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[40] Senghor MW, Niang AA, Depaquit J, Ferté H, Faye MN, Elguero E, Gaye O, Alten B, Perktas U, Cassan C, Faye B, Bañuls AL. Transmission of *Leishmania infantum* in the canine leishmaniasis focus of Mont-Rolland, Senegal: Ecological, parasitological and molecular evidence for a possible role of *Sergentomyia* sand flies. PLoS Neglected Tropical Diseases.

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[43] Tall K. Etude épidémio-clinique et prise en charge de la leishmaniose cutanée à Bamako et dans deux villages endémiques du Mali [thesis]. Faculty of Medicine and Dentistry;

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[45] WHO. Weekly Epidemiological Record. No. 38; 2017, Vol. 92. pp. 557-572. Available at: http://www.who.int/leishmaniasis/resources/REH\_38\_TABLEAU\_S1\_S2\_Version\_fi-

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**Section 3**

**Immune Evasion in Leishmaniasis**

**Immune Evasion in Leishmaniasis**

**Chapter 4**

**Provisional chapter**

**Immune Evasion Strategies**

**Immune Evasion Strategies**

DOI: 10.5772/intechopen.75169

*Leishmania* is the causative protozoan parasite of leishmaniasis. Distinct species provoke localized/diffuse cutaneous leishmaniasis or visceral leishmaniasis. *Leishmania* parasites have developed diverse strategies to evade the host immune response expressed through various cells, especially macrophages, NK cells, and dendritic cells. Participating in some of these strategies are *Leishmania* surface molecules, such as lipophosphoglycan (LPG) and protease gp63, which are thus considered virulence factors. LPG has been shown to modulate proinflammatory responses. For example, *L. major* LPG activates NK cells through tolllike receptor-2 (TLR2), while *L. mexicana* LPG elicits a differential production of cytokines in human dendritic cells and monocytes. Moreover, *L. mexicana* LPG activates MAP kinases in macrophages, which in turn enhance proinflammatory cytokine production through TLRs. Additionally, *Leishmania* exosomes have been found to strongly affect macrophage signaling and functions. Furthermore, proteins secreted by *Leishmania* promastigotes and amastigotes modulate the production of proinflammatory cytokines in human macrophages. Since *Leishmania* is an obligate intracellular parasite, its promastigotes utilize several mechanisms to survive and duplicate inside host cells, including the inhibition of apoptosis. It is now clear that MAPK p38, JNK, ERK 1/2, and PI3K/Akt participate in the inhibition of both

natural and induced apoptosis of macrophages, neutrophils, and dendritic cells.

Leishmaniasis is a complex of neglected tropical diseases (NTDs) caused by protozoan parasites of the genus *Leishmania*. Epidemiological studies have revealed that 12 million people are

**Keywords:** *Leishmania*, cytokines, TLR, inflammasome, apoptosis, NO

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

M. Magdalena Aguirre-Garcia, Alma R. Escalona-Montaño, Arturo A. Wilkins-Rodríguez and

M. Magdalena Aguirre-Garcia, Alma R. Escalona-Montaño, Arturo A. Wilkins-Rodríguez and

http://dx.doi.org/10.5772/intechopen.75169

Laila Gutiérrez-Kobeh

Laila Gutiérrez-Kobeh

**Abstract**

**1. Introduction**

#### **Chapter 4 Provisional chapter**

#### **Immune Evasion Strategies Immune Evasion Strategies**

M. Magdalena Aguirre-Garcia, Alma R. Escalona-Montaño, Arturo A. Wilkins-Rodríguez and Laila Gutiérrez-Kobeh M. Magdalena Aguirre-Garcia, Alma R. Escalona-Montaño, Arturo A. Wilkins-Rodríguez and Laila Gutiérrez-Kobeh

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75169

#### **Abstract**

*Leishmania* is the causative protozoan parasite of leishmaniasis. Distinct species provoke localized/diffuse cutaneous leishmaniasis or visceral leishmaniasis. *Leishmania* parasites have developed diverse strategies to evade the host immune response expressed through various cells, especially macrophages, NK cells, and dendritic cells. Participating in some of these strategies are *Leishmania* surface molecules, such as lipophosphoglycan (LPG) and protease gp63, which are thus considered virulence factors. LPG has been shown to modulate proinflammatory responses. For example, *L. major* LPG activates NK cells through tolllike receptor-2 (TLR2), while *L. mexicana* LPG elicits a differential production of cytokines in human dendritic cells and monocytes. Moreover, *L. mexicana* LPG activates MAP kinases in macrophages, which in turn enhance proinflammatory cytokine production through TLRs. Additionally, *Leishmania* exosomes have been found to strongly affect macrophage signaling and functions. Furthermore, proteins secreted by *Leishmania* promastigotes and amastigotes modulate the production of proinflammatory cytokines in human macrophages. Since *Leishmania* is an obligate intracellular parasite, its promastigotes utilize several mechanisms to survive and duplicate inside host cells, including the inhibition of apoptosis. It is now clear that MAPK p38, JNK, ERK 1/2, and PI3K/Akt participate in the inhibition of both natural and induced apoptosis of macrophages, neutrophils, and dendritic cells.

DOI: 10.5772/intechopen.75169

**Keywords:** *Leishmania*, cytokines, TLR, inflammasome, apoptosis, NO

### **1. Introduction**

Leishmaniasis is a complex of neglected tropical diseases (NTDs) caused by protozoan parasites of the genus *Leishmania*. Epidemiological studies have revealed that 12 million people are

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

infected worldwide, with 2 million new cases each year. Approximately 350 million people are currently at risk of contracting leishmaniasis, mostly in developing countries.

The term "secretome" was introduced for the first time in the global study of the genome of proteins secreted by *Bacillus subtilis.* The authors defined the secretome as a subset of the proteome consisting of secreted proteins and the components of the cellular machinery involved in protein secretion. They predicted all exported *B. subtilis* proteins by employing computational methods to search for signal peptides and cellular retention signals in protein sequences [12]. In *Plasmodium falciparum*, the secretome refers to all proteins exported to the host erythrocyte and mediated by an endoplasmic reticulum signal sequence, along with one export element of this parasite [13, 14]. Until very recently, there was very little information about the proteins secreted by protozoan parasites. Given the role of these proteins as virulence factors and their capacity to modulate host cells, this scant information represented an important scientific limitation.

Immune Evasion Strategies

41

http://dx.doi.org/10.5772/intechopen.75169

Regarding trypanosomatids, the term secretome was introduced by Silverman in a proteomics approach used to identify a large number of extracellular proteins in a culture media conditioned by *L. donovani* [15]. Several studies on trypanosomatids have aimed to identify and characterize excreted/secreted factors due to their potential for the development of vac-

*Leishmania* and other intracellular pathogens have developed strategies to invade and persist within the respective host cell. In some cases, the mechanisms entail the export of virulence factors to the cytosol of this same cell [15]. Almost a decade has passed since the first report of the secretion proteins by *L. donovani*. By means of a stable isotope label of amino acids in a culture called SILAC, the authors identified 151 proteins secreted by this species into the culture media. Interestingly, few of these are secreted through classical mechanisms [15]. Additionally, the bioinformatic analysis in the same study showed that none of the histidine

The authors found various proteins with several possible functions. For instance, some proteins take part in the vesicular transport process, essential for the survival of the parasite, and may thus be virulence factors. Among the proteins participating in signal transduction are those encoded by the gene LmjF 25.0750 of *L. major*, including a phosphatase serine threonine type phosphoprotein, a metal-dependent phosphatase (PPM) called PP2C [15]. This protein was cloned from the DNA genome of *L. major* and localized in the pocket and flagellum of the parasite through fluorescence microscopy assays and transmission electron microscopy [3].

Since the flagellum of *L. major* represents an important structure for differentiation in trypanosomatids, this location of PP2C suggests a role in a vital biological process of the parasite [3]. The same authors have shown that *L. mexicana* promastigotes and amastigotes secrete proteins with phosphatase activity into the culture medium. Such activity was more pronounced in the promastigote than amastigote secretion medium. Both media stimulated the production of various cytokines in human macrophages: tumor necrosis factor (TNF)-α, interleukin

Exosomes are organelles (30–100 nm) released by numerous mammalian cells, including reticulocytes, B cells, T cells, dendritic cells, and macrophages [18]. Bioactive exosomes are released

phosphatase proteins result from the classical mechanism of secretion.

cines and/or new drugs [16].

(IL)-1β, IL-12p70, and IL-10 [17].

**2.1. Exosomes secreted by** *Leishmania*

*Leishmania* is a dimorphic protozoan parasite that completes its life cycle in two organisms: the sand fly vector and a mammalian host (a rodent, canid, or human). In the vector, the parasite enters the insect as aflagellated amastigotes through a blood meal from a mammal. In the sand fly midgut, this form changes into motile extracellular flagellated promastigotes that divide by binary fission. After being injected into the bloodstream of a mammalian host, the promastigotes are quickly engulfed by macrophages, where they differentiate into aflagellated intracellular amastigotes that can survive in this acidic environment. In macrophages, the amastigotes replicate by binary fission, causing the lysis of the cell and the invasion of other cells [1].

The outcome of a *Leishmania* infection depends on a wide array of factors, especially the species of the parasite and the host immune response. Species such as *L. major*, *L. mexicana* and *L. guyanensis* induce cutaneous leishmaniasis, while *L. amazonensis* and *L. braziliensis* cause the mucocutaneous form. *L. donovani* and *L. chagasi*, on the other hand, cause visceral leishmaniasis (VL) [2].

Whether *Leishmania* parasites manage to establish themselves in mammalian cells depends on their capacity to surpass host defense mechanisms. The survival strategies of the protozoan are based on the manipulation of distinct host cell functions, including modulation of cell signaling pathways through phosphorylation and dephosphorylation mechanisms [3]. The different strategies of *Leishmania* to evade the host immune response during the process of infection involve macrophages, NK cells, and dendritic cells. One of the most successful survival strategies displayed by *Leishmania* is the inhibition of apoptosis of host cells through the activation or silencing of proapoptotic or antiapoptotic signaling pathways [4, 5]. Among the *Leishmania* surface components participating in such evasion strategies is lipophosphoglycan (LPG), an abundant molecule that exerts its activity by binding to TLR2. Although the ability of *Leishmania* to inhibit inflammatory signaling pathways has been proposed as a virulence mechanism, the molecular events underlying this process have still not been fully elucidated [6].

## **2. Parasite molecules that regulate host cell signaling pathways**

Two of the most studied molecules of *Leishmania* spp. are LPG and glycoprotein 63 (gp63), postulated as possible virulence factors for some species. LPG covers the surface of the parasite and the flagellum, forming a glycocalyx. The structure of LPG, which differs between the distinct species of *Leishmania*, is mainly constituted by repeating units of a disaccharide and a phosphate bound to the membrane by glycosylphosphatidylinositol (GPI). This molecule is more abundant in promastigotes than amastigotes [7]. Contrarily, gp63 is more frequently expressed in amastigotes than promastigotes. The absence of LPG in amastigotes emphasizes the relevance of gp63 in protozoan survival, as well as in the regulation of signaling pathways of host cells [8, 9].

Other important molecules for *Leishmania* are glycosylinositolphospholipids (GPILs), a class of glycolipids bound by GPIs and expressed 10 times more frequently than LPG. Their small size keeps them close to the parasite membrane [10, 11].

The term "secretome" was introduced for the first time in the global study of the genome of proteins secreted by *Bacillus subtilis.* The authors defined the secretome as a subset of the proteome consisting of secreted proteins and the components of the cellular machinery involved in protein secretion. They predicted all exported *B. subtilis* proteins by employing computational methods to search for signal peptides and cellular retention signals in protein sequences [12]. In *Plasmodium falciparum*, the secretome refers to all proteins exported to the host erythrocyte and mediated by an endoplasmic reticulum signal sequence, along with one export element of this parasite [13, 14]. Until very recently, there was very little information about the proteins secreted by protozoan parasites. Given the role of these proteins as virulence factors and their capacity to modulate host cells, this scant information represented an important scientific limitation.

Regarding trypanosomatids, the term secretome was introduced by Silverman in a proteomics approach used to identify a large number of extracellular proteins in a culture media conditioned by *L. donovani* [15]. Several studies on trypanosomatids have aimed to identify and characterize excreted/secreted factors due to their potential for the development of vaccines and/or new drugs [16].

*Leishmania* and other intracellular pathogens have developed strategies to invade and persist within the respective host cell. In some cases, the mechanisms entail the export of virulence factors to the cytosol of this same cell [15]. Almost a decade has passed since the first report of the secretion proteins by *L. donovani*. By means of a stable isotope label of amino acids in a culture called SILAC, the authors identified 151 proteins secreted by this species into the culture media. Interestingly, few of these are secreted through classical mechanisms [15]. Additionally, the bioinformatic analysis in the same study showed that none of the histidine phosphatase proteins result from the classical mechanism of secretion.

The authors found various proteins with several possible functions. For instance, some proteins take part in the vesicular transport process, essential for the survival of the parasite, and may thus be virulence factors. Among the proteins participating in signal transduction are those encoded by the gene LmjF 25.0750 of *L. major*, including a phosphatase serine threonine type phosphoprotein, a metal-dependent phosphatase (PPM) called PP2C [15]. This protein was cloned from the DNA genome of *L. major* and localized in the pocket and flagellum of the parasite through fluorescence microscopy assays and transmission electron microscopy [3].

Since the flagellum of *L. major* represents an important structure for differentiation in trypanosomatids, this location of PP2C suggests a role in a vital biological process of the parasite [3]. The same authors have shown that *L. mexicana* promastigotes and amastigotes secrete proteins with phosphatase activity into the culture medium. Such activity was more pronounced in the promastigote than amastigote secretion medium. Both media stimulated the production of various cytokines in human macrophages: tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-12p70, and IL-10 [17].

### **2.1. Exosomes secreted by** *Leishmania*

infected worldwide, with 2 million new cases each year. Approximately 350 million people are

*Leishmania* is a dimorphic protozoan parasite that completes its life cycle in two organisms: the sand fly vector and a mammalian host (a rodent, canid, or human). In the vector, the parasite enters the insect as aflagellated amastigotes through a blood meal from a mammal. In the sand fly midgut, this form changes into motile extracellular flagellated promastigotes that divide by binary fission. After being injected into the bloodstream of a mammalian host, the promastigotes are quickly engulfed by macrophages, where they differentiate into aflagellated intracellular amastigotes that can survive in this acidic environment. In macrophages, the amastigotes replicate by binary fission, causing the lysis of the cell and the invasion of other cells [1].

The outcome of a *Leishmania* infection depends on a wide array of factors, especially the species of the parasite and the host immune response. Species such as *L. major*, *L. mexicana* and *L. guyanensis* induce cutaneous leishmaniasis, while *L. amazonensis* and *L. braziliensis* cause the mucocutaneous form. *L. donovani* and *L. chagasi*, on the other hand, cause visceral leishmaniasis (VL) [2].

Whether *Leishmania* parasites manage to establish themselves in mammalian cells depends on their capacity to surpass host defense mechanisms. The survival strategies of the protozoan are based on the manipulation of distinct host cell functions, including modulation of cell signaling pathways through phosphorylation and dephosphorylation mechanisms [3]. The different strategies of *Leishmania* to evade the host immune response during the process of infection involve macrophages, NK cells, and dendritic cells. One of the most successful survival strategies displayed by *Leishmania* is the inhibition of apoptosis of host cells through the activation or silencing of proapoptotic or antiapoptotic signaling pathways [4, 5]. Among the *Leishmania* surface components participating in such evasion strategies is lipophosphoglycan (LPG), an abundant molecule that exerts its activity by binding to TLR2. Although the ability of *Leishmania* to inhibit inflammatory signaling pathways has been proposed as a virulence mechanism, the

molecular events underlying this process have still not been fully elucidated [6].

**2. Parasite molecules that regulate host cell signaling pathways**

size keeps them close to the parasite membrane [10, 11].

Two of the most studied molecules of *Leishmania* spp. are LPG and glycoprotein 63 (gp63), postulated as possible virulence factors for some species. LPG covers the surface of the parasite and the flagellum, forming a glycocalyx. The structure of LPG, which differs between the distinct species of *Leishmania*, is mainly constituted by repeating units of a disaccharide and a phosphate bound to the membrane by glycosylphosphatidylinositol (GPI). This molecule is more abundant in promastigotes than amastigotes [7]. Contrarily, gp63 is more frequently expressed in amastigotes than promastigotes. The absence of LPG in amastigotes emphasizes the relevance of gp63 in protozoan survival, as well as in the regulation of signaling pathways of host cells [8, 9].

Other important molecules for *Leishmania* are glycosylinositolphospholipids (GPILs), a class of glycolipids bound by GPIs and expressed 10 times more frequently than LPG. Their small

currently at risk of contracting leishmaniasis, mostly in developing countries.

40 Leishmaniases as Re-emerging Diseases

Exosomes are organelles (30–100 nm) released by numerous mammalian cells, including reticulocytes, B cells, T cells, dendritic cells, and macrophages [18]. Bioactive exosomes are released by cells infected with viruses and bacteria as well as some tumor cells [19, 20]. The release of exosomes also constitutes a mechanism for the secretion of proteins by *Leishmania*, and these vesicles allow for communication with the host cell [15]. Among the proteins released through exosomes, the metalloprotease 63 kDa and the elongation factor 1 alpha (EF-1 α) are known to have a substantial role in deregulating certain signaling pathways. These two molecules, contained in microvesicles, are responsible for the activation of tyrosine phosphatases (SHP-1) in the host cell [21]. A study of *L. mexicana* demonstrated a type of vesicle induced at a temperature of 37°C that did not correspond to exosomes, as evidenced by its size.

by bacterial toxins, bacterial RNA, ATP, nigericin, uric acid, and silica crystals, is an essential

Immune Evasion Strategies

43

http://dx.doi.org/10.5772/intechopen.75169

*Leishmania* parasites have developed diverse strategies to evade the immune response, especially in the form of macrophages, NK cells, and dendritic cells. Becker analyzed the interaction between *Leishmania* LPG and TLR2 receptors on human NK cells, finding that LPG purified from metacyclic and procyclic promastigotes of *L. major* stimulates these host cells. The consequent activation of NK cells leads to an upregulation of TLR2 expression, the nuclear translocation of NF-κB, and an increased production of IFN-γ and TNF-α. Indeed, the activation of NK cells turned out to be greater with the infective metacyclic form than the

*L. mexicana* LPG elicits a differential production of proinflammatory cytokines, such as IL-12, TNF-α and IL-10, as well as the nuclear translocation of NF-κB in monocytes and dendritic cells [27]. It also activates ERK and p38 MAP kinase in macrophages and induces proinflam-

After infecting the THP1 human cell line with *L. donovani*, the production of IL-10, TNF-α and IFN-γ was measured, and the expression of TLR2, TLR4, and TLR9 was determined in blood samples and the THP1 cell line. IL-10 levels were higher in controls positive to the leishmanin skin test (LST+) compared to patients with VL. TNF-α was moderately produced, exhibiting no variation between patients, controls, and THP1 cells. TLR4 and TLR9 expression was elevated in patients with VL. *L. donovani* increased the expression of TLR4 and TLR9 in patients with VL, and of TLR2 in THP1 cells, which suggests a link between TLRs and the generation

Interestingly, other authors analyzed the expression of some components related to the inflammasome pathway in murine macrophages infected with *L. major*. At 6 and 18 h post infection, they evaluated the mRNA expression levels in control and infected macrophages of two NLRs (NLRP3 and NAIP5), the inflammasome adaptor molecule ASC, proinflammatory caspase-1, and proinflammatory cytokines IL-1β and IL-18. The components related to the inflammasome pathway (NLRP3, ASC, IPAF, IL-1β, and IL-18) were upregulated in murine macrophages infected with *L. major*. The activity of caspase-1 was more pronounced in infected than noninfected macrophages. Infected (versus uninfected) macrophages also showed significantly greater caspase-1 activity in harvested cells and a significantly higher

It has been documented, based on *in vitro* (in macrophages) and *in vivo* studies, that a *Leishmania* infection activates the NLRP3 inflammasome and that the latter is key to the inhibition of parasite replication. For example, the capacity of inflammasome-deficient mice to resist infection with *L. amazonensis, L. braziliensis, L. infantum,* and *L. chagasi* was favored by IL-1β production resulting from inflammasome activation. The mechanism involved in such activation was the increase in the level of nitric oxide (NO), which in turn was mediated by the elevated availability of nitric oxide synthase NOS2 resulting from signaling through the IL-1 receptor and MyD88. Lima-Junior et al. previously showed that the NLR3 inflammasome is vital for the host response to *L. amazonensis* infection, having proven to restrict

matory cytokine production through TLR2 and TLR4 signaling [28].

concentration of IL-1β in the supernatant of the cultured media [30].

component of the host immune response against bacterial and viral pathogens [25].

non-infective procyclic form of LPG [26].

of a mixed cytokine response [29].

Other studies confirm a nonclassical route of secretion for *Leishmania* proteins. Two-dimensional electrophoresis displayed 270 secreted protein spots originating from *L. braziliensis*, of which 42 were identified. About 57% of these proteins presented non-classical secretion mechanisms [22].

In *L. infantum*, distinct proteins were observed in exovesicles of the parasites in the different phases of their growth. Ribosomal proteins were detected in the logarithmic phase of growth, thus indicating their crucial role in protein turnover. In the stationary phase, contrarily, there was a specific enrichment of vesicles with properties similar to apoptotic vesicles [23].

Diverse *Leishmania* species have been analyzed to explore the effect exerted by their secreted molecules on the host immune response. For example, in infection by *L. donovani* and interferon (IFN)-γ treatment, the exposure of human monocytes to the exosomes secreted by this parasite led to an alteration in the cytokine response. The resulting inhibition of IL-8 and TNF-α production combined with enhanced levels of IL-10 caused an anti-inflammatory effect. This immune suppression induced during *Leishmania* infection suggests that the secretion of exosomes by *Leishmania* likely plays a major role in the establishment of infection. Indeed, exosomes may be a mechanism of immune modulation used more generally by intracellular and extracellular pathogens [24].

## **3.** *Leishmania* **modulates proinflammatory cytokines, inflammasomes, and TLR expression**

To detect *Leishmania* infections, the innate immune system utilizes different sets of germlineencoded receptors, including the TLRs found on cell membranes and in the endosome. Other receptors that have only been detected in the cytoplasm, such as the NOD-like receptor (NLR) family, also play a role in defending the host from a *Leishmania* invasion. Encompassing 34 members in all, NLRs sense pathogen- and danger-associated molecular patterns (PAMPs and DAMPs, respectively).

There is a subset of NLRs that assembles large multiprotein complexes known as inflammasomes. The latter trigger inflammatory caspase 1, which in turn promotes the conversion of pro–IL-1β and pro–IL-18 into their bioactive forms. For instance, NLRP3 (the best-characterized inflammasome) consists of the NLRP3 protein, the bipartite adaptor protein ASC, and caspase 1 (in its minimum form). It has been demonstrated that this inflammasome, activated by bacterial toxins, bacterial RNA, ATP, nigericin, uric acid, and silica crystals, is an essential component of the host immune response against bacterial and viral pathogens [25].

by cells infected with viruses and bacteria as well as some tumor cells [19, 20]. The release of exosomes also constitutes a mechanism for the secretion of proteins by *Leishmania*, and these vesicles allow for communication with the host cell [15]. Among the proteins released through exosomes, the metalloprotease 63 kDa and the elongation factor 1 alpha (EF-1 α) are known to have a substantial role in deregulating certain signaling pathways. These two molecules, contained in microvesicles, are responsible for the activation of tyrosine phosphatases (SHP-1) in the host cell [21]. A study of *L. mexicana* demonstrated a type of vesicle induced at a tem-

Other studies confirm a nonclassical route of secretion for *Leishmania* proteins. Two-dimensional electrophoresis displayed 270 secreted protein spots originating from *L. braziliensis*, of which 42 were identified. About 57% of these proteins presented non-classical secretion

In *L. infantum*, distinct proteins were observed in exovesicles of the parasites in the different phases of their growth. Ribosomal proteins were detected in the logarithmic phase of growth, thus indicating their crucial role in protein turnover. In the stationary phase, contrarily, there

Diverse *Leishmania* species have been analyzed to explore the effect exerted by their secreted molecules on the host immune response. For example, in infection by *L. donovani* and interferon (IFN)-γ treatment, the exposure of human monocytes to the exosomes secreted by this parasite led to an alteration in the cytokine response. The resulting inhibition of IL-8 and TNF-α production combined with enhanced levels of IL-10 caused an anti-inflammatory effect. This immune suppression induced during *Leishmania* infection suggests that the secretion of exosomes by *Leishmania* likely plays a major role in the establishment of infection. Indeed, exosomes may be a mechanism of immune modulation used more generally by intra-

To detect *Leishmania* infections, the innate immune system utilizes different sets of germlineencoded receptors, including the TLRs found on cell membranes and in the endosome. Other receptors that have only been detected in the cytoplasm, such as the NOD-like receptor (NLR) family, also play a role in defending the host from a *Leishmania* invasion. Encompassing 34 members in all, NLRs sense pathogen- and danger-associated molecular patterns (PAMPs

There is a subset of NLRs that assembles large multiprotein complexes known as inflammasomes. The latter trigger inflammatory caspase 1, which in turn promotes the conversion of pro–IL-1β and pro–IL-18 into their bioactive forms. For instance, NLRP3 (the best-characterized inflammasome) consists of the NLRP3 protein, the bipartite adaptor protein ASC, and caspase 1 (in its minimum form). It has been demonstrated that this inflammasome, activated

was a specific enrichment of vesicles with properties similar to apoptotic vesicles [23].

perature of 37°C that did not correspond to exosomes, as evidenced by its size.

mechanisms [22].

42 Leishmaniases as Re-emerging Diseases

cellular and extracellular pathogens [24].

and DAMPs, respectively).

**inflammasomes, and TLR expression**

**3.** *Leishmania* **modulates proinflammatory cytokines,**

*Leishmania* parasites have developed diverse strategies to evade the immune response, especially in the form of macrophages, NK cells, and dendritic cells. Becker analyzed the interaction between *Leishmania* LPG and TLR2 receptors on human NK cells, finding that LPG purified from metacyclic and procyclic promastigotes of *L. major* stimulates these host cells. The consequent activation of NK cells leads to an upregulation of TLR2 expression, the nuclear translocation of NF-κB, and an increased production of IFN-γ and TNF-α. Indeed, the activation of NK cells turned out to be greater with the infective metacyclic form than the non-infective procyclic form of LPG [26].

*L. mexicana* LPG elicits a differential production of proinflammatory cytokines, such as IL-12, TNF-α and IL-10, as well as the nuclear translocation of NF-κB in monocytes and dendritic cells [27]. It also activates ERK and p38 MAP kinase in macrophages and induces proinflammatory cytokine production through TLR2 and TLR4 signaling [28].

After infecting the THP1 human cell line with *L. donovani*, the production of IL-10, TNF-α and IFN-γ was measured, and the expression of TLR2, TLR4, and TLR9 was determined in blood samples and the THP1 cell line. IL-10 levels were higher in controls positive to the leishmanin skin test (LST+) compared to patients with VL. TNF-α was moderately produced, exhibiting no variation between patients, controls, and THP1 cells. TLR4 and TLR9 expression was elevated in patients with VL. *L. donovani* increased the expression of TLR4 and TLR9 in patients with VL, and of TLR2 in THP1 cells, which suggests a link between TLRs and the generation of a mixed cytokine response [29].

Interestingly, other authors analyzed the expression of some components related to the inflammasome pathway in murine macrophages infected with *L. major*. At 6 and 18 h post infection, they evaluated the mRNA expression levels in control and infected macrophages of two NLRs (NLRP3 and NAIP5), the inflammasome adaptor molecule ASC, proinflammatory caspase-1, and proinflammatory cytokines IL-1β and IL-18. The components related to the inflammasome pathway (NLRP3, ASC, IPAF, IL-1β, and IL-18) were upregulated in murine macrophages infected with *L. major*. The activity of caspase-1 was more pronounced in infected than noninfected macrophages. Infected (versus uninfected) macrophages also showed significantly greater caspase-1 activity in harvested cells and a significantly higher concentration of IL-1β in the supernatant of the cultured media [30].

It has been documented, based on *in vitro* (in macrophages) and *in vivo* studies, that a *Leishmania* infection activates the NLRP3 inflammasome and that the latter is key to the inhibition of parasite replication. For example, the capacity of inflammasome-deficient mice to resist infection with *L. amazonensis, L. braziliensis, L. infantum,* and *L. chagasi* was favored by IL-1β production resulting from inflammasome activation. The mechanism involved in such activation was the increase in the level of nitric oxide (NO), which in turn was mediated by the elevated availability of nitric oxide synthase NOS2 resulting from signaling through the IL-1 receptor and MyD88. Lima-Junior et al. previously showed that the NLR3 inflammasome is vital for the host response to *L. amazonensis* infection, having proven to restrict parasite replication in both isolated macrophages and *in vivo*. As can be appreciated, IL-1β production is involved in the host resistance to infection. The signaling that triggers the production of this cytokine takes place through the IL-1 receptor and MyD88, contributing to elevated levels of NOS2. An increase in the latter enzyme leads to a greater generation of NO, a major host defense mechanism against *Leishmania* spp. [31].

urea and L-ornithine. The latter is a basic source for the synthesis of polyamines, essential nutrients for the growth and surveillance of *Leishmania* [38, 39]. Hence, whether L-arginine metabolism takes place through ARG-1 or NOS2 is decisive for the life or death of *Leishmania*

Immune Evasion Strategies

45

http://dx.doi.org/10.5772/intechopen.75169

To establish infection and avoid host surveillance, *Leishmania* parasites have developed different strategies to hijack L-arginine metabolism in order to promote the production of polyamines rather than NO. During its development inside the vector, for instance, the parasite generates a mucin-rich gel that sand flies deliver into the host skin when transmitting *Leishmania* promastigotes [41]. This gel, called promastigote secretory gel, is known to modulate L-arginine metabolism in macrophages [42]. Accordingly, promastigote secretory gel stimulates the recruitment of macrophages and promotes their alternative activation, causing an increased expression of arginase-1 along with its greater capacity to metabolize L-arginine

The parasite-induced upregulation of arginase-1 can affect the production of NO through substrate (L-arginine) competition [41]. Additionally, the generation of some polyamines resulting from L-arginine metabolism via arginase-1 (e.g., spermine, spermidine and putrescine) inhibit NOS2 function [38, 39, 43]. The modulation of L-arginine metabolism is relevant not only during the onset of the infection but throughout the course the disease. The success of host immunity or the pathology of leishmaniasis depends mainly on the balance of the immune response. Since the formation of either NOS2 or arginase leads to the inhibition of the other, these two metabolic states are competitive and tightly regulated [44], determining the levels of NO and therefore the outcome of *Leishmania* infection (survival versus elimina-

Besides the production of polyamines and the resulting enhancement of *Leishmania* intracellular growth in alternatively activated macrophages, recently findings have shown that *Leishmania*-induced L-arginine metabolism via ARG-1 polarization is advantageous to the parasite in yet another way. A substantial accumulation of alternatively activated macrophages causes an elevated demand, consumption, and depletion of L-arginine in the microenvironment [41]. Since T lymphocytes are very sensitive to L-arginine starvation, a greater consumption of this amino acid via ARG-1 limits its availability to T cells, which in turn notably impairs the development and function of these cells that are required for the control

*Leishmania* is an obligate intracellular parasite that invades a variety of host cells, but it is in dendritic cells and macrophages where it can survive and replicate inside the phagosome. The condition of being obligate intracellular parasites presupposes the utilization by *Leishmania* of mechanisms to manipulate host cells in order to evade the immune response and survive inside cells. Along the evolutionary history of this parasite, diverse survival strategies have

to afford polyamines, which in turn enhance the growth of *Leishmania* [42].

during infection [40].

tion) in the host.

been developed.

of a *Leishmania* infection [45, 46].

**5. Inhibition of apoptosis**

In the case of *L. major*, secreted antigens suppressed the proliferation of BALB/c mice lymphocytes *in vitro*. After semi-purifying these secreted antigens, they were found to suppress 60% of lymphocyte proliferation and prevent the stimulation of lymphocytes. The fractions obtained decreased the production of IFN-γ and increased the level of IL-4 in lymphocytes, whereas they downregulated the formation of NO by activated macrophages. Hence, proteins secreted by *L. major* may function as immunosuppressive factors that downregulate the immune system [32]. On the other hand, the immunomodulatory effect of proteins excreted/ secreted by *L. infantum* was described in the context of differentiation and maturation of human dendritic cells [33].

Regarding *L. donovani*, an immunomodulatory role has also been established for leishmanial excretory-secretory antigens (LESAs) released by promastigotes to the culture medium. The separation of fractions from LESAs revealed proteins of different molecular weights. Both fractions were highly immunogenic, as they significantly enhanced the activity of NADPH oxidase and SOD, as well as the production of NO, TNF-α, IFN-γ, and IL-12 in stimulated RAW 264.7 macrophages. These results strongly suggest the potential role of LESAs in the modulation of macrophage effector functions and Th1 responses, which could possibly be used in the development of a potent vaccine for visceral leishmaniasis [34]. Similarly, Kumar reported a potential immunostimulatory effect of soluble exogenous antigens of *L. donovani*, which may be instrumental in developing a subunit vaccine against VL [35].

## **4.** *Leishmania* **modulates L-arginine metabolism via NOS2 and arginase-1**

Among other strategies developed by *Leishmania* parasites to avoid elimination by the host immune response, regulation of L-arginine metabolism via NOS2 and arginase-1 (ARG-1) enzymes has emerged as a crucial mechanism for parasite survival. Macrophages, the main host cells that battle *Leishmania*, can be instructed to kill or host intracellular amastigote forms of this parasite, depending on their ability to express NOS2 or ARG-1. The expression of these enzymes, which share L-arginine as a substrate, is regulated in macrophages by their perception of the environmental balance of cytokines.

Proinflammatory cytokines (e.g., TNF-α and IFN-γ) induce the classical activation of macrophages, upregulating NOS2 expression. This enzyme catalyzes the conversion of L-arginine into L-citrulline and NO, the latter molecule being considered the most potent leishmanicidal agent for the elimination of intracellular *Leishmania* parasites [36]. On the other hand, antiinflammatory cytokines (e.g., IL-4, IL-10 and IL-13) elicit an alternative activation of macrophages that upregulates ARG-1 [37], which in turn catalyzes the conversion of L-arginine into urea and L-ornithine. The latter is a basic source for the synthesis of polyamines, essential nutrients for the growth and surveillance of *Leishmania* [38, 39]. Hence, whether L-arginine metabolism takes place through ARG-1 or NOS2 is decisive for the life or death of *Leishmania* during infection [40].

To establish infection and avoid host surveillance, *Leishmania* parasites have developed different strategies to hijack L-arginine metabolism in order to promote the production of polyamines rather than NO. During its development inside the vector, for instance, the parasite generates a mucin-rich gel that sand flies deliver into the host skin when transmitting *Leishmania* promastigotes [41]. This gel, called promastigote secretory gel, is known to modulate L-arginine metabolism in macrophages [42]. Accordingly, promastigote secretory gel stimulates the recruitment of macrophages and promotes their alternative activation, causing an increased expression of arginase-1 along with its greater capacity to metabolize L-arginine to afford polyamines, which in turn enhance the growth of *Leishmania* [42].

The parasite-induced upregulation of arginase-1 can affect the production of NO through substrate (L-arginine) competition [41]. Additionally, the generation of some polyamines resulting from L-arginine metabolism via arginase-1 (e.g., spermine, spermidine and putrescine) inhibit NOS2 function [38, 39, 43]. The modulation of L-arginine metabolism is relevant not only during the onset of the infection but throughout the course the disease. The success of host immunity or the pathology of leishmaniasis depends mainly on the balance of the immune response. Since the formation of either NOS2 or arginase leads to the inhibition of the other, these two metabolic states are competitive and tightly regulated [44], determining the levels of NO and therefore the outcome of *Leishmania* infection (survival versus elimination) in the host.

Besides the production of polyamines and the resulting enhancement of *Leishmania* intracellular growth in alternatively activated macrophages, recently findings have shown that *Leishmania*-induced L-arginine metabolism via ARG-1 polarization is advantageous to the parasite in yet another way. A substantial accumulation of alternatively activated macrophages causes an elevated demand, consumption, and depletion of L-arginine in the microenvironment [41]. Since T lymphocytes are very sensitive to L-arginine starvation, a greater consumption of this amino acid via ARG-1 limits its availability to T cells, which in turn notably impairs the development and function of these cells that are required for the control of a *Leishmania* infection [45, 46].

## **5. Inhibition of apoptosis**

parasite replication in both isolated macrophages and *in vivo*. As can be appreciated, IL-1β production is involved in the host resistance to infection. The signaling that triggers the production of this cytokine takes place through the IL-1 receptor and MyD88, contributing to elevated levels of NOS2. An increase in the latter enzyme leads to a greater generation of NO,

In the case of *L. major*, secreted antigens suppressed the proliferation of BALB/c mice lymphocytes *in vitro*. After semi-purifying these secreted antigens, they were found to suppress 60% of lymphocyte proliferation and prevent the stimulation of lymphocytes. The fractions obtained decreased the production of IFN-γ and increased the level of IL-4 in lymphocytes, whereas they downregulated the formation of NO by activated macrophages. Hence, proteins secreted by *L. major* may function as immunosuppressive factors that downregulate the immune system [32]. On the other hand, the immunomodulatory effect of proteins excreted/ secreted by *L. infantum* was described in the context of differentiation and maturation of

Regarding *L. donovani*, an immunomodulatory role has also been established for leishmanial excretory-secretory antigens (LESAs) released by promastigotes to the culture medium. The separation of fractions from LESAs revealed proteins of different molecular weights. Both fractions were highly immunogenic, as they significantly enhanced the activity of NADPH oxidase and SOD, as well as the production of NO, TNF-α, IFN-γ, and IL-12 in stimulated RAW 264.7 macrophages. These results strongly suggest the potential role of LESAs in the modulation of macrophage effector functions and Th1 responses, which could possibly be used in the development of a potent vaccine for visceral leishmaniasis [34]. Similarly, Kumar reported a potential immunostimulatory effect of soluble exogenous antigens of *L. donovani*,

which may be instrumental in developing a subunit vaccine against VL [35].

**4.** *Leishmania* **modulates L-arginine metabolism via NOS2 and**

Among other strategies developed by *Leishmania* parasites to avoid elimination by the host immune response, regulation of L-arginine metabolism via NOS2 and arginase-1 (ARG-1) enzymes has emerged as a crucial mechanism for parasite survival. Macrophages, the main host cells that battle *Leishmania*, can be instructed to kill or host intracellular amastigote forms of this parasite, depending on their ability to express NOS2 or ARG-1. The expression of these enzymes, which share L-arginine as a substrate, is regulated in macrophages by their percep-

Proinflammatory cytokines (e.g., TNF-α and IFN-γ) induce the classical activation of macrophages, upregulating NOS2 expression. This enzyme catalyzes the conversion of L-arginine into L-citrulline and NO, the latter molecule being considered the most potent leishmanicidal agent for the elimination of intracellular *Leishmania* parasites [36]. On the other hand, antiinflammatory cytokines (e.g., IL-4, IL-10 and IL-13) elicit an alternative activation of macrophages that upregulates ARG-1 [37], which in turn catalyzes the conversion of L-arginine into

a major host defense mechanism against *Leishmania* spp. [31].

human dendritic cells [33].

44 Leishmaniases as Re-emerging Diseases

**arginase-1**

tion of the environmental balance of cytokines.

*Leishmania* is an obligate intracellular parasite that invades a variety of host cells, but it is in dendritic cells and macrophages where it can survive and replicate inside the phagosome. The condition of being obligate intracellular parasites presupposes the utilization by *Leishmania* of mechanisms to manipulate host cells in order to evade the immune response and survive inside cells. Along the evolutionary history of this parasite, diverse survival strategies have been developed.

Although the inhibition of the phagosome-lysosome fusion comprises one such strategy, one of the most intriguing is the inhibition of apoptosis. The latter process is a type of programmed cell death characterized by a very orderly and immunologically silent dismantling of a cell [47, 48]. The activation or inhibition of several signaling pathways is required for apoptosis to occur [49–53]. Whereas the initiation of apoptosis involves gene activation and transduction pathways, the executioner phase requires the activation of the cellular machinery necessary for the dismantling of the cell.

cellular survival processes [72]. MAPKs [73, 74] and PI3K [75] are activated during *Leishmania*

Immune Evasion Strategies

47

http://dx.doi.org/10.5772/intechopen.75169

Diverse signaling pathways have been implicated in the inhibition of apoptosis by *Leishmania*, such as NF-κB, PI3K, and p38 MAPK PI3K. However, only the inhibition of PI3K resulted in the abrogation of the antiapoptotic phenotype, whose activation confers apoptosis inhibition in infected macrophages [79] and dendritic cells [5]. ERK1/2 is activated in neutrophils infected with *L. major* and modulates multiple apoptotic pathways [80]. Contrarily, p38 and JNK are deactivated to prevent apoptosis [4, 5]. Survival is mediated by signaling pathways (e.g., PI3K/Akt), as well as by the expression of antiapoptotic proteins of the Bcl-2 family (e.g., Bcl-2, Bcl-xL, MCL-1, and A1). We have demonstrated that *L. mexicana* promastigotes and amastigotes also inhibit apoptosis in dendritic cells [62, 63] through the downregulation of proapoptotic pathways (e.g., MAPK p38 and JNK), as well as the activation of antiapoptotic

*Leishmania* parasites have developed diverse strategies to evade the immune response: elicitation of a differential production of proinflammatory cytokines, upregulation of TLR2 and TLR4 expression, the nuclear translocation of NF-κB, and modulation of inflammasome. These parasites have a dual relation with host cells. Whereas host cells provide them with nutrients and a place to survive and replicate, these same cells exhibit an immune response aimed at the destruction of the parasites. Hence, the latter must display a wide array of strategies to overcome host cells defense mechanisms. One of the most successful strategies utilized

Apoptosis is a type of programmed cell death involving a precisely orchestrated series of steps that culminate in the orderly dismantling of the cell. This process encompasses the activation and silencing of a wide variety of signaling pathways, among which a leading role is played by MAPK, PI3K/Akt and proapoptotic/antiapoptotic proteins of the Bcl-2 family. *Leishmania* has the capacity to inhibit apoptosis of different cells, especially macrophages, monocytes, neutrophils, and dendritic cells. Although the precise mechanisms have not been fully elucidated, it is now clear that MAPK p38, JNK, ERK 1/2, and PI3K/Akt participate in the inhibition of apoptosis in macrophages, neutrophils, and dendritic cells. The inhibition of apoptosis is a key strategy for the survival and replication of *Leishmania* in host cells and may have implications for its pathogenesis because of favoring the invasion of the host and the persistence of the parasite in host cells. Further research is needed on the mechanisms of activation and regulation of the inflammasome pathway to provide insights into the patho-

The authors are especially grateful to Bruce Allan Larsen for proofreading the manuscript.

infections and participate in the apoptosis or survival cells [76–78].

by *Leishmania* inside host cells is the inhibition of apoptosis.

physiology of chronic diseases and reveal new therapeutic targets.

routes such as PI3K/Akt [4, 5].

**6. Conclusion**

**Acknowledgements**

Apoptosis is a crucial defense mechanism against intracellular pathogens [54]. However, many pathogenic microorganisms such as virus [55], bacteria [56], and protozoan parasites [57, 58] have developed mechanisms to persist within host cells without inducing apoptosis. It has been widely documented that *Leishmania* inhibits apoptosis of different cells such as macrophages [59–61], monocytes [64] and neutrophils [66].

Recently, it has been demonstrated that *L. mexicana* promastigotes and amastigotes also inhibit apoptosis in dendritic cells [62, 63]. In some of these studies, monocytes, dendritic cells, and macrophages were grown under apoptogenic conditions and infected with different species of *Leishmania*, resulting in the inhibition of normal apoptosis.

In particular, infection with *L. donovani* or a stimulus with its LPG inhibits apoptosis in macrophages. Cellular activation caused by infection increases the production of TNF-α, TGFβ, IL-6, and GM-CSF, while decreasing the secretion of M-CSF and IL-1β [60]. Additionally, *L. major* delayed apoptosis by inhibiting the release of mitochondrial cytochrome C in infected macrophages grown in the presence of staurosporine [59]. Studies performed on other cell lines report a similar outcome, such as the inhibition of actinomycin D-induced apoptosis in the monocyte cell line U937 infected with *L. infantum* [64]. In macrophages from the cell line RAW 264.7 infected with *L. major*, apoptosis diminished even in the presence of cycloheximide [65]. Exposing neutrophils to *L. major* led to reduced caspase-3 activity, thus inhibiting spontaneous apoptosis [66]. Moreover, amastigotes and promastigotes of *L. mexicana* inhibited camptothecininduced apoptosis in monocyte-derived dendritic cells [62, 63]. In the majority of reports, the antiapoptotic effect has been associated with a significant decline in caspase-3 activity in cells.

## **5.1. Signaling pathways involved in** *Leishmania***-induced inhibition of host cell apoptosis**

Although *Leishmania* infection is known to inhibit apoptosis in several cells, the mechanism(s) through which this process takes place in infected cells is not fully clear. One signaling pathway involved in apoptosis is that of MAPKs, a family of serine/threonine kinases. Four major pathways have been identified in mammalian cells for signaling by MAPKs: extracellular signal-related kinases (ERK1/2), c-Jun amino–terminal kinases (JNK1/2/3), p38 (α, β, γ, δ), and ERK5 [67–70]. MAPKs respond to a wide variety of stimuli, such as proinflammatory cytokines, environmental stress, DNA damage, and growth factors [50, 71]. The p38 pathway is associated with cytokine production, inflammation, cell growth and differentiation, and cell death. JNK participates in the control of cell death and is encoded by three genes: JNK1/SAPKγ, JNK2/SAPKα, and JNK3/SAPKβ. Contrarily, the signaling pathway of PI3K/ AKT has an antiapoptotic role through the phosphorylation of PI3K/AKT that conducts to the downstream activation of multiple signaling pathways related to growth, development, and cellular survival processes [72]. MAPKs [73, 74] and PI3K [75] are activated during *Leishmania* infections and participate in the apoptosis or survival cells [76–78].

Diverse signaling pathways have been implicated in the inhibition of apoptosis by *Leishmania*, such as NF-κB, PI3K, and p38 MAPK PI3K. However, only the inhibition of PI3K resulted in the abrogation of the antiapoptotic phenotype, whose activation confers apoptosis inhibition in infected macrophages [79] and dendritic cells [5]. ERK1/2 is activated in neutrophils infected with *L. major* and modulates multiple apoptotic pathways [80]. Contrarily, p38 and JNK are deactivated to prevent apoptosis [4, 5]. Survival is mediated by signaling pathways (e.g., PI3K/Akt), as well as by the expression of antiapoptotic proteins of the Bcl-2 family (e.g., Bcl-2, Bcl-xL, MCL-1, and A1). We have demonstrated that *L. mexicana* promastigotes and amastigotes also inhibit apoptosis in dendritic cells [62, 63] through the downregulation of proapoptotic pathways (e.g., MAPK p38 and JNK), as well as the activation of antiapoptotic routes such as PI3K/Akt [4, 5].

## **6. Conclusion**

Although the inhibition of the phagosome-lysosome fusion comprises one such strategy, one of the most intriguing is the inhibition of apoptosis. The latter process is a type of programmed cell death characterized by a very orderly and immunologically silent dismantling of a cell [47, 48]. The activation or inhibition of several signaling pathways is required for apoptosis to occur [49–53]. Whereas the initiation of apoptosis involves gene activation and transduction pathways, the executioner phase requires the activation of the cellular machinery necessary

Apoptosis is a crucial defense mechanism against intracellular pathogens [54]. However, many pathogenic microorganisms such as virus [55], bacteria [56], and protozoan parasites [57, 58] have developed mechanisms to persist within host cells without inducing apoptosis. It has been widely documented that *Leishmania* inhibits apoptosis of different cells such as

Recently, it has been demonstrated that *L. mexicana* promastigotes and amastigotes also inhibit apoptosis in dendritic cells [62, 63]. In some of these studies, monocytes, dendritic cells, and macrophages were grown under apoptogenic conditions and infected with different species

In particular, infection with *L. donovani* or a stimulus with its LPG inhibits apoptosis in macrophages. Cellular activation caused by infection increases the production of TNF-α, TGFβ, IL-6, and GM-CSF, while decreasing the secretion of M-CSF and IL-1β [60]. Additionally, *L. major* delayed apoptosis by inhibiting the release of mitochondrial cytochrome C in infected macrophages grown in the presence of staurosporine [59]. Studies performed on other cell lines report a similar outcome, such as the inhibition of actinomycin D-induced apoptosis in the monocyte cell line U937 infected with *L. infantum* [64]. In macrophages from the cell line RAW 264.7 infected with *L. major*, apoptosis diminished even in the presence of cycloheximide [65]. Exposing neutrophils to *L. major* led to reduced caspase-3 activity, thus inhibiting spontaneous apoptosis [66]. Moreover, amastigotes and promastigotes of *L. mexicana* inhibited camptothecininduced apoptosis in monocyte-derived dendritic cells [62, 63]. In the majority of reports, the antiapoptotic effect has been associated with a significant decline in caspase-3 activity in cells.

**5.1. Signaling pathways involved in** *Leishmania***-induced inhibition of host cell**

Although *Leishmania* infection is known to inhibit apoptosis in several cells, the mechanism(s) through which this process takes place in infected cells is not fully clear. One signaling pathway involved in apoptosis is that of MAPKs, a family of serine/threonine kinases. Four major pathways have been identified in mammalian cells for signaling by MAPKs: extracellular signal-related kinases (ERK1/2), c-Jun amino–terminal kinases (JNK1/2/3), p38 (α, β, γ, δ), and ERK5 [67–70]. MAPKs respond to a wide variety of stimuli, such as proinflammatory cytokines, environmental stress, DNA damage, and growth factors [50, 71]. The p38 pathway is associated with cytokine production, inflammation, cell growth and differentiation, and cell death. JNK participates in the control of cell death and is encoded by three genes: JNK1/SAPKγ, JNK2/SAPKα, and JNK3/SAPKβ. Contrarily, the signaling pathway of PI3K/ AKT has an antiapoptotic role through the phosphorylation of PI3K/AKT that conducts to the downstream activation of multiple signaling pathways related to growth, development, and

for the dismantling of the cell.

46 Leishmaniases as Re-emerging Diseases

**apoptosis**

macrophages [59–61], monocytes [64] and neutrophils [66].

of *Leishmania*, resulting in the inhibition of normal apoptosis.

*Leishmania* parasites have developed diverse strategies to evade the immune response: elicitation of a differential production of proinflammatory cytokines, upregulation of TLR2 and TLR4 expression, the nuclear translocation of NF-κB, and modulation of inflammasome. These parasites have a dual relation with host cells. Whereas host cells provide them with nutrients and a place to survive and replicate, these same cells exhibit an immune response aimed at the destruction of the parasites. Hence, the latter must display a wide array of strategies to overcome host cells defense mechanisms. One of the most successful strategies utilized by *Leishmania* inside host cells is the inhibition of apoptosis.

Apoptosis is a type of programmed cell death involving a precisely orchestrated series of steps that culminate in the orderly dismantling of the cell. This process encompasses the activation and silencing of a wide variety of signaling pathways, among which a leading role is played by MAPK, PI3K/Akt and proapoptotic/antiapoptotic proteins of the Bcl-2 family. *Leishmania* has the capacity to inhibit apoptosis of different cells, especially macrophages, monocytes, neutrophils, and dendritic cells. Although the precise mechanisms have not been fully elucidated, it is now clear that MAPK p38, JNK, ERK 1/2, and PI3K/Akt participate in the inhibition of apoptosis in macrophages, neutrophils, and dendritic cells. The inhibition of apoptosis is a key strategy for the survival and replication of *Leishmania* in host cells and may have implications for its pathogenesis because of favoring the invasion of the host and the persistence of the parasite in host cells. Further research is needed on the mechanisms of activation and regulation of the inflammasome pathway to provide insights into the pathophysiology of chronic diseases and reveal new therapeutic targets.

## **Acknowledgements**

The authors are especially grateful to Bruce Allan Larsen for proofreading the manuscript.

## **Financial support**

This work was supported by grant IN220816 from DGAPA-PAPIIT to MMAG.

## **Author details**

M. Magdalena Aguirre-Garcia\*, Alma R. Escalona-Montaño, Arturo A. Wilkins-Rodríguez and Laila Gutiérrez-Kobeh

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**Section 4**

**Vaccine Candidates: Past, Present and Future**

**Vaccine Candidates: Past, Present and Future**

**Chapter 5**

**Provisional chapter**

**Vaccines for Human Leishmaniasis: Where Do We**

**Vaccines for Human Leishmaniasis: Where Do We**

DOI: 10.5772/intechopen.75000

Responsible for up to 30,000 deaths annually, leishmaniasis is a complex spectrum of diseases endemic in 97 countries around the globe. Disease control relies heavily on the early diagnosis and treatment of the active cases (relevant for anthroponotic disease), although it is widely accepted that a prophylactic vaccine for human leishmaniasis is the way to achieve the successful elimination of human disease (taking in consideration the vast list of non-human reservoirs that enable the perpetuation of parasites all around the globe). The notion that infection leads to strong and long-lasting immunity against leishmaniasis supports vaccination as an achievable goal. However, and in spite of the different candidates tested along the years, till date, we still do not have an approved vaccine for humans. In this chapter, we will explore the last advances made in the field of vaccines against *Leishmania* without forgetting the historical perspective, essential to the understanding of the road already undergone. We will then discuss the correlates of disease and protection, still neither consensual nor definitive, as well as the issue of pre-clinical to clinical translation. The complete understanding of these issues will be

essential for the approval of a successful vaccine for human leishmaniasis.

**Keywords:** leishmaniasis, human vaccines, correlates of protection, cellular immunity,

Vaccination is undoubtedly one of the greatest achievements of modern medicine, responsible, together with the use of antimicrobials and access to clean water and sanitation, for the global human demographics transformation in the past two centuries [1–3]. The apparently

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Stand and What Is Still Missing?**

**Stand and What Is Still Missing?**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Pedro Cecílio, Fabiano Oliveira and

Pedro Cecílio, Fabiano Oliveira and

http://dx.doi.org/10.5772/intechopen.75000

Anabela Cordeiro da Silva

Anabela Cordeiro da Silva

**Abstract**

cross-protection

**1. Introduction**

#### **Vaccines for Human Leishmaniasis: Where Do We Stand and What Is Still Missing? Vaccines for Human Leishmaniasis: Where Do We Stand and What Is Still Missing?**

DOI: 10.5772/intechopen.75000

Pedro Cecílio, Fabiano Oliveira and Anabela Cordeiro da Silva Pedro Cecílio, Fabiano Oliveira and Anabela Cordeiro da Silva

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75000

#### **Abstract**

Responsible for up to 30,000 deaths annually, leishmaniasis is a complex spectrum of diseases endemic in 97 countries around the globe. Disease control relies heavily on the early diagnosis and treatment of the active cases (relevant for anthroponotic disease), although it is widely accepted that a prophylactic vaccine for human leishmaniasis is the way to achieve the successful elimination of human disease (taking in consideration the vast list of non-human reservoirs that enable the perpetuation of parasites all around the globe). The notion that infection leads to strong and long-lasting immunity against leishmaniasis supports vaccination as an achievable goal. However, and in spite of the different candidates tested along the years, till date, we still do not have an approved vaccine for humans. In this chapter, we will explore the last advances made in the field of vaccines against *Leishmania* without forgetting the historical perspective, essential to the understanding of the road already undergone. We will then discuss the correlates of disease and protection, still neither consensual nor definitive, as well as the issue of pre-clinical to clinical translation. The complete understanding of these issues will be essential for the approval of a successful vaccine for human leishmaniasis.

**Keywords:** leishmaniasis, human vaccines, correlates of protection, cellular immunity, cross-protection

#### **1. Introduction**

Vaccination is undoubtedly one of the greatest achievements of modern medicine, responsible, together with the use of antimicrobials and access to clean water and sanitation, for the global human demographics transformation in the past two centuries [1–3]. The apparently

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

insignificant proportion of the world population, whose lives are spared annually, thanks to vaccines (0.04 or 0.1%, if we include deaths avoided by smallpox eradication), is equivalent to up to 3 (or 8) million lives spared per year and a cumulative of more than half a billion deaths avoided just in the twentieth century [4, 5]. Nevertheless, and notwithstanding the significant and successful global efforts toward the goal of universal health protection/promotion, the picture could be much better. On the one hand, just by improving global vaccination coverage, an additional 1.5 million deaths could be avoided yearly [5]. On the other hand, there are still many deadly infectious diseases, whose prevention through vaccination is theoretically possible but for which there are no vaccines approved [6–8]. There are different compatible explanations/hypothesis that together justify it. The first one has to do with legal and ethical reasons: to test/approve/administer a pharmaceutical product nowadays is harder than it was 100 years ago [9]. Also in a chronologic point of view, it is not surprising that there are still no vaccines available for emerging diseases (e.g., Zika or MERS-CoV) [10, 11]. Other reasons have to do directly with the convergence of the nature of the pathogens with the evolution of vaccine technologies [12]: (i) almost all vaccines available till date are humoral based, which is not the best option against intracellular pathogens (e.g., *Leishmania* spp., *Trypanosoma cruzi*) [13, 14] and (ii) there are pathogens with immune-evasion strategies dependent on high antigenic variability that poses a challenge in vaccine development [6, 15]. Lastly, but not least important, there are diseases more relevant from an economic standpoint than others: many diseases for which there are still no vaccines available affect almost exclusively the poorest of the poor (neglected tropical diseases—NTDs) [8, 16, 17].

With this chapter, we propose to explore the broad anti-*Leishmania* vaccines field, with humans as the focus population. Starting from a historical perspective, we will clarify where we stand today by discussing the different candidates and approaches followed along the years, situating them in the vaccine development pipeline. Additionally, we will debate what is missing (focusing mainly, but not only, on the correlates of protection and the disease models) as a way to substantiate why currently there are no vaccines against leishmaniasis approved for humans.

Vaccines for Human Leishmaniasis: Where Do We Stand and What Is Still Missing?

http://dx.doi.org/10.5772/intechopen.75000

61

**2. Vaccines for human leishmaniasis: where do we stand?**

**2.1. Leishmanization as the proof of principle of vaccines against leishmaniasis**

The close relation of the human host and *Leishmania* parasites is quite ancient: there is evidence of parasite genetic material (identified retrospectively) in mummies from the year 2000 B.C. [23, 27]. However, the major breakthroughs in the leishmaniasis field were only achieved starting from the beginning of the twentieth century, with the identification of the causative agent(s), the incrimination of the vector(s), and consequently the understanding of parasite(s) life cycle and the distinct physiopathologic mechanisms that characterize each of the leishmaniasis forms [23, 28]. The definitive allocation of leishmaniasis within the infectious (or communicable) diseases, in convergence with the "success of variolation" and the birth of vaccination [29], boosted the investigation of the anti-*Leishmania* immune response envisioning the development of an effective prophylactic approach. The first reports date from early 1900 and are based on either contemporary common "medical practices" from Old World Cutaneous Leishmaniasis (CL) endemic countries or directly on evidence produced in human clinical trial-like studies [30, 31]. The general conclusions of these pioneer "vaccine studies" that used as inoculum either material from CL patient's ulcers or live parasites collected from *in vitro* cultures (*L. tropica*) were (i) only the individuals that developed a lesion and then selfhealed were resistant to reinfection and (ii) reinoculation of immune individuals led to what is nowadays known as Type I delayed type hypersensitivity (DTH) reaction [30, 31]. Such studies established the dogma accepted today by the scientific community—"previous infection leads to robust immunity against *Leishmania*"—and were the proof of principle of the only prophylactic approach clinically used against leishmaniasis known as leishmanization. Leishmanization was no more than the controlled induction of the cutaneous disease to prevent the consequences of natural infection, such as the scarification of exposed body parts (particularly the face) and the consequent life-long psychosocial impact and simultaneously to decrease the disease incidence in hyperendemic areas [32, 33]. In the 1970s and 1980s, several trials were performed using live virulent *L. major* parasites with promising results (up to 80% efficacy, **Table 1**) [32, 34–36]. This vaccine approach was accepted in countries such as the former Soviet Union, Iran, Israel, and Uzbekistan [32, 36, 37]. However, it was generally abandoned (with the exception of Uzbekistan, where it is still a licensed approach according to the most recent reviews on the field [34, 36, 38]) due to a number of concerns such as: (i) some individuals (1–2/10,000 inoculations) developed non-healing lesions, hard to resolve with chemotherapy [32, 39]; (ii) live vaccines (even the attenuated) are contraindicated to immuno-suppressed individuals [40] (whose worldwide prevalence has increased in the modern days, due not only to the HIV pandemic but also, for instance, to the increase of organ transplantation

Fortunately, with the arrival of the new millennium, WHO/UN initiatives such as the Millennium Development Goals (Goal 6, Target 3) and more recently the Sustainable Development Goals (Goal 3, Target 3.3) contributed to an increase in the awareness on the NTDs and consequently the investment on strategies to control them [18, 19]. The best example of concrete measures undertaken to "end the neglect" is given by the London Declaration on NTDs, signed in 2012 by 20 parties (including governmental organizations, non-profits and pharmaceutical companies) and endorsed thereafter by many others, that proposes to meet the goals set by the WHO Roadmap to overcome the global burden of NTDs (2012– 2020), that include the elimination of five diseases and the control of five others. One of the potential short-term controllable NTDs is the fatal form of leishmaniasis [20].

Endemic in 97 countries around the globe, leishmaniasis is a complex spectrum of diseases [21, 22]. The first layer of complexity is given by its vector-borne nature, which introduces an extra variable (the phlebotomine vector) to the binomium host pathogen. The second one is given by the 20 *Leishmania* species known to cause human diseases (usually in a species/ disease-manifestation-specific fashion), which is mostly but not exclusively of zoonotic origin (there are no animal reservoirs recognized for *L. donovani*) [23]. The third one relates to the infectious process, which frequently does not lead to an overt disease but instead to a chronic and "benign" asymptomatic state [24]. These are some of the main challenges to consider within the topic of disease control, which relies heavily on the early diagnosis and treatment of the active cases (whose influence in the diminishment of disease incidence should be relevant in anthroponotic *versus* zoonotic leishmaniasis) [25, 26]. Although till date there is no vaccine available against human leishmaniasis, not only is it widely accepted that the development of an effective vaccine is possible but also it is recognized that vaccination is the only viable option to achieve zoonotic disease elimination [25].

With this chapter, we propose to explore the broad anti-*Leishmania* vaccines field, with humans as the focus population. Starting from a historical perspective, we will clarify where we stand today by discussing the different candidates and approaches followed along the years, situating them in the vaccine development pipeline. Additionally, we will debate what is missing (focusing mainly, but not only, on the correlates of protection and the disease models) as a way to substantiate why currently there are no vaccines against leishmaniasis approved for humans.

## **2. Vaccines for human leishmaniasis: where do we stand?**

insignificant proportion of the world population, whose lives are spared annually, thanks to vaccines (0.04 or 0.1%, if we include deaths avoided by smallpox eradication), is equivalent to up to 3 (or 8) million lives spared per year and a cumulative of more than half a billion deaths avoided just in the twentieth century [4, 5]. Nevertheless, and notwithstanding the significant and successful global efforts toward the goal of universal health protection/promotion, the picture could be much better. On the one hand, just by improving global vaccination coverage, an additional 1.5 million deaths could be avoided yearly [5]. On the other hand, there are still many deadly infectious diseases, whose prevention through vaccination is theoretically possible but for which there are no vaccines approved [6–8]. There are different compatible explanations/hypothesis that together justify it. The first one has to do with legal and ethical reasons: to test/approve/administer a pharmaceutical product nowadays is harder than it was 100 years ago [9]. Also in a chronologic point of view, it is not surprising that there are still no vaccines available for emerging diseases (e.g., Zika or MERS-CoV) [10, 11]. Other reasons have to do directly with the convergence of the nature of the pathogens with the evolution of vaccine technologies [12]: (i) almost all vaccines available till date are humoral based, which is not the best option against intracellular pathogens (e.g., *Leishmania* spp., *Trypanosoma cruzi*) [13, 14] and (ii) there are pathogens with immune-evasion strategies dependent on high antigenic variability that poses a challenge in vaccine development [6, 15]. Lastly, but not least important, there are diseases more relevant from an economic standpoint than others: many diseases for which there are still no vaccines available affect almost exclusively the poorest of

Fortunately, with the arrival of the new millennium, WHO/UN initiatives such as the Millennium Development Goals (Goal 6, Target 3) and more recently the Sustainable Development Goals (Goal 3, Target 3.3) contributed to an increase in the awareness on the NTDs and consequently the investment on strategies to control them [18, 19]. The best example of concrete measures undertaken to "end the neglect" is given by the London Declaration on NTDs, signed in 2012 by 20 parties (including governmental organizations, non-profits and pharmaceutical companies) and endorsed thereafter by many others, that proposes to meet the goals set by the WHO Roadmap to overcome the global burden of NTDs (2012– 2020), that include the elimination of five diseases and the control of five others. One of the

Endemic in 97 countries around the globe, leishmaniasis is a complex spectrum of diseases [21, 22]. The first layer of complexity is given by its vector-borne nature, which introduces an extra variable (the phlebotomine vector) to the binomium host pathogen. The second one is given by the 20 *Leishmania* species known to cause human diseases (usually in a species/ disease-manifestation-specific fashion), which is mostly but not exclusively of zoonotic origin (there are no animal reservoirs recognized for *L. donovani*) [23]. The third one relates to the infectious process, which frequently does not lead to an overt disease but instead to a chronic and "benign" asymptomatic state [24]. These are some of the main challenges to consider within the topic of disease control, which relies heavily on the early diagnosis and treatment of the active cases (whose influence in the diminishment of disease incidence should be relevant in anthroponotic *versus* zoonotic leishmaniasis) [25, 26]. Although till date there is no vaccine available against human leishmaniasis, not only is it widely accepted that the development of an effective vaccine is possible but also it is recognized that vaccination is the only

potential short-term controllable NTDs is the fatal form of leishmaniasis [20].

the poor (neglected tropical diseases—NTDs) [8, 16, 17].

60 Leishmaniases as Re-emerging Diseases

viable option to achieve zoonotic disease elimination [25].

#### **2.1. Leishmanization as the proof of principle of vaccines against leishmaniasis**

The close relation of the human host and *Leishmania* parasites is quite ancient: there is evidence of parasite genetic material (identified retrospectively) in mummies from the year 2000 B.C. [23, 27]. However, the major breakthroughs in the leishmaniasis field were only achieved starting from the beginning of the twentieth century, with the identification of the causative agent(s), the incrimination of the vector(s), and consequently the understanding of parasite(s) life cycle and the distinct physiopathologic mechanisms that characterize each of the leishmaniasis forms [23, 28]. The definitive allocation of leishmaniasis within the infectious (or communicable) diseases, in convergence with the "success of variolation" and the birth of vaccination [29], boosted the investigation of the anti-*Leishmania* immune response envisioning the development of an effective prophylactic approach. The first reports date from early 1900 and are based on either contemporary common "medical practices" from Old World Cutaneous Leishmaniasis (CL) endemic countries or directly on evidence produced in human clinical trial-like studies [30, 31]. The general conclusions of these pioneer "vaccine studies" that used as inoculum either material from CL patient's ulcers or live parasites collected from *in vitro* cultures (*L. tropica*) were (i) only the individuals that developed a lesion and then selfhealed were resistant to reinfection and (ii) reinoculation of immune individuals led to what is nowadays known as Type I delayed type hypersensitivity (DTH) reaction [30, 31]. Such studies established the dogma accepted today by the scientific community—"previous infection leads to robust immunity against *Leishmania*"—and were the proof of principle of the only prophylactic approach clinically used against leishmaniasis known as leishmanization.

Leishmanization was no more than the controlled induction of the cutaneous disease to prevent the consequences of natural infection, such as the scarification of exposed body parts (particularly the face) and the consequent life-long psychosocial impact and simultaneously to decrease the disease incidence in hyperendemic areas [32, 33]. In the 1970s and 1980s, several trials were performed using live virulent *L. major* parasites with promising results (up to 80% efficacy, **Table 1**) [32, 34–36]. This vaccine approach was accepted in countries such as the former Soviet Union, Iran, Israel, and Uzbekistan [32, 36, 37]. However, it was generally abandoned (with the exception of Uzbekistan, where it is still a licensed approach according to the most recent reviews on the field [34, 36, 38]) due to a number of concerns such as: (i) some individuals (1–2/10,000 inoculations) developed non-healing lesions, hard to resolve with chemotherapy [32, 39]; (ii) live vaccines (even the attenuated) are contraindicated to immuno-suppressed individuals [40] (whose worldwide prevalence has increased in the modern days, due not only to the HIV pandemic but also, for instance, to the increase of organ transplantation procedures [41]); (iii) batch-to-batch variability issues of such complex immunogens raise reproducibility concerns [36, 42]; and (iv) complex logistics are usually associated with live vaccines [42].

## **2.2. An overview of the vaccine candidates against human leishmaniasis explored since leishmanization until the present day**

The knowledge produced by leishmanization trials and campaigns conducted at the end of the last century is the most important evidence that the development of a vaccine against leishmaniasis is quite far from being impossible. The quest for such an essential pharmaceutical, indispensable for the achievement of global disease control, has been continuous (in a scale proportional to the funding for NTD research) and fruitful if we consider the number of candidates and different approaches tested. Here we will separate them into five major groups: live vaccines ("leishmanization like"), first-, second-, and third-generation vaccines, and vector-derived vaccines. **Table 1** compiles the information to be discussed in the next subheadings, presenting not only the different candidates/approaches tested along the years but also the disease form they were destined to prevent, their placement in the vaccine development pipeline, and the main findings reported.

#### *2.2.1. Live vaccine candidates*

The success of leishmanization is still used to support the investigation of vaccine approaches based on live parasites (called by some as leishmanization revisitation [38]), that according to the authors have the advantage of at least partially reproducing the normal infectious process (and consequently induce a "close-to-natural" anti-*Leishmania* memory) [43]. This includes for some candidates the long-term parasite persistence in the site of inoculation that will continuously boost the immune system and prevent the loss of immunity to reinfection [44–46]. The first two approaches explored, relying on parasite persistence as the key to effectiveness, are readaptations of leishmanization directed toward the prevention of visceral disease and proposed the controlled infection with either virulent *L. major* parasites or with a virulent but dermotropic *L. donovani* strain to promote heterologous or homologous protection against visceral disease caused by viscerotropic *L. infantum* or *L. donovani* strains, respectively [38, 47–49]. Still, both approaches, although shown effective in the pre-clinical context, will unlikely proceed in the vaccine development pipeline, mainly due to the safety concerns always raised by the use of virulent pathogens. As a way to partially overcome this barrier, different live vaccine approaches proposed the use of attenuated parasites, that would still mimic the natural infection (although in a sub-clinical form) and induce anti-*Leishmania* memory but in most of the cases would then be completely eliminated. In the pre-genomic era (but not only) chemically and physically attenuated parasites were shown to be effective, in pre-clinical trials, against CL, muco-cutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL) [50–52]. These attenuation approaches that did not assure a homogeneous parasite population (with an unpredictable potential of reversion to the virulent form) were almost completely replaced by the genetically modified parasites in the post-genomic era. Two main groups of genetically modified *Leishmania* parasites were used in the pre-clinical context: loss-of-function mutants

**Type**

**Approach/vaccine candidate(s)**

Inoculation of live, virulent *L. major* parasites:

Leishmanization

**Disease**

**Vaccine**

**Efficacy/outcome**

**Reference**

**form**

**development**

**pipeline**

CL

Effective clinical

About 80% efficacy

[31, 34, 36]

use in the former

Soviet Union, Israel

and Middle East\*

(discontinued)

Heterologous protection mediated by inoculation of

VL

Pre-clinical studies

No effect in BALB/c mice; protection in

[46–48]

in mice

C57Bl/6 mice

live, virulent *L. major*

Inoculation of a dermotropic *L. donovani*

Physically attenuated parasites

Non-defined

Chemically attenuated parasites (N-nitrosamines/

composition; live,

antibiotic pressure)

attenuated and/

or drug-sensitive

parasites (through

culture, chemical,

Genetically attenuated parasites (**Lmaj**dhfr-ts,

CL/

Pre-clinical studies in

Homologous protection for *L. major*

[51–55]

in mice, but not monkeys; *L. mexicana*

in mice and hamsters; *L. infantum* in

Vaccines for Human Leishmaniasis: Where Do We Stand and What Is Still Missing?

mice and *L. donovani* in mice, hamsters

and dogs; heterologous protection for

*L. major* in mice, *L. braziliensis* in mice

and hamsters and *L. infantum* in dogs

(mediated by *L. donovani* KO parasites)

Homologous long-term protection

[50, 56, 57]

http://dx.doi.org/10.5772/intechopen.75000

(lesion free) in mice for *L. major*;

heterologous protection against *L.*

*donovani* mediated by *L. amazonensis*:

99% reduction in parasite loads and

suppression of disease

63

mice/hamsters/dogs/

MCL/

VL

macaques

**Lmex**CystProt, **Lmaj**LPG2, **Lmaj**PPM, **Ld**Cen1,

**Li**HSP70-II, **Ld**p27, **Ld**ALO and **Ld**BT1 null mutants;

Genetically modified parasites (gain of function)—

CL/VL

Pre-clinical studies in

mice and hamsters

suicide mutants: *L. major* tk-cd+/+ (susceptible to

Ganciclovir and 5-flurocytosine), *L. amazonensis*

alad-pbgd+/+ (used in the context of photodynamic

vaccination)

radiation or genetic

manipulation)

**Li**SIR2 sKO)

CL/

Pre-clinical studies in

Homologous protection for *L. major*, *L.*

[49–51]

*tropica*, *L. amazonensis*, *L. donovani* and

*L. braziliensis*; no effect for *L.*

Homologous protection for *L. major*

[51]

and *L. mexicana* in BALB/c mice;

promising results for *L. infantum* in

dogs

 *Infantum*

mice and hamsters

MCL/

VL

CL/

Pre-clinical studies in

MCL/

mice and dogs

VL

VL

Pre-clinical studies

Protection against challenge with

[37]

viscerotropic *L. donovani* in BALB/c

mice

in mice

Live vaccines

procedures [41]); (iii) batch-to-batch variability issues of such complex immunogens raise reproducibility concerns [36, 42]; and (iv) complex logistics are usually associated with live

The knowledge produced by leishmanization trials and campaigns conducted at the end of the last century is the most important evidence that the development of a vaccine against leishmaniasis is quite far from being impossible. The quest for such an essential pharmaceutical, indispensable for the achievement of global disease control, has been continuous (in a scale proportional to the funding for NTD research) and fruitful if we consider the number of candidates and different approaches tested. Here we will separate them into five major groups: live vaccines ("leishmanization like"), first-, second-, and third-generation vaccines, and vector-derived vaccines. **Table 1** compiles the information to be discussed in the next subheadings, presenting not only the different candidates/approaches tested along the years but also the disease form they were destined to prevent, their placement in the vaccine develop-

The success of leishmanization is still used to support the investigation of vaccine approaches based on live parasites (called by some as leishmanization revisitation [38]), that according to the authors have the advantage of at least partially reproducing the normal infectious process (and consequently induce a "close-to-natural" anti-*Leishmania* memory) [43]. This includes for some candidates the long-term parasite persistence in the site of inoculation that will continuously boost the immune system and prevent the loss of immunity to reinfection [44–46]. The first two approaches explored, relying on parasite persistence as the key to effectiveness, are readaptations of leishmanization directed toward the prevention of visceral disease and proposed the controlled infection with either virulent *L. major* parasites or with a virulent but dermotropic *L. donovani* strain to promote heterologous or homologous protection against visceral disease caused by viscerotropic *L. infantum* or *L. donovani* strains, respectively [38, 47–49]. Still, both approaches, although shown effective in the pre-clinical context, will unlikely proceed in the vaccine development pipeline, mainly due to the safety concerns always raised by the use of virulent pathogens. As a way to partially overcome this barrier, different live vaccine approaches proposed the use of attenuated parasites, that would still mimic the natural infection (although in a sub-clinical form) and induce anti-*Leishmania* memory but in most of the cases would then be completely eliminated. In the pre-genomic era (but not only) chemically and physically attenuated parasites were shown to be effective, in pre-clinical trials, against CL, muco-cutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL) [50–52]. These attenuation approaches that did not assure a homogeneous parasite population (with an unpredictable potential of reversion to the virulent form) were almost completely replaced by the genetically modified parasites in the post-genomic era. Two main groups of genetically modified *Leishmania* parasites were used in the pre-clinical context: loss-of-function mutants

**2.2. An overview of the vaccine candidates against human leishmaniasis explored**

vaccines [42].

62 Leishmaniases as Re-emerging Diseases

**since leishmanization until the present day**

ment pipeline, and the main findings reported.

*2.2.1. Live vaccine candidates*

**Approach/vaccine candidate(s)**

Fucose-Manose ligand adjuvanted with saponin

VL

Pre-clinical studies in

Protection in mice and hamsters

[66, 75]

challenged with *L. donovani*

(homologous); effective heterologous

protection (against *L.*

in dogs; transmission blocking

potential; commercialized as a

canine anti-*Leishmania* vaccine

with the name Leishmune in Brazil

(commercialization license suspended

in 2014)

Significant, long-lasting protection

[54, 66, 76,

77]

against canine VL in a field trial in an

endemic area (**Li**); promising results

in terms of heterologous protection

against *Leishmania infantum* (**La**)**;**

commercialized as a canine anti-*Leishmania* vaccine with the name

CaniLeish in Europe (**Li**)

Promising results regarding

[50, 54, 71,

83–85]

homologous protection in mice;

dubious protection in monkeys against

*L. major* challenge (gp63)

Promising results in mice and

[36, 50, 54,

Vaccines for Human Leishmaniasis: Where Do We Stand and What Is Still Missing?

67, 84, 85,

87–90]

hamsters; a major limitation is that

most of the antigens were not tested

in superior models; positive response

*vivo* human studies for **Ld**elF-2;

partial homologous and heterologous

http://dx.doi.org/10.5772/intechopen.75000

(*L. infantum*) protection in dogs and

heterologous protection in mice

challenged with *L. infantum* and *L.*

*amazonensis* (**Ld**A2: licensed veterinary

product in Brazil—LeishTec)

65

in *ex*

*L. infantum* or *L. amazonensis* excreted-secreted antigens

VL

Pre-

clinical/"Clinical"

studies in dogs

adjuvanted with saponin

Second generation

Membrane proteins: native **Ld**Dp-72, gp63 and PSA-2

CL/VL

Pre-clinical studies

in mice, dogs and

macaques

and recombinant **Li**LCR1, **Ld**HASPB1, KMP-11 and

gp63; adjuvanted (BCG, CpG-ODN, MPL-SE, IL-12,

saponin, cationic nanoparticles, liposomes)

"Soluble proteins": recombinant **Ld**A2, **Li**PHB, **Ld**F14,

CL/

Pre-clinical studies in

mice, hamsters and

dogs; *ex vivo* human

studies

MCL/

VL

**Ld**p27, **Ld**pSP **Ld**P45, **Ld**PDI, **Ld**TPI, **Ld**TPR, **Li**P0,

**Lmaj**STM1, **Li**TDR-1, **Lb**Hyp, EiF5a, eIF-2, NH, CPA

and CPB, SMT, PEPCK, Histone H1, Heat shock

proteins (HSP), **Li**Ribosomal proteins, **Li**Hypothetical

amastigote-specific protein, cysteine proteinases,

LACK; adjuvanted (BCG, ALD, *P. acnes*, CpG-ODN,

MPL-SE, IL-12, saponin, cationic nanoparticles)

vaccines

*Infantum*)

mice and hamsters;

"clinical" studies in

dogs

**Disease**

**Vaccine**

**Efficacy/outcome**

**Reference**

**form**

**development**

**pipeline**

**Approach/vaccine candidate(s)**

Immunization with non-pathogenic *L. tarentolae* (wild

type or genetically modified strains producing LPG3,

**Ld**A2 or **Ld**A2/CPA/CPB)

ALM adjuvanted with BCG

Alum-ALM adjuvanted with BCG

Autoclaved *L. donovani*

Phenol or Heat inactivated *L. guyanensis*, *L. braziliensis*

CL/

Human clinical

52% Efficacy in endemic area

[65, 67]

(phenol inactivation); no protection

against *L. amazonensis* infection (heat

inactivation)

MCL

studies

and *L. amazonensis* adjuvanted with BCG

Merthiolate-killed *L. amazonensis* (with/without BCG)

Non-defined

Sonicated *L. donovani* (whole cell or soluble antigens)

VL

Pre-clinical studies

Good homologous protection in all

[36, 54, 71,

72]

species; liposomal formulation elicits

the best protection in mice

Significant levels of homologous

[36]

protection

in mice hamsters and

monkeys

adjuvanted (MPL-A, BCG, liposomes)

Liposomal *L. major* soluble antigen adjuvanted with

CL

Pre-clinical studies

in mice

CpG

composition; whole

killed parasites or

parasite fractions

CL

Pre-clinical and

Protection in mice not reproduced in

[65, 73, 74]

Human clinical

humans

studies

VL

Pre-clinical studies

in mice

CL/VL

Pre-clinical and

human clinical

studies

CL/VL

Pre-clinical and

Protection in macaques against *L.*

[54, 65, 67,

68]

*donovani* challenge; poor efficacy in

humans. Protection in mice against *L.*

*major* infection; clinical studies with

disappointing results

Immunogenic and safe in humans;

[54, 56, 69]

protective (single dose) in macaques

challenged with *L. donovani*; moderate

efficacy against canine visceral

leishmaniasis; protection in BALB/c

mice against challenge with *L. major*

Significant levels of homologous

[70]

protection

human clinical

studies

First generation

vaccines

**Disease**

**Vaccine**

**Efficacy/outcome**

**Reference**

**form**

**development**

**pipeline**

VL

Pre-clinical studies in

Promising results in mice and dogs

[50, 60, 61]

64 Leishmaniases as Re-emerging Diseases

mice and dogs

**Approach/vaccine candidate(s)**

Live recombinant bacterial vectors: *Lactococcus*

*lactis* expressing A2 and LACK+IL-12; recombinant

*S. typhimurium* vaccine strains expressing gp63,

LinJ08.1190 and LinJ23.0410; recombinant *L.*

*monocytogenes* (attenuated) expressing LACK

Th1 immunity inducing sand fly salivary proteins:

CL/

Pre-clinical studies in

Evidences or described effect in

[128–135]

protection from (natural) infection in

all animal models, except with PpSP-44 which leads to exacerbation of

cutaneous disease

86% reduction of sand fly-midgut

[136–137]

*L. major* infection; impairment of

metacyclogenesis

mice, hamsters, dogs

MCL/

VL

and macaques

recombinant or DNA encoding LJM-19 (SALO), PdSP-15, PpSP15 (also *L. tarentolae* based), PpSP-44, LJM-143, LJM-17, LJM-11 (also *L. monocytogenes* based); alone, or

in combination with common anti-*Leishmania* vaccine

Insect-based transmission blocking vaccine: anti *P.*

CL

*In vitro* and *in vivo*

insect studies

(artificial feeding)

A2, amastigote specific protein 2; ALM, autoclaved *L. major*; ALO, arabino-1,4-lactone oxidase; Cen, centrine; BCG, Bacillus Calmette–Guérin; CL, cutaneous Leishmaniasis;

CPA/B, cysteine peptidase A/B; CystProt, cysteine proteinase; dhfr-ts, dihydrofolate reductase-thymidylate synthase; elF, elongation factor; GCS, glutamylcysteine

synthetase; GLA, glucopyranosyl lipid A; gp, glycoprotein; HASP, hydrophilic acylated surface protein; HbR, hemoglobin receptor; HSP, heat shock protein; IL,

interleukin; KMP, kinetoplastid membrane protein; LACK, *Leishmania* homolog of receptors for activated c-kinase; Ld, *L. donovani*; Li, *L. infantum*; LJL, *Lutzomyia longipalpis*

Jacobina large; LJM, *Lutzomyia longipalpis* Jacobina medium; Lmaj, *L. major*; Lmex, *L. mexicana*; LPG, lipophosphoglycan; MIDGE, minimalistic immunogenically defined

Vaccines for Human Leishmaniasis: Where Do We Stand and What Is Still Missing?

http://dx.doi.org/10.5772/intechopen.75000

67

gene expression; MCL, mucocutaneous leishmaniasis; MPL, monophosphoryl lipid A; NH, nucleoside hydrolase; ODN, oligodeoxynucleotides; ORFF, open reading frame

fragment; P0, acidic ribosomal protein P0; PDI, protein disulphide-isomerase; PdSP, *Phlebotomus duboscqi* salivary protein; PEPCK, phosphoenolpyruvate carboxykinase;