Vaccines for leishmaniasis and the implications of their development for American tegumentary leishmaniasis

ABSTRACT

The leishmaniases are a collection of vector-borne parasitic diseases caused by a number of different Leishmania species that are distributed worldwide. Clinical and laboratory research have together revealed several important immune components that control Leishmania infection and indicate the potential of immunization to prevent leishmaniasis. In this review we introduce previous and ongoing experimental research efforts to develop vaccines against Leishmania species. First, second and third generation vaccine strategies that have been proposed to counter cutaneous and visceral leishmaniasis (CL and VL, respectively) are summarized. One of the major bottlenecks in development is the transition from results in animal model studies to humans, and we highlight that although American tegumentary leishmaniasis (ATL; New World CL) can progress to destructive and disfiguring mucosal lesions, most research has been conducted using mouse models and Old World Leishmania species. We conclude that assessment of vaccine candidates in ATL settings therefore appears merited.

KEYWORDS:

• Leishmaniasis
• American Tegumentary Leishmaniasis
• Leishmania spp
• immune response
• vaccines

Introduction

Through their residence in endemic regions, approximately 350 million people are currently at risk of infection with protozoan parasites of the genus Leishmania and subsequent development of leishmanaisis.^1,2 Estimates suggest that the disease is present in 98 countries and about 2 million new cases occur each year. Three different clinical manifestations can be observed: cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL). The presenting form is determined by which of the more than 20 Leishmania species that can infect humans is actually manifesting disease,^3,4 with many different Leishmania species associated with CL. CL is associated with a lower mortality than VL, but it is more widespread and afflicts a higher number of individuals. In the Old World L. major and L. tropica are the major causes of CL, while in the New World the disease is more commonly referred to as American tegumentary leishamaniasis (ATL) and is caused by infection with L. amazonensis, L. braziliensis, L. guyanensis, L. panamensis, L. mexicana, among others. Although ATL is usually relatively benign and can cure spontaneously if given enough time, in some cases symptoms exacerbate to cause MCL that presents with disfigurement and causes loss of productivity.⁴ Currently, the strategies to reduce leishmaniasis are limited to vector control and treatment of patients with outdated and toxic antimonial drugs for which there are increasing reports of resistance.⁵ Thus, there is both an opening and an urgent need for a safe and effective vaccine against leishmaniasis.

Although a considerable amount has been revealed with regard to the development of immunity during Leishmania infection, most of the knowledge, especially of the parasite-host relationship, has been derived from experimental studies in animal models.^6,7 Leishmania infection is established when promastigotes, the flagellated form of the parasite, are transmitted during the bite of an infected sand fly on mammalian hosts. Once in the skin, the parasites are phagocytosed by different cell types, including inflammatory monocytes and dendritic cells (DCs) that are recruited to the site of infection. Over the first few days following infection, these cells become the predominant foci of Leishmania.⁸ Among the most important innate mechanisms that control the infection are the production of reactive oxygen species (ROS), generated by the respiratory burst during phagocytosis, and production of nitric oxide (NO), generated by inducible NO synthase (iNOS), followed by the activation of interferon gamma (IFN-γ) mediated cells.⁹ The interaction of parasites and DCs is one of the main factors that determines the outcome of Leishmania infection with DC-mediated development of Leishmania-specific CD4 and CD8 T lymphocytes well established as being important for protection.^10,11 In murine models, the activation of Th1 and CD8 T cells depends upon antigen presentation through major histocompatibility complexes/human leukocyte antigen (MHC/HLA) complexes on the surface of infected DC in the presence of interleukin (IL)-12, driving the T cells to produce IFN-γ that in turn activates macrophages to produce NO. This cascade of events contributes to the elimination of the parasites, whereas T cell production of IL-4 and TGF-β in the local microenvironment inhibits DC secretion of IL-12 and favors parasite survival.⁹

The observation that recovery from primary infection with L. major, and to some extent with L. braziliensis is typically associated with long term protection against reinfection, indicates that vaccines can be developed against CL.^12,13 Spontaneous cure has been documented in endemic areas for L. braziliensis infection (usually occurring with a clinical evolution of less than 3 months) and this suggests that an immune response that controls the infection has developed.¹⁴ Carvalho et al.¹⁵ discovered that patients with rapidly self-healing L. braziliensis ATL exhibited positive intradermal skin test results, lymphocyte proliferation and IFN-γ production, indicating a strong T-cell response to Leishmania antigens. Infection with L. braziliensis is often difficult to heal, however, and sometimes produces mutilating lesions in the nasopharyngeal mucosa with responses significantly higher in healed patients than in those with active disease.

It is noteworthy that majority of our understanding of Leishmania infections has been generated in mice using tightly controlled L. major infection models, and the better understanding of experimental immunity against L. major might be one of the reasons why most current vaccine candidates are focused on Old World species instead of those found throughout the New World. Until recently it was believed that long-lived memory cells generated during chronic L. major infection were required for durable protection, and vaccination strategies based on generating such cells were therefore desirable. Peters et al demonstrated that for T helper 1 (Th1) concomitant immunity, in which protection against reinfection coincides with a persisting primary infection, preexisting CD44(+)CD62L(-)T-bet(+)Ly6C+ effector T cells that are short-lived in the absence of infection and are not derived from memory cells reactivated by secondary challenge, mediate immunity.¹⁶ Such gaps in our knowledge regarding immunity against Leishmania infections in humans may be confounding vaccine development, and it is noteworthy that the New World Leishmania species induce a response pattern distinct form that induced by Old World Leishmania species.¹⁷ IL-17 has been linked to a massive influx of inflammatory cells that lead to disease exacerbation,¹⁸ while IL-10 has been linked with strong immunosuppression and exacerbated pathology.¹⁹ The immune response against Leishmania in humans is not fully understood, and a far more complex response appears to occur that likely involves important interactions of a wide variety of cytokines and cells that dictate clinical outcome. It remains unclear how to best generate the immune memory that prevents reinfection and the immune correlates that would be beneficial in determining this are also not fully defined.^9,20,21 Nevertheless, we believe that identifying antigens that appropriately target of the anti-Leishmania response is fundamental for the development of an effective vaccine against CL/ATL. Indeed, many different peptides and antigens from New World Leishmania species have been screened using modern bioinformatics tools and appear to have potential within vaccines for ATL.^22,23 Most, however, remain in research stages and have not been fully developed as vaccines ready for clinical trials.

Currently, five vaccines have been licensed and approved against Leishmania species. Two of these are approved for administration to humans, with one in Brazil using killed L. amazonensis parasites for immunotherapy of CL and the other in Uzbekistan using live L. major parasites. The others use recombinant proteins for prophylactic immunization of dogs in Brazil.^5,24–27 Although these vaccines were approved by the respective national regulatory agencies, their effectiveness remain controversial. For example, the vaccines used for immunotherapy cannot be used alone but rather in combination with conventional chemotherapies.²⁸ Whole parasites need to be used with caution since they can induce chronic lesions; and although the vaccine for dogs was proven to be effective in the short term, its long term impact remains unknown and further studies are needed to determine if its use can reduce the spread of the disease.^25,29 To date, there are no licensed vaccines for human CL but those that have advanced to clinical trials include two killed L. amazonensis vaccines,^30,31 each of which generated inconsistent results between vaccine and placebo groups, and the defined polyprotein-containing subunit vaccine LEISH-F1+ MPL-SE, which was demonstrated to be safe while generating an antigen-specific Th1 response.³²

Vaccine development

A considerable challenge in developing a vaccine is identifying the best type or set of antigens against which to direct an appropriate immune response. Additional variables such as the specific gene or protein targeted, the amount of antigen/vector used, the number of immunizations, the Leishmania species prioritized for evaluation and the type of experimental challenge, can confound results between studies and somewhat confuse the vaccine landscape. As an example, it became understood only recently that components within sand fly saliva can alternatively inhibit or assist in promoting the anti-Leishmania Th1 response.³³ It is clear, however, that a variety of different targets and strategies can be used to develop a vaccine for leishmaniasis, and in this regard the likelihood that an effective vaccine will emerge should be considered as high.

In a simplified form, the composition of a vaccine consists of two key elements: antigen(s) to generate a pathogen-specific response and the adjuvant to initiate and direct the immune response.³⁴ In this sense, to direct and adequately stimulate immunity against Leishmania, the vaccines require specific parasite antigens and immunostimulatory molecules. In first generation vaccines the antigen component is derived from use of the whole parasite, either in a live attenuated manner or inactivated and killed by chemical or physical processes. In first generation vaccines the adjuvant component is typically also inherent to the parasite, with multiple pathogen-associated molecular patterns (PAMP) established and defined for Leishmania species.^35,36 Second generation vaccines are composed of molecularly defined components, using recombinant antigens, which can be one or more proteins, and specified adjuvants. The panel of defined adjuvants that can be used with recombinant proteins to tailor the vaccine-induced response continues to expand,^36–39 and a variety of defined adjuvants have now been used in studies with vaccines against Leishmania.^32,40,41 Regarding third generation vaccines, these include vaccines that use the pathogen-specific DNA or RNA, or a platform/carrier that contains genetic component(s) of the pathogen to target the immune response (Figure 1).

Figure 1. Maturation and expansion of Leishmania vaccine development.

Antigen target selection

Concurrent with the development of new adjuvants, advances in molecular and in silico tools in recent years has led to a dramatic increase in the number of Leishmania-specific targets that have been evaluated in vaccine studies.^22,42,43 Genome sequencing revolutionized vaccine development because the availability of pathogen genomes has informed discovery of novel antigens while the exclusion of targets that have homology with human genes. The number of Leishmania species genomes that are available in public data banks has grown over time, making it now possible to infer the complete proteome of different species (predicted proteome). The use of these genome sequences to make in silico predictions of suitable targets (reverse vaccinology)⁴⁴ provides an efficient means with which to identify important epitopes of both CD4+ and/or CD8 + T cells.^45,46 Numerous research groups are now applying reverse vaccinology to identify antigens that are common, or highly homologous, across the published genomes of various Leishmania spp. One approach has used linear methods to search for epitopes in different Leishmania species proteomes,⁴⁷ while another has focused on identifying epitopes within already known antigens.⁴⁸ The latter approach led to the development of a refined vaccine candidate containing multi-epitope peptides of Leishmania Cysteine Protease A (CPA160–189) that provided partial protection against L. infantum infection in BALB/c mice.⁴⁹ Freitas-Silva et al., used a combination of in silico methods to identify epitopes of CD4 and CD8 T cells within the predicted proteome of Leishmania (Viannia) braziliensis.²² These peptides had a high binding affinity to both MHC/HLA complexes and the immunogenicity of some was demonstrated by stimulation of peripheral blood mononuclear cells (PBMC) from healthy controls and post-treatment ATL patients. To validate the use of bioinformatics tools for epitope prediction, protein-protein interaction networks and metabolic pathways, Brito et al., integrated data from experimental and in silico studies and demonstrated that some of these methods correlated with protection observed in murine models.²³ This indicates that reverse vaccinology may not only be important for screening of potential candidates, but suggest that it may also help us better understand parasite virulence mechanisms and how the host immune response can curtail them.

Phage display, a high-throughput method where mimotopes are presented on the surface of phages and are recognized by specific target ligands, provides another strategy to rapidly select potential candidates.⁵⁰ A recent study used phage display and PBMC from ATL patients to identify T cell-specific mimotopes, then to evaluate the immunogenicity of two of the identified clones in mice after challenge with L. amazonensis.⁵¹ The results were promising as polarized Th1 responses were generated and immunized mice had significant lower numbers of parasites when compared with controls, but further testing is needed to determine if these can advance. It is also noteworthy that despite being selected by screening among ATL patients, animal testing was conducted against L. amazonensis, a strain also associated with VL.

First generation vaccines

First generation vaccines consist of live-attenuated or killed parasites that are used to generate broad immunity. Leishmanization (LZ; the inoculation of a low dose of live Leishmania parasites to generate a minor lesion but also a natural anti-Leishmania response) was the first vaccine strategy proven to be effective against leishmaniasis in humans and it was widely used among soldiers in the Soviet Union.⁵² Clinical trials in humans based on LZ were performed due to its high efficacy, and motivated the return of this practice in high-incidence regions.⁵³ In Venezuela a relatively large number of ATL patients received immunotherapy with monthly intradermal injections of a combined vaccine containing autoclaved promastigotes of L. mexicana amazonensis MHOM/VE/84/MEL and viable Bacillus Calmette- Guerin (BCG) during the 1980s and 90s.⁵⁴ Clinical healing varied from 91.2 to 98.7%, with an average of 95.7%. The high percentage of clinical cures achieved with this immunotherapy (> 90%) supported further use in the routine treatment of localized ATL, and the immunotherapy was modified to use promastigotes of L. braziliensis strain MHOM/BR/84/LTB 300 killed by pasteurization.^54,55

Although first generation vaccines are attractive for developing countries because they can be produced at a relative low cost,⁵⁶ sustaining a consistent product can present a barrier. There can be difficulties in standardizing culture conditions to obtain the immunogen and progressive declines in infectivity can occur when subculturing the parasites.^40,57 This situation is analogous to the Bacille-Calmette Guerin (BCG) vaccines used to prevent tuberculosis, where numerous substrains, each of which provide varying degrees of efficacy, are now used worldwide.⁵⁸

L. major LZ can provide protection against infection with different Leishmania species in mice and suggests the possibility of using L. major LZ to prevent VL.⁵⁹ Among the important considerations when using live Leishmania parasites in human populations is not only safety in the vaccinee but also safety of introducing parasites into the local population. Ideally, live parasites should induce a protective immune response in the vaccinated individual but should be cleared and not persist in the long term.⁶⁰ LZ has the capacity of inducing non-healing lesions in immunocompromised patients, and persistent parasites could support recrudescence in the event of immune suppression while also potentially serving as a reservoir for transmission to susceptible individuals and the introduction of this species into a non-endemic region. Murine studies have shown that although heat-killed Leishmania can stimulate an early Th1 response and protection, protective immunity is not sustained and wanes with time.^61–63 One hypothesis is that a long-term anti-Leishmania response is not generated because mice immunized in this way are not able to induce effector memory cells following a secondary challenge. When repeated boosts of killed parasites were provided, however, both effector and central memory T cells (TEM and TCM, respectively) were produced and a prolonged protection against virulent challenge could be observed.⁶⁴ A number of strategies are being developed to enhance LZ, including the concomitant inoculation of adjuvant molecules (such as CpG-ODNs) to prompt the innate immune response to rapidly kill the parasites or generate long term immunity.⁶⁴ Attempts to improve the efficacy of attenuated/killed Leishmania vaccines against CL using different types of immunization, different immunization schemes and different ways of attenuation (i.e. photosensitization;⁶⁵ impairing the mannose activation pathway⁶⁶) and challenge have indicated promise but have not yet reached advanced phases of clinical development.^67–69

Targeted genetic modification to attenuate or delete specific virulence genes within Leishmania is considered a powerful strategy with the potential to provide cross-protective parasites with improved safety profiles, examples are described on Table 1. While the first attempts using attenuated Leishmania parasites rendered protection in murine models, lack of knowledge regarding potential reversion to a wild type genotype left the parasites unsuitable for use in human populations.^73–77 Advances in genetic manipulation have provided new methods with which to address these issues, however, and various reverse genetic modifications of Leishmania parasites have now been characterized.⁷⁸ Genetically engineered parasites lacking essential genes such as dhfr-ts (L. major), lpg2 (L. mexicana and L. major), SIR2 (L. infantum), P27 (L. major), Centrin1 (L. donovani) and ΔCPB (L. mexicana) have been developed and evaluated in animal models.^{70–72,79–82} In addition to targeted genetic attenuation, use of naturally attenuated Leishmania species (i.e., those species that are nonpathogenic in humans) also appears possible as indicated by the use of, L. tarentolae, a species with the genus Sauroleishmania⁸³ that typically infects lizards. L. tarentolae is advancing on the basis that it could also potentially be used as a prophylactic or immunotherapeutic vaccine in immunocompromised patients.^84–87

Table 1. Examples of first generation vaccine candidates using genetically modified parasites against CL associated with protection.

SPECIES	MODIFICATION	OLD WORLD OUTCOME	NEW WORLD OUTCOME	REFERENCE
L. mexicana	Mannose pathway impairment	Not tested	Protection (live parasites challenge) – No protection (killed parasites challenge)	66
L. mexicana	Lack of the dhfr-ts gene	Not tested	No protection	70
L. major	Lack of the dhfr-ts gene	Protection	Not tested	71
L. major	Lack of the P27 gene	Partial protection	Not tested	72

Second generation vaccines

In contrast to first generation vaccines, second generation vaccines use defined products to generate the desired immune responses. Native proteins from Leishmania have been used either crudely or in a purified manner to generate protective responses and the majority of Leishmania vaccines currently being explored include antigenic proteins from the parasite or different individual antigens produced as recombinant products.⁸⁸ The search for antigenic proteins of Leishmania is becoming more common and different strategies are being used to characterize these antigens, including genomic or proteomic approaches using serum samples or T cells from Leishmania-infected individuals^28,88 and bioinformatics tools, which have facilitated the recombinant production of a variety of targets.^62,89 The most common production methods are prokaryotic expression systems using Escherichia coli or those using yeast.^90,91 Proteins alone are insufficient as vaccines, however, and they need to be co-injected with adjuvants to induce a satisfactory T cell response.⁶³ Experimental evidence also suggests that multiple injections of most sub-unit vaccines are required to generate lasting immunity. Despite this, in addition to a high degree of control and reproducibility in production, one of the major advantages of the second-generation vaccine approach is overall cost. Mathematical modeling indicates that a vaccine dose costing 2 USD or less would be much more economically beneficial then the currently available leishmaniasis treatments.⁹²

Many Leishmania proteins have now been purified or recombinantly expressed for evaluation as defined subunit vaccine candidates (Table 2). Due to the fact that it is expressed in both the insect and vertebrate host phases of the parasite lifecycle, Leishmania homolog of receptors for activated C kinase (LACK) has been widely evaluated and mice immunized with LACK resist L. major infection.^105,106 L. major H2B histone protein, and its divergent N-terminal region, have also been used to confer protection against experimental L. major infection.¹⁰⁷ When histone H1 was tested in conjunction with the Montanide ISA 720 adjuvant in monkeys, it reduced the lesions formed after L. major infection and these self-healed over time.¹⁰⁸ Among the purified proteins, the parasite cell surface metalloprotease GP63 mediated a robust protection against challenge with both L. mexicana and L. major in mice but conferred only partial protection in monkeys.^109–111 With regard to using proteins from one Leishmania species to protect against an alternate species, sterol methyl transferase (SMT) of L. infantum, protected mice against L. major challenge when formulated with MPL-SE.¹¹² Another example of a protein that could potentially yield cross-protection due to high sequence homology between Leishmania species is L. donovani nucleoside hydrolase (NH36), which protected mice against both L. (L.) amazonensis and L. (V.) braziliensis infection.^113,114 Alves-Silva et al. recently extended this initial finding by assessing the efficacies of NH36, its F1 and F3 domains, and the recombinant chimera F1F3, each formulated with Riedel de Haen saponin, against L. braziliensis mouse infection, observing some variance in the onset and magnitude of multifunctional IL-2⁺TNF-α⁺IFN-γ⁺ antigen-specific CD4 and CD8 T cells but finding that the F1F3 chimera resulted in the greatest reduction of the ear lesions sizes.¹¹² Similarly, L. major recombinant ribosomal proteins L3 and L5 combined with CpG-ODNs induced a Th1 response in BALB/c mice that conferred protection against L. major and L. braziliensis challenge.¹¹⁵

Table 2. Examples of second generation candidate antigens tested in mice.¹⁸

SPECIES	CANDIDATE ANTIGEN	FUNCTION	OLD WORLD OUTCOME	NEW WORLD OUTCOME	REFERENCE
L. braziliensis	Thiol-specific antioxidant (TSA) LeiF LACK	Tryparedoxin peroxidase	Protection against L. major	No protection	93
		Leishmania putative eukaryotic initiation factor	Protection against L. major	No protection	93
		Leishmania homolog of receptors for activated C-kinase	Protection against L. major	Partial protection	93
L. amazonensis	P4 nuclease Cysteine proteinase HSP20 GP46	Endonuclease activity Cysteine-type peptidase Activity Heat-shock protein Membrane glycoprotein	Not tested Not tested Not tested Not tested	Protection Partial protection No protection Protection	94 95 96 97
L. mexicana	GP63	Metalloendopeptidase activity	Not tested	Protection	98
L. major	LmTSI GP63 PSA2 TSA Histone H1 LACK	Stress-Induced protein sti1 Metalloendopeptidase activity Promastigote surface antigen protein 2 Typaredoxin peroxidase DNA binding Leishmania homolog of receptors for activated C-kinase	Protection Protection No protection No protection Protection Partial protection	No protection (L. braziliensis) Protection (L. mexicana) Protection (L. mexicana) Not tested Not tested Not tested	99 100 101 102 103 104

Interestingly, the impact of sand fly saliva has been systematically investigated over the last few years and it is now understood that sand fly components inoculated during feeding can modify the bite site environment.¹¹⁶ Several proteins found in the saliva of Lutzomyia longipalpis (Lu. longipalpis), one of the main vectors of the disease in the New World, appear to enhance the parasite’s pathogenesis¹¹⁷ and based on these observations, vaccine candidates containing sand fly salivary proteins have been proposed. Indeed, evaluation of these salivary proteins either alone or in conjunction with Leishmania proteins has generated promising results in animal models.^117–120 Mice immunized with the LJM11 protein from Lu. longipalpis have significantly reduced parasite numbers in the ear and lymph node following challenge with L. braziliensis plus Lu. longipalpis salivary gland sonicate, but not when the parasites were inoculated with salivary gland sonicate from an alternate sand fly.¹²¹

Recombinant methods also make it possible to manipulate and combine proteins and/or complementary epitopes into a single polyepitope/polyprotein product. Leish-111f, an antigen made of 3 fused proteins (L. major thiol-specific antioxidant [TSA], L. major stress-inducible protein-1 [STI1], and L. braziliensis elongation and initiation factor [LeIF]) provided protection against experimental L. major infection.^41,122 The combination of Leish-111f with the adjuvant monophosphoryl lipid A plus squalene (MPL-SE) became the first defined vaccine candidate to advance to phase 1 and phase 2 clinical trials, where it was found to be safe and immunogenic.⁴⁰ When injected with the Toll-like receptor (TLR)-4 agonist glucopyranosyl lipid A (GLA), KSAC, a recombinant protein made of KMP-11, SMT, A2 and CPB, conferred protection in susceptible BALB/c mice against sand-fly transmission of L. major.¹²³ A fusion protein made of L. major cysteine proteinases (CP) A and B and L. pifanoi cysteine protease has also been shown to provide partial protection against L. amazonensis in mice.^124,125 It is not clear, however, if these vaccines can provide protection against ATL-causing Leishmania species.

Third generation vaccines

In the last few years, genetic immunization consisting of either nucleic acids alone or as genes added into delivery vectors, has emerged as another alternative, namely third generation vaccine strategy. These nucleic acid-based vaccines also have a significant logistical advantage because their typically high stability appears to make them practical for use in tropical areas. Additional advantages provided by this method include the selective expression of proteins that are assumed to be folded close to their native/natural conformation; production or persistence of the antigen over several days; and induction of antigen-specific memory cells.¹²⁶ Both DNA and RNA platforms have been developed, but those using DNA need to access the nucleus and currently appear to work better in small animal models. To date, higher doses of DNA have been required in larger animals and humans and this has raised some safety concerns.^127,128 Furthermore, the low immunogenicity of DNA vaccines observed in dogs and humans has limited their advance and has necessitated the use of approaches such as in vivo electroporation, microneedle-based delivery and DNA encapsulation to increase their immunogenicity.⁵² Despite this, a wide repertoire of antigens has been investigated in the context of DNA vaccines.¹²⁹ The gene coding for GP63 was the first DNA vaccine developed against leishmaniasis, mediating solid protection against L. major infection in mice.^130-132 LACK is perhaps the most extensively studied DNA vaccine against Leishmania and in clinical trials pairing of LACK with IL-12 increased the protection achieved relative to that observed with LACK alone.¹⁰⁶ DNA-encoding the A2 protein has also mediated protection in mice infected with L. amazonensis.¹³³ In a recent report, a TSA-based DNA vaccine was successful in promoting a Th1 immune response and protection against L. major challenge.¹³⁴ Experiments using the iron superoxide dismutase of L. donovani demonstrated protection against L. amazonensis in BALB/c mice by inducing IFN-γ that reduced the parasite burden.¹³⁵ Studies involving the pcDNA3H3H4 plasmid expressing L. major histone proteins H3 and H4 resulted in a partial resistance to L. major associated with the development of Th1/Th2-type responses and a reduced number of parasite-specific regulatory T cells at the infection site.¹³⁶ L. infantum histone genes H2A, H2B, H3, and H4 have also been able to control both L. major and L. braziliensis infections in BALB/c mice.^137,138 Similar to the ability to produce chimeric fusion proteins, multiple genes can be fused together for use within third generation vaccines. A recent study compared the responses generated by genes encoding either LACK alone, TSA alone or a fusion of these two genes named LACK-TSA, and found that LACK-TSA triggered a stronger protective response against challenge with L. major than the individual genes.¹³⁹

In contrast to DNA, RNA platforms need access only to the cytoplasm and have been demonstrated to be effective in both small and large animal models.^140–143 RNA can be generated in non-replicating platforms optimized and/or modified to avoid their detection by the immune system, or in self-replicating platforms that use viral replication machinery. Self-replicating RNA vaccines can engage the innate immune system in a manner similar to the parent virus and essentially provide a self-adjuvanting function.¹⁴⁴ Unlike the responses generated with second generation vaccines that typically consist of antigen-specific CD4 T cells exclusive of CD8 T cells, an important feature of third generation vaccines is the induction of both antigen-specific CD4 and CD8 T cells.¹⁴⁵ A fusion of the kinetoplastid membrane protein 11 (KMP11) and hydrophilic acylated surface protein B (HASPB) genes was cloned into a lentiviral vector (pCDH-cGFP) that generated a protective response with significant increases in both pro- and anti-inflammatory markers (IFN-γ and IgG2a versus IL-10 and IgG1).¹⁴⁶ The higher levels of IFN-γ and IgG2a indicated skewing toward a desirable Th1 response.

The influenza virus has also been used as a vector, and a recombinant influenza virus expressing the single MHCII-restricted peptide LACK_158–173 achieved protection against L. major while generating considerable levels of peptide-specific IFN-γ.¹⁴⁷ Recently, the first human trial using a third-generation vaccine designed for VL and post-kala azar dermal leishmaniasis (PKDL) was safely conducted and confirmed the ability of a simian adenovirus (ChAd63) that encodes two Leishmania proteins (KMP11 and HASPB) to generate antigen-specific responses.¹⁴⁸ Similar studies in primates and/or humans have not yet been conducted against CL-causing Leishmania species.

In addition to the use of viral vectors, a number of bacteria including Listeria monocytogenes, Mycobacterium bovis BCG and Salmonella enterica serovar Typhimurium, have been used as delivery systems for Leishmania antigens.^62,131 An example of this approach was shown in a study where novel Leishmania antigens were selected through a proteomic/in silico approach then expressed in Salmonella Typhimurium SL3261. Immunization of mice with individual serovars of Salmonella expressing the antigens LinJ08.1190 and LinJ23.0410, or a pool of these constructs, significantly delayed progression of L. major infection and increased resistance against L. donovani.¹⁴⁹ Some examples of these candidates are listed on Table 3.

Table 3. Examples of third generation (DNA) vaccine candidates against CL associated with protection.

SPECIES	GENE	OLD WORLD OUTCOME	NEW WORLD OUTCOME	REFERENCE
L. major	GP63	Protection	Not tested	131
L. major	LACK	Protection	Not tested	106
L. amazonensis	A2	Protection (L. chagasi)	Protection (L. amazonensis)	133
L. major	TSA	Protection	Not tested	134
L. major	H3, H4	Partial protection	Not tested	136
L. infantum	H2A, H2B, H3, H4	Protection against L. major	Protection against L. braziliensis	134
L. major	KMP11+ HASPB	Protection	Not tested	146

Conclusions

Cutaneous leishmaniasis (CL) and American tegumentary leishmaniasis (ATL) remain important neglected tropical diseases that directly or indirectly impact millions each year. In addition to sand fly vector control efforts, the current control strategies for leishmaniasis rely upon early and accurate diagnosis (when attainable) coupled with chemotherapeutic approach to limit disease symptoms and ongoing parasite transmission. These efforts are having more success on the Indian subcontinent (against VL) than in South America, where VL- and CL-causing Leishmania species co-exist.

It is our belief that integrating a vaccine within leishmaniasis control strategies would have the greatest and most sustainable impact. It is quite conceivable that a combination of approaches may be used to achieve effective immunization. Indeed, we modeled such an approach against VL-causing Leishmania species, providing mice with a heterologous immunization regimen that involved second and third-generation vaccines with the same target proteins/inserts. This heterologous immunization scheme stimulated robust antigen-specific T cell responses and was capable of protecting against experimental L. donovani infection.¹⁵⁰ This approach could potentially provide the same benefit against CL-causing Leishmania species. Until now, most attempts to develop a vaccine against CL have been based on Old World Leishmania species, however, and unfortunately these species appear to have a response pattern that differs from the species that predominate in the New World and especially so from L. braziliensis. Even though most of the preclinical evaluations have used experimental L. major infection to determine vaccine efficacy, immunization to prevent ATL should be attainable given the plethora of targets and variety of platforms under preclinical investigation. For this to happen, however, it will be necessary to ensure that both ATL and its complications remain a priority among public health policy decision makers and that vaccine development efforts evaluate promising candidates against ATL-causing species that are endemic throughout South America.

Disclosure of potential conflicts of interest

The authors declare that their research is conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

BCO is the recipient of a PhD scholarship from the Foundation of Science and Technology Support of Pernambuco (FACEPE) and a fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES – Doutorado Sanduíche) – Finance code 001.

Leishmania research at IDRI has been funded by grants from CRDF Global (#62966), National Institute of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R01AI025038, the Bill and Melinda Gates Foundation (#631 and #39129) and The Global Health Innovative Technology Fund (G2018-111).

Leishmania research at FIOCRUZ Pernambuco is funded by Tesouro Fiocruz and the National Council for Science and Technology Development (CNPq), Brazil. Universal, Process number 14/2014.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding bodies.

Additional information

Funding

This work was supported by the CRDF Global [62966];Conselho Nacional de Desenvolvimento Científico e Tecnológico [14/2014];Coordenação de Aperfeiçoamento de Pessoal de Nível Superior [001];Fundação Oswaldo Cruz [Tesouro];Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco;National Institute of Allergy and Infectious Diseases [R01AI025038];Global Health Innovative Technology Fund [G2018-111].