites, which induced substantial protection against both L major and L amazonensis infections in mice but failed to prevent infection in monkeys.22–24 Null mutant of linJhsp70-II−/− (heat shock protein 70-II) of L infantum protected Balb/C mice against L major infection, induced NO production, and triggered a TH1 immune response. In addition, this strain failed to form a lesion in immunodeficient mice, suggesting that it is a safe vaccine candidate.25 More recently, ldcen−/− and ldp27−/−, lacking CENTRIN or P27 gene, respectively, were tested against L mexicana infection and found to be effective in protecting Balb/C mice.26 However, LdCen−/− conferred only partial protection against L braziliensis.27 Cysteine proteinase–deficient mutants of L mexicana (CP-deficient L mexicana) were effective in protecting hamsters against homologous challenge by eliciting significantly lower levels of TH2-associated cytokines IL-10 and TGF-β than the corresponding wild type.28 None of these null mutant strains has been tested in other animal models yet. Despite encouraging results, null mutants as vaccine candidates may revert to a virulent form and thus create a true concern regarding their safety.71
“Suicidal” parasites as vaccination tools
“Suicidal” parasites are transgenic lines of Leishmania that are designed so they can be killed either by physical methods or by application of a specific drug. Therefore, their growth within a host can be precisely controlled, making these strains safer than their live virulent counterparts. Delta-aminolevulinate dehydratase and porphobilinogen deaminase are absent in Leishmania, and thus, expression of these enzymes render the transgenic parasites sensitive to UV irradiation.29 Vaccination with such a transgenic line led to a 99% reduction in parasitic load.30 Another study demonstrated that transgenic parasites lmtkcd+/+, expressing thymidine kinase and cytosine deaminase, become sensitive to the drugs ganciclovir and 5-fluorocytosine. Balb/C mice lesions were cured in 2 weeks in the presence of these drugs, and the transgenic line mediated complete protection when wild-type L major was injected 8 days after vaccination.72 However, development of drug resistance is a plausible risk associated with the latter vaccination protocol.
Purified Antigens and Recombinant Subunits
In recent studies, more than 30 different Leishmania recombinant subunits and purified antigens have been identified and tested in animal models (reviewed in the study by Okwor and Uzonna6), but most of these models were assessed against L donovani, the causative agent of visceral leishmaniasis, the most severe form of the disease. Recombinant subunits or antigens are very safe and relatively easy to produce in large quantities but need to be co-injected with an adjuvant to stimulate the immune system. In addition, several injections (boosts) may be required to induce a satisfactory immune response. Recombinant proteins are typically expressed using a heterologous microbial system, whereas others, known as synthetic vaccines, are produced in vitro as short polypeptides that are predicted to be immunogenic. Synthetic vaccines are considered much safer than vaccines originating from a parasite. Purified antigens originate from the parasite, and their isolation protocol may be difficult to upscale or may contain contaminants. Regarding purified antigens, much work has been done with parasite cell surface metalloprotease GP63, which conferred only partial protection in monkeys but mediated robust protection in mice against challenge with both L mexicana and L major.31–33Leishmania homolog of receptors for activated C kinase (LACK) has also attracted much interest as a vaccine candidate because it is expressed in both insect and vertebrate host form of the parasite.34 Mice vaccinated with LACK became resistant to L major infection.35 Other examples of antigens are L major H2B histone protein and its divergent N-terminal region, which were tested for their ability to protect against CL and visceral leishmaniasis in the presence of the adjuvant CpG.36 Immunization with sterol 24-C-methyltransferase sterol methyl transferase of L infantum, formulated with monophosphoryl lipid A as adjuvant, cross-protected mice against CL caused by L major.37
Examples of recombinant antigens include histone H1, which was tested in vervet monkeys in the presence of the Montanide ISA 720 adjuvant and showed reduced lesion formation after L major infection that self-healed with time,38 suggesting a good potential for human vaccination. Fusion protein made of CP A and B from L major and cysteine protease from L pifanoi mediated only partial protection in mice.39,40 KSAC is a recombinant protein made of KMP-11, SMT, A2 and CPB that when injected with the toll-like receptor (TLR)-4 agonist glucopyranosyl lipid A protected against cutaneous disease following sandfly transmission of L major in susceptible Balb/C mice.41 With the idea to develop a pan-Leishmania vaccine, L major recombinant ribosomal proteins L3 and L5 combined with CpG-oligo-deoxynucleotides conferred protection against L major and L braziliensis challenge in Balb/C mice by inducing a TH1 response.42 Leish-111F, an antigen made of 3 fused proteins (L major thiol-specific antioxidant thiol-specific antioxidant [TSA], L major stress-inducible protein-1 [STI1], and L braziliensis elongation and initiation factor) rendered mice resistant to L major infection.43,44 This antigen, combined with the adjuvant monophosphoryl lipid A plus squalene (MPL-SE), was the first defined vaccine candidate that was tested in human phase 1 and 2 clinical trials and was found to be safe and immunogenic.45 It is still unclear whether Leish-111F confers protection in humans; however, optimization of Leish-111F system is currently underway.1 The C-terminal and N-terminal domains of L donovani nucleoside hydrolase vaccines also decreased the footpad lesion formation caused by L amazonensis.73 Although numerous purified and recombinant antigens have been tested successfully in animals, no human trials have yet been attempted. It is encouraging to observe that cross-species reactivity exists with several species-specific antigens, opening the possibility of a “pan” anti-Leishmania vaccine. Although there is no lack of antigen candidates, the challenge lies in identifying the proper adjuvant(s) that will induce a robust protective immunity.
DNA Vaccines
DNA vaccines, also referred to as third-generation vaccines, are the newest approach in vaccine development. The main advantage of DNA vaccines is that they induce a stronger immune response against the encoded antigen74 by providing a constant source of antigen in its native configuration. Furthermore, they are safe, relatively easy to administer, and preferentially induce a TH1 immune response.75 Similar to purified or recombinant antigens, DNA vaccines may require the co-injection of adjuvants and several boosts to induce a satisfactory protective immune response.
The gene coding for surface metalloprotease GP63 was the first DNA vaccine developed against leishmaniasis. Expression of GP63 in mice mediated solid protection against L major infection when DNA was injected or when GP63 was expressed in attenuated Salmonella typhimurium.46–48Leishmania homolog of receptors for activated C kinase antigen is the most extensively studied DNA vaccine against Leishmania. In clinical trials, inclusion of IL-12 increased the protection of LACK compared with LACK alone.35 DNA-encoding A2 protein mediated protection against L amazonensis infection in mice in contrast to heat shock protein 20 (HSP20) and surface protein 2.49,50 More recently, the TSA-based DNA vaccine was successful in controlling L major challenge via a TH1 immune response.76 Iron superoxide dismutase of L donovani protected Balb/C mice against L amazonensis infection by inducing IFN-γ production which led to reduced parasitism.51 Further studies that involved vaccination with plasmid pcDNA3H3H4 expressing L major histone proteins H3 and H4 resulted in partial resistance to L major challenge associated with the development of mixed TH1/TH2-type response and a reduction in the number of parasite-specific regulatory T cells at the site of infection.77 Vaccination with DNA-encoding L infantum histone genes H2A, H2B, H3, and H4 also controlled both L major and L braziliensis infections in Balb/C mice.52,53 Addition of KMP-11 (kinetoplastid membrane protein-11), A2, and HSP70 genes to H2A, H2B, H3, and H4 in the form of HisAK70 DNA vaccine was successful in clearing parasites from the liver in a mouse model of visceral leishmaniasis and resulted in 100% inhibition of parasite visceralization in the CL model.54 Therefore, the enhanced DNA vaccine provided cross-protection against both CL and visceral leishmaniasis (L major and L infantum).54 The overall efficacy of this vaccine was attributed to the ability of the immunized mice to control key factors such as IFN-γ, IL-10, and IL-4 activity. The promising nature of the HisAK70 once again reinforces the common belief that development of an effective antileishmanial vaccine is entirely possible, and that HisAK70 may play an integral role in such development. Although the results were more than promising in the mouse model, such success has yet to be translated in primates and humans.
Sandfly Saliva Components
During the infection process, a sandfly injects parasites as well as saliva components, which have been shown to help the establishment of infection.78 Similar to immunization with parasite antigens, immunization with sandfly saliva components is very safe. In addition, previous studies have examined sandfly saliva as a transmission blocking vaccine candidate. Pre-exposure to the saliva of the P papatasi sandfly mediated protection against L major challenge by inducing strong IFN-γ production.55,56 Furthermore, P papatasi PpSP15, a component of the sandfly saliva, when expressed and secreted by the nonpathogenic strain L tarentolae in combination with CpG as a prime boost conferred resistance to L major infection in mice.57 Similarly, immunization with recombinant Ljm11 or salivary gland extracts from L longipalpis saliva mediated protection against L major infected sandfly bites and L braziliensis, respectively, via induction of IFN-γ.58 In contrast, L intermedia salivary gland sonicate failed to control L braziliensis infection and even increased disease progression due to low IFN-γ to IL-4 ratio.59 These conflicting results suggest that (1) salivary gland components have the ability to change the immune response of mice by either increasing susceptibility or resistance and (2) use of sandfly saliva components may not be a suitable strategy for all strains of Leishmania. Because leishmanization seems to be an effective procedure that does not involve the sandfly, sandfly components may not be essential to the development of an effective Leishmania vaccine but may still be useful against certain strains of Leishmania.
Immunotherapy
Leishmania is an intracellular parasite; thus, control of its infection is T-cell mediated. Both CD4+ and CD8+ T cells are important for primary immunity against L major, even though their contributions vary depending on the strain of Leishmania (reviewed in the study by Glennie and Scott18). CD4+ TH1 cells produce IFN-γ and TNF-α that activate macrophages, resulting in parasite elimination in resistant mice.13 In contrast, the early production of IL-4 promotes differentiation and proliferation of TH2 cells, resulting in disease progression in susceptible mice.79 The amount of IL-12 produced by DC at the initial phase of infection determines the outcome of the infection. Low levels of IL-12 lead to a TH2 immune response, whereas high amounts of IL-12 result in a TH1 immune response.80 However, IL-4 and IL-13 synergize to mediate susceptibility to L major infection.81,82 Other important cytokines that regulate the disease progression are IL-10 and IL-17, which favor parasite survival and disease progression.83,84
Recovery from Leishmania infection leads to infection-induced resistance, which is the underlying principle of leishmanization and by extension, of lifelong immunity. A thorough understanding of the molecular processes involved in infection-induced immunity is critical for vaccine development. In mice, infection-induced immunity is characterized by IFN-γ producing CD4+ TH1 cells. Stimulation and maintenance of TH1 cells are mediated by IL-12, which is secreted by antigen-presenting cells such as DCs. Because CD8+ cells can also produce IFN-γ, they are believed to contribute to L major immunity by suppressing the early CD4+ TH2 cell development. IL 12 promotes a TH1 response in a mouse of model of CL for long-term immunity.85,86 Consistent with this concept, inclusion of IL-12 as part of a DNA vaccine cocktail improved protection against L major challenge.87–89 However, administration of IL-12 in humans is toxic; thus, this strategy is not suitable for human vaccination.90,91
Several studies have demonstrated that complete clearance of the parasite by a TH1 immune response, which is desirable for the safety of a patient, is, however, not sufficient in mediating long-term immunity.6,92 It is also well accepted that sustained controlled stimulation of IFN-γ–producing long-lived memory CD4+ T cells is necessary to confer long-term immunity.18 This can be accomplished by having a small population of persistent parasites or by “boosting” several times to maintain protection.93–95 Alternatively, adjuvants need to be added to the vaccine cocktail to elicit the proper immune response.
Targeting TLRs for vaccine development
Toll-like receptors are a collection of 13 eleven-transmembrane proteins that recognize structurally conserved molecules derived from pathogens and play a role in innate immune system. Many adjuvants are TLR antagonists and thus amplify the response of the immune system. Adjuvants improve the efficacy of Leishmania vaccine candidates by triggering high levels of IL-12 and IFN-γ expression, both of which play vital roles in long-term immunological memory.85,86,96 The TLR7 agonist Aldara and TLR9 agonist CpG DNA exhibited therapeutic antileishmanial properties (reviewed in the study by Raman et al97). In contrast, TLR2 agonist Pam3CSK4 led to conflicting results depending on the mouse model used.63,98 These encouraging results support the idea that targeting the proper TLRs for vaccine development is a feasible strategy.
DCs containing vaccines
Dendritic cells are one of the first phagocytic and antigen-presenting cells that phagocytose Leishmania parasites shortly after the inoculation. Due to their unique ability to initiate and moderate immune responses, typically in the generation of a protective TH1 cell immune response (reviewed in the study by Bagirova et al99), DC may serve as a central target for the eventual development of proficient vaccines against Leishmania. This idea was exploited by several laboratories, and results from these studies have been summarized in the recent review by Bagirova et al.99 In a typical DC vaccination protocol, DCs are isolated and stimulated with the antigens of interest (soluble Leishmania antigen, subunit, recombinant proteins, or DNA vaccine) before being injected into an animal followed by parasite challenge. Dendritic cell vaccination, with most antigens tested, provided protection against L major in a mouse model of infection.99 However, DC vaccine is not applicable against L amazonensis and L mexicana as these species poorly activate DCs.99 Instead, L amazonensis parasites activate natural killer (NK) cells, which promote IL-12 secretion similar to DCs.100 Thus, modulation of NK cells may offer an alternative vaccine strategy against this stain of Leishmania. Dendritic cell vaccines are extremely safe. Their success depends on the choice of antigen (native versus denatured antigens and recombinant proteins) as certain antigens exacerbate the disease rather than mediate protection.101 In addition, the inclusion of a suitable adjuvant is critical in optimizing the efficiency of such protocol, as well as the use of proper subtypes of DC.102,103
Conclusions
Cutaneous form of leishmaniasis remains a serious global health problem. Currently, no effective vaccines exist against this disease despite much effort from numerous research groups over several decades. Various approaches have been tested, from live whole parasites to attenuated cell lines and from the use of individual antigens/recombinant proteins to DNA vaccines. Several suitable antigens have been identified so far and delivered promising results in animal models. One of the main challenges is to transfer results from animal model studies to humans. More recently, immunotherapy has presented itself as a promising strategy for Leishmania vaccination. However, a better knowledge of the CL immunology is needed to uncover suitable points of intervention. This will provide a platform for the identification of suitable adjuvants, reagents, and methodologies needed to induce the maturation and proliferation of the proper memory T cells that are “pretrained” to recognize and clear Leishmania parasites on an ulterior infection. Overcoming these challenges will lead to the development of an effective vaccine for prevention and treatment of not only CL but also the most severe visceral leishmaniasis.
Five peer reviewers contributed to the peer review report. Reviewers’ reports totaled 722 words, excluding any confidential comments to the academic editor.
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: R.Z. was supported by the NIH SC3GM113743 grant.
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
DCW and RZ wrote, reviewed, and approved the final manuscript.
ReferencesWHO. Status of vaccine research and development of vaccines for leishmaniasis. http://www.who.int/immunization/research/meetings_workshops/Leishmaniasis_vaccineRD_Sept2014.pdf. Published 2014.DavidCVCraftN. Cutaneous and mucocutaneous leishmaniasis. . 2009;22:491–502.KayePScottP. Leishmaniasis: complexity at the host-pathogen interface. . 2011;9:604–615.CarlsenEDLiangYSheliteTRWalkerDHMelbyPCSoongL. Permissive and protective roles for neutrophils in leishmaniasis. . 2015;182:109–118.Tacchini-CottierFZweifelCBelkaidY. An immunomodulatory function for neutrophils during the induction of a CD4+ Th2 response in BALB/c mice infected with Leishmania major. . 2000;165:2628–2636.OkworIUzonnaJ. Vaccines and vaccination strategies against human cutaneous leishmaniasis. . 2009;5:291–301.KorbelDSFinneyOCRileyEM. Natural killer cells and innate immunity to protozoan pathogens. . 2004;34:1517–1528.FeijoDTiburcioRAmpueroMBrodskynCTavaresN. Dendritic cells and Leishmania infection: adding layers of complexity to a complex disease. . 2016;2016:3967436.GorakPMEngwerdaCRKayePM. Dendritic cells, but not macrophages, produce IL-12 immediately following Leishmania donovani infection. . 1998;28:687–695.LeonBLopez-BravoMArdavinC. Monocyte-derived dendritic cells formed at the infection site control the induction of protective T helper 1 responses against Leishmania. . 2007;26:519–531.KonecnyPStaggAJJebbariHEnglishNDavidsonRNKnightSC. Murine dendritic cells internalize Leishmania major promastigotes, produce IL-12 p40 and stimulate primary T cell proliferation in vitro. . 1999;29:1803–1811.MarovichMAMcDowellMAThomasEKNutmanTB. IL-12p70 production by Leishmania major-harboring human dendritic cells is a CD40/CD40 ligand-dependent process. . 2000;164:5858–5865.LiewFYLiYMillottS. Tumor necrosis factor-alpha synergizes with IFN-gamma in mediating killing of Leishmania major through the induction of nitric oxide. . 1990;145:4306–4310.NovaisFOSantiagoRCBaficaA. Neutrophils and macrophages cooperate in host resistance against Leishmania braziliensis infection. . 2009;183:8088–8098.BhattacharyaPAliN. Involvement and interactions of different immune cells and their cytokines in human visceral leishmaniasis. . 2013;46:128–134.CostaDLCarregaroVLima-JuniorDS. BALB/c mice infected with antimony treatment refractory isolate of Leishmania braziliensis present severe lesions due to IL-4 production. . 2011;5:e965.ZaphCUzonnaJBeverleySMScottP. Central memory T cells mediate long-term immunity to Leishmania major in the absence of persistent parasites. . 2004;10:1104–1110.GlennieNDScottP. Memory T cells in cutaneous leishmaniasis. . 2016;309:50–54.WHO. Control of Leishmaniasis. . http://apps.who.int/iris/bitstream/10665/22438/1/A60_10-en.pdf. Published 2007.NoazinSKhamesipourAMoultonLH. Efficacy of killed whole-parasite vaccines in the prevention of leishmaniasis: a meta-analysis. . 2009;27:4747–4753.VelezIDGilchristKArbelaezMP. Failure of a killed Leishmania amazonensis vaccine against American cutaneous leishmaniasis in Colombia. . 2005;99:593–598.TitusRGGueiros-FilhoFJde FreitasLABeverleySM. Development of a safe live Leishmania vaccine line by gene replacement. . 1995;92:10267–10271.VerasPBrodskynCBalestieriF. A dhfr-ts- Leishmania major knockout mutant cross-protects against Leishmania amazonensis. . 1999;94:491–496.KhamesipourARafatiSDavoudiNMaboudiFModabberF. Leishmaniasis vaccine candidates for development: a global overview. . 2006;123:423–438.CarrionJFolgueiraCSotoMFresnoMRequenaJM. Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation. . 2011;4:150.DeyRNatarajanGBhattacharyaP. Characterization of cross-protection by genetically modified live-attenuated Leishmania donovani parasites against Leishmania mexicana. . 2014;193:3513–3527.SelvapandiyanADeyRNylenSDuncanRSacksDNakhasiHL. Intracellular replication-deficient Leishmania donovani induces long lasting protective immunity against visceral leishmaniasis. . 2009;183:1813–1820.SaraviaNGEscorciaBOsorioY. Pathogenicity and protective immunogenicity of cysteine proteinase-deficient mutants of Leishmania mexicana in non-murine models. . 2006;24:4247–4259.SahJFItoHKolliBKPetersonDASassaSChangKP. Genetic rescue of Leishmania deficiency in porphyrin biosynthesis creates mutants suitable for analysis of cellular events in uroporphyria and for photodynamic therapy. . 2002;277:14902–14909.GhaffarifarFJorjaniOMirshamsMMiranbaygiMHHosseiniZK. Photodynamic therapy as a new treatment of cutaneous leishmaniasis. . 2006;12:902–908.OloboJOAnjiliCOGicheruMM. Vaccination of vervet monkeys against cutaneous leishmaniosis using recombinant Leishmania “major surface glycoprotein” (gp63). . 1995;60:199–212.AbdelhakSLouzirHTimmJ. Recombinant BCG expressing the leishmania surface antigen Gp63 induces protective immunity against Leishmania major infection in BALB/c mice. . 1995;141:1585–1592.GonzalezCRNoriegaFRHuertaS. Immunogenicity of a Salmonella typhi CVD 908 candidate vaccine strain expressing the major surface protein gp63 of Leishmania mexicana mexicana. . 1998;16:1043–1052.MougneauEAltareFWakilAE. Expression cloning of a protective Leishmania antigen. . 1995;268:563–566.GurunathanSSacksDLBrownDR. Vaccination with DNA encoding the immunodominant LACK parasite antigen confers protective immunity to mice infected with Leishmania major. . 1997;186:1137–1147.ChenikMLouzirHKsontiniHDilouAAbdmoulehIDellagiK. Vaccination with the divergent portion of the protein histone H2B of Leishmania protects susceptible BALB/c mice against a virulent challenge with Leishmania major. . 2006;24:2521–2529.GotoYBhatiaARamanVS. Leishmania infantum sterol 24-c-methyltransferase formulated with MPL-SE induces cross-protection against L. major infection. . 2009;27:2884–2890.MasinaSGicheruMDemotzSOFaselNJ. Protection against cutaneous leishmaniasis in outbred vervet monkeys using a recombinant histone H1 antigen. . 2003;188:1250–1257.SoongLDuboiseSMKimaPMcMahon-PrattD. Leishmania pifanoi amastigote antigens protect mice against cutaneous leishmaniasis. . 1995;63:3559–3566.Zadeh-VakiliATaheriTTaslimiYDoustdariFSalmanianAHRafatiS. Immunization with the hybrid protein vaccine, consisting of Leishmania major cysteine proteinases Type I (CPB) and Type II (CPA), partially protects against leishmaniasis. . 2004;22:1930–1940.PetersNCBertholetSLawyerPG. Evaluation of recombinant Leishmania polyprotein plus glucopyranosyl lipid A stable emulsion vaccines against sand fly-transmitted Leishmania major in C57BL/6 mice. . 2012;189:4832–4841.RamirezLSantosDMSouzaAP. Evaluation of immune responses and analysis of the effect of vaccination of the Leishmania major recombinant ribosomal proteins L3 or L5 in two different murine models of cutaneous leishmaniasis. . 2013;31:1312–1319.ColerRNSkeikyYABernardsK. Immunization with a polyprotein vaccine consisting of the T-cell antigens thiol-specific antioxidant, Leishmania major stress-inducible protein 1, and Leishmania elongation initiation factor protects against leishmaniasis. . 2002;70:4215–4225.SkeikyYAColerRNBrannonM. Protective efficacy of a tandemly linked, multi-subunit recombinant leishmanial vaccine (Leish-111f) formulated in MPL adjuvant. . 2002;20:3292–3303.DuthieMSRamanVSPiazzaFMReedSG. The development and clinical evaluation of second-generation leishmaniasis vaccines. . 2012;30:134–141.XuDLiewFY. Protection against leishmaniasis by injection of DNA encoding a major surface glycoprotein, gp63, of L. major. . 1995;84:173–176.XuDMcSorleySJChatfieldSNDouganGLiewFY. Protection against Leishmania major infection in genetically susceptible BALB/c mice by gp63 delivered orally in attenuated Salmonella typhimurium (AroA- AroD-). . 1995;85:1–7.WalkerPSScharton-KerstenTRowtonED. Genetic immunization with glycoprotein 63 cDNA results in a helper T cell type 1 immune response and protection in a murine model of leishmaniasis. . 1998;9:1899–1907.Montalvo-AlvarezAMFolgueiraCCarrionJMonzote-FidalgoLCanavateCRequenaJM. The Leishmania HSP20 is antigenic during natural infections, but, as DNA vaccine, it does not protect BALB/c mice against experimental L. amazonensis infection. . 2008;2008:695432.ZaninFHCoelhoEATavaresCA. Evaluation of immune responses and protection induced by A2 and nucleoside hydrolase (NH) DNA vaccines against Leishmania chagasi and Leishmania amazonensis experimental infections. . 2007;9:1070–1077.CamposBLSilvaTNRibeiroSP. Analysis of iron superoxide dismutase-encoding DNA vaccine on the evolution of the Leishmania amazonensis experimental infection. . 2015;37:407–416.IborraSSotoMCarrionJAlonsoCRequenaJM. Vaccination with a plasmid DNA cocktail encoding the nucleosomal histones of Leishmania confers protection against murine cutaneous leishmaniosis. . 2004;22:3865–3876.CarneiroMWSantosDMFukutaniKF. Vaccination with L. infantum chagasi nucleosomal histones confers protection against new world cutaneous leishmaniasis caused by Leishmania braziliensis. . 2012;7:e52296.Dominguez-BernalGHorcajoPOrdenJA. HisAK70: progress towards a vaccine against different forms of leishmaniosis. . 2015;8:629.KamhawiSBelkaidYModiGRowtonESacksD. Protection against cutaneous leishmaniasis resulting from bites of uninfected sand flies. . 2000;290:1351–1354.MorrisRVShoemakerCBDavidJRLanzaroGCTitusRG. Sandfly maxadilan exacerbates infection with Leishmania major and vaccinating against it protects against L. major infection. . 2001;167:5226–5230.KatebiAGholamiETaheriT. Leishmania tarentolae secreting the sand fly salivary antigen PpSP15 confers protection against Leishmania major infection in a susceptible BALB/c mice model. . 2015;67:501–511.GomesROliveiraFTeixeiraC. Immunity to sand fly salivary protein LJM11 modulates host response to vector-transmitted leishmania conferring ulcer-free protection. . 2012;132:2735–2743.de MouraTROliveiraFNovaisFO. Enhanced Leishmania braziliensis infection following pre-exposure to sandfly saliva. . 2007;1:e84.GreenblattCL. The present and future of vaccination for cutaneous leishmaniasis. . 1980;47:259–285.McCallLIZhangWWRanasingheSMatlashewskiG. Leishmanization revisited: immunization with a naturally attenuated cutaneous Leishmania donovani isolate from Sri Lanka protects against visceral leishmaniasis. . 2013;31:1420–1425.LaabsEMWuWMendezS. Vaccination with live Leishmania major and CpG DNA promotes interleukin-2 production by dermal dendritic cells and NK cell activation. . 2009;16:1601–1606.HuangLHinchmanMMendezS. Coinjection with TLR2 agonist Pam3CSK4 reduces the pathology of leishmanization in mice. . 2015;9:e0003546.WuWWeigandLBelkaidYMendezS. Immunomodulatory effects associated with a live vaccine against Leishmania major containing CpG oligodeoxynucleotides. . 2006;36:3238–3247.MendezSTabbaraKBelkaidY. Coinjection with CpG-containing immunostimulatory oligodeoxynucleotides reduces the pathogenicity of a live vaccine against cutaneous Leishmaniasis but maintains its potency and durability. . 2003;71:5121–5129.PinheiroROPintoEFLopesJRGuedesHLFentanesRFRossi-BergmannB. TGF-beta-associated enhanced susceptibility to leishmaniasis following intramuscular vaccination of mice with Leishmania amazonensis antigens. . 2005;7:1317–1323.PrattiJERamosTDPereiraJC. Efficacy of intranasal LaAg vaccine against Leishmania amazonensis infection in partially resistant C57Bl/6 mice. . 2016;9:534.da Silva-CoutoLRibeiro-RomaoRPSaavedraAF. Intranasal vaccination with leishmanial antigens protects golden hamsters (Mesocricetus auratus) against Leishmania (Viannia) Braziliensis infection. . 2015;9: e3439.RivierDShahRBovayPMauelJ. Vaccine development against cutaneous leishmaniasis. Subcutaneous administration of radioattenuated parasites protects CBA mice against virulent Leishmania major challenge. . 1993;15:75–84.TurcoSDescoteauxARyanKGarrawayLBeverleyS. Isolation of virulence genes directing GPI synthesis by functional complementation of Leishmania. . 1994;27:133–138.SpathGFLyeLFSegawaHTurcoSJBeverleySM. Identification of a compensatory mutant (lpg2-REV) of Leishmania major able to survive as amastigotes within macrophages without LPG2-dependent glycoconjugates and its significance to virulence and immunization strategies. . 2004;72:3622–3627.DavoudiNKhamesipourAMahboudiFMcMasterWR. A dual drug sensitive L. major induces protection without lesion in C57BL/6 mice. . 2014;8:e2785.NicoDGomesDCAlves-SilvaMV. Cross-protective immunity to Leishmania amazonensis is mediated by CD4+ and CD8+ epitopes of Leishmania donovani nucleoside hydrolase terminal domains. . 2014;5:189.LiuMAWahrenBKarlsson HedestamGB. DNA vaccines: recent developments and future possibilities. . 2006;17:1051–1061.DumonteilE. DNA vaccines against protozoan parasites: advances and challenges. . 2007;2007:90520.TabatabaieFMahdaviMFaeziS. Th1 platform immune responses against Leishmania major induced by Thiol-specific antioxidant-based DNA vaccines. . 2014;7:e8974.CarrionJ. Mechanisms of immunity to Leishmania major infection in mice: the contribution of DNA vaccines coding for two novel sets of histones (H2A-H2B or H3-H4). . 2011;34:381–386.TitusRGRibeiroJM. Salivary gland lysates from the sand fly Lutzomyia longipalpis enhance Leishmania infectivity. . 1988;239:1306–1308.ReinerSLLocksleyRM. The regulation of immunity to Leishmania major. . 1995;13:151–177.HimmelrichHParra-LopezCTacchini-CottierFLouisJALaunoisP. The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor beta 2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12. . 1998;161:6156–6163.MatthewsDJEmsonCLMcKenzieGJJolinHEBlackwellJMMcKenzieAN. IL-13 is a susceptibility factor for Leishmania major infection. . 2000;164:1458–1462.HurdayalRBrombacherF. The role of IL-4 and IL-13 in cutaneous Leishmaniasis. . 2014;161:179–183.KaneMMMosserDM. The role of IL-10 in promoting disease progression in leishmaniasis. . 2001;166:1141–1147.Lopez KostkaSDingesSGriewankKIwakuraYUdeyMCvon StebutE. IL-17 promotes progression of cutaneous leishmaniasis in susceptible mice. . 2009;182:3039–3046.PakpourNZaphCScottP. The central memory CD4+ T cell population generated during Leishmania major infection requires IL-12 to produce IFN-gamma. . 2008;180:8299–8305.StobieLGurunathanSPrussinC. The role of antigen and IL-12 in sustaining Th1 memory cells in vivo: iL-12 is required to maintain memory/effector Th1 cells sufficient to mediate protection to an infectious parasite challenge. . 2000;97:8427–8432.MaspiNGhaffarifarFSharifiZDalimiA. Codelivery of DNA vaccination encoding LeIF gene and IL-12 increases protection against Leishmania major infection in BALB/c mice. . 2016;38:228–235.HugentoblerFDi RobertoRBGillardJCousineauB. Oral immunization using live Lactococcus lactis co-expressing LACK and IL-12 protects BALB/c mice against Leishmania major infection. . 2012;30:5726–5732.HugentoblerFYamKKGillardJMahbubaROlivierMCousineauB. Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling. . 2012;7:e30945.CohenJ. IL-12 deaths: explanation and a puzzle. . 1995;270:908.WrightAKBrilesDEMetzgerDWGordonSB. Prospects for use of interleukin-12 as a mucosal adjuvant for vaccination of humans to protect against respiratory pneumococcal infection. . 2008;26:4893–4903.AmaralVFTevaAOliveira-NetoMP. Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease. . 2002;97:1041–1048.TuboNJJenkinsMK. CD4+ T cells: guardians of the phagosome. . 2014;27:200–213.BelkaidYPiccirilloCAMendezSShevachEMSacksDL. CD4+CD25+ regulatory T cells control Leishmania major persistence and immunity. . 2002;420:502–507.UzonnaJEWeiGYurkowskiDBretscherP. Immune elimination of Leishmania major in mice: implications for immune memory, vaccination, and reactivation disease. . 2001;167:6967–6974.SakaiSTakashimaYMatsumotoYReedSGHayashiYMatsumotoY. Intranasal immunization with Leish-111f induces IFN-gamma production and protects mice from Leishmania major infection. . 2010;28:2207–2213.RamanVSDuthieMSFoxCBMatlashewskiGReedSG. Adjuvants for Leishmania vaccines: from models to clinical application. . 2012;3:144.PandeySPChandelHSSrivastavaS. Pegylated bisacycloxypropylcysteine, a diacylated lipopeptide ligand of TLR6, plays a host-protective role against experimental Leishmania major infection. . 2014;193:3632–3643.BagirovaMAllahverdiyevAMAbamorES. Overview of dendritic cell-based vaccine development for leishmaniasis. . 2016;38:651–662.SanabriaMXVargas-InchausteguiDAXinLSoongL. Role of natural killer cells in modulating dendritic cell responses to Leishmania amazonensis infection. . 2008;76:5100–5109.TsagozisPKaragouniEDotsikaE. Dendritic cells pulsed with peptides of gp63 induce differential protection against experimental cutaneous leishmaniasis. . 2004;17:343–352.CarrionJNietoASotoMAlonsoC. Adoptive transfer of dendritic cells pulsed with Leishmania infantum nucleosomal histones confers protection against cutaneous leishmaniosis in BALB/c mice. . 2007;9:735–743.RemerKAApetreiCSchwarzTLindenCMollH. Vaccination with plasmacytoid dendritic cells induces protection against infection with Leishmania major in mice. . 2007;37:2463–2473.