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Immunity to visceral leishmaniasis: implications for immunotherapy

Forough Khadem1 & Jude E Uzonna*,2 ABSTRACT: Visceral leishmaniasis, caused by Leishmania donovani, L. infantum (syn. Leishmania chagasi), is a globally widespread disease with a burden of about 400,000 new infections reported annually. It is the most dangerous form of human leishmaniasis in terms of mortality and morbidity and is spreading to several nonendemic areas because of migration, global traveling and military conflicts. The emergence of Leishmania-HIV co-infection and increased prevalence of drug-resistant strains have worsened the impact of the disease. The traditional low-cost drugs are often toxic with several adverse effects, highlighting the need for development of new therapeutic and prophylactic strategies. Therefore, a detailed understanding of mechanisms of protective immunity is extremely important in order to develop new therapeutics in the form of vaccines or immunotherapies. This review gives an overview of visceral leishmaniasis, with particular emphasis on the innate and adaptive immune responses, vaccine and vaccination strategies and their potentials for immunotherapy against the disease. Leishmaniasis is a disease caused by several members of the genus Leishmania, which are intracellular protozoan parasites. The disease is endemic in 98 countries and three territories [1] , and is mostly associated with social imbalance related to poverty including malnutrition, population displacement, poor housing and lack of social resources [2] . Leishmaniasis is mainly manifested as one of the three forms: cutaneous leishmaniasis (CL), which is the most common form; visceral leishmaniasis (VL), which is the most serious form or mucocutaneous leishmaniasis [3] . CL is mainly caused by Leishmania major, L. tropica and L. aethiopica in the Old World and by L. mexicana in the New World. Leishmania amazonensis and L. braziliensis are the primary causative agents of mucocutaneous leishmaniasis in the new world. VL is caused by L. donovani, L. infantum (syn. L. chagasi) in the Old World and by L. chagasi in the New World [4] . The prevalence of leishmaniasis is dependent on several risk factors including man-made factors (such as migration, deforestation and urbanization), changes in the human host’s susceptibility to infection (immunosuppression and malnutrition) and natural environmental changes [5] . Due to relatively incomplete epidemiological data, official figures are likely to underestimate the real prevalence of the disease. According to WHO, it is estimated that 1.3 million new cases of Leishmaniasis occur annually leading to 20,000–30,000 deaths [2] . The disease is rapidly spreading to nonendemic areas of the world owing to increased international travel, globalization, military conflicts (soldiers returning from duties in the endemic areas) [6] , lack of effective vaccines [7] and difficulties in controlling the vectors [8] . In addition, Leishmania-HIV co-infection [9] and drug resistance [10] are

KEYWORDS 

• adaptive immunity • chemotherapy • immunotherapy • innate immunity • leishmaniasis • vaccines • visceral

leishmaniasis

Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada Parasite Vaccines Development Laboratory, Departments of Immunology & Medical Microbiology, University of Manitoba, 750 McDermot Avenue, Winnipeg, Manitoba, R3E 0T5, Canada *Author for correspondence: [email protected] 1 2

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Review  Khadem & Uzonna increasingly emerging and complicate an already difficult problem. Over 90% of cases of VL occur in seven countries: Bangladesh, Brazil, Ethiopia, India, Nepal, South Sudan, and Sudan. VL is highly endemic in the Indian subcontinent and in East Africa and about 200,000–400,000 new cases of VL occur worldwide annually [2] . L. donovani is the primary cause of VL in India, Pakistan, China and Africa, while L. infantum is the causative agent in the Mediterranean region and L. chagasi in the new world [3] . There is however a controversy as to whether L. infantum and L. chagasi are the same [11] or different species. VL has a very variable incubation period that ranges from 6 weeks to 9 years. If not treated, death may occur within few weeks to 2–3 years depending on whether the disease is acute, subacute or chronic [12] . The symptoms of VL comprise headache, fever, chills, sweating, cough, diarrhea, dizziness, vomiting, bleeding of gums, pains in the limbs, weight loss, enlargement of spleen (splenomegaly) and liver (hepatomegaly), anemia with leukopenia and lymphadenopathy [13] . The skin may become discolored, particularly dark gray, hence the name Kala-Azar or black sickness in India [14] . In some cases, VL mimicking or exacerbating various autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, autoimmune hepatitis and antimitochondrial antibody positive primary b­iliary cirrhosis have been reported [15] . Because VL is a systemic infection and affects most of the host’s internal organs, particularly the spleen, liver and bone marrow [16], latent cases may remain undiagnosed for years to decades until the individual becomes immunocompromised. Since the parasites invade macrophages [17] , which constitute a major part of the host immune defense mechanisms, VL patients are invariably immunosuppressed and thus easily fall prey to secondary opportunistic infections [18] . Heterogeneous complications associated with immunosuppression including transplantation, rheumatology, hematology and oncology disorders have emerged to contribute to VL immunopathology [19] . In addition, VL-HIV co-infections are a major health issue where VL and HIV are both endemic [9] . Furthermore, both HIV and non-HIV cases of immunosuppression pose significant diagnostic and therapeutic challenges for VL. Treatment of VL Parenteral antimonials are still the major drugs of choice for treatment of leishmaniasis,

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including visceral disease. Treatment is highly recommended in VL because untreated disease ultimately leads to death. Sodium stibogluconate [20] and meglumine antimoniate administered as 20 mg/kg daily for 28 days has been shown to be effective in treating VL [21] . Amphotericin B (AB; 1 mg/kg alternate days for 30 days) [22] , liposomal AB (LaB; 2 mg/kg for 5 days) [23] , miltefosine (150 mg/day for 28 days) [22] , and paromomycin (aminosidine, 11–20 mg/kg/day for 28 days) [24] have all been effectively used in various parts of the world including South Sudan and Europe [21] , East Africa [25] , USA [20] , Brazil [26] and India [22] . Several potentially new compounds for treatment of VL are being developed and these have previously been extensively reviewed [27] . In order to increase site-specific drug delivery and reduce side effects, several new delivery systems including liposomes, emulsomes, niosomes, polymeric particles, conjugates and plant products such as alkaloids, terpenes, phenolics and chalcones are being developed and their potential applications in VL treatment have been reviewed previously [28] . Combination therapies such as the administration of fluconazole, miltefosine and picroliv [29] have been examined and proven to be useful in order to reduce treatment costs, increase treatment efficacy and tolerance, reduce treatment duration, reduce burden on the health system and limit the emergence of drug resistance [30] . Most drugs used for treatment of VL are associated with undesirable side effects such as gastrointestinal symptoms, ototoxicity and hepatotoxicity [21] as well as variable cure rates, which necessitate an urgent need for d­evelopment of novel treatment options. Host immune response to VL The protective immune response to Leishmania infection is cell-mediated immunity. Th1 response correlates with resistance whereas Th2 response is associated with susceptibility to infection [31] . Several factors influence resistance or susceptibility to leishmaniasis including parasite strains, host genetics and environmental factors [16] . In general, the host determined delayed-type hypersensitivity (DTH), antigenspecific T-cell reactivity and cytokine secretion, parasite species and virulence of the infecting Leishmania species determine the ultimate clinical presentation of the disease. An overview of the host immune response to Leishmania is ­presented in Figure 1.

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Immunity to visceral leishmaniasis: implications for immunotherapy 

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Parasite lysis Resistance

IFN-γ

Complement NK MQ

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Leishmania infected sand fly

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Figure 1. Innate and adaptive immune mechanisms underlying resistance/susceptibility to leishmaniasis. During a blood meal, an infected sand fly deposits saliva-containing metacyclic promastigotes into the dermis of a mammalian host. In addition to containing anticoagulant properties, some salivary proteins may initiate local inflammatory responses that could either enhance parasite survival or enhance host immune response. The majority of parasites are lysed by the membrane attack complex of the complement system. The remaining parasites are phagocytosed by MQ where they transform into amastigotes. In addition to MQ, some parasites may be taken up by neutrophils, DC and mast cells. Infection of neutrophils may facilitate escape from complement-mediated lysis and serve as a means (Trojan horses) for delivering parasites to MQ. In resistant animals, infected DCs produce IL-12 that primes CD4+ T cells toward Th1 response and IFN-γ production leading to activation of infected MQs, the production of NO and ROI and ultimately parasite destruction and resistance. IL-12 may also prime NK and NK T cells to produce IFN-γ that further promote CD4+ Th1 and cytotoxic CD8+ T-cell responses. The pathways leading to induction of CD4+ Th2 cells that results in susceptibility are not very well defined although IL-4 produced by non-T cells (including mast cells) are thought to contribute to this process. Once CD4+ Th2 cells are induced, their cytokines (including IL-4, IL-5, IL-10 and IL-13) deactivate infected MQ and downregulate NO and ROI production leading to unregulated parasite replication and persistence. In addition, IL-10 and TGF-β produced by CD4+CD25+Foxp3+ Tregs contribute to the downregulation of parasticidal activities of infected MQ. Furthermore, during infection, Leishmania-specific B cells are also activated to produce anti-Leishmania antibodies. The uptake of antibody-coated parasites by MQ enhances their IL-10 and TGF-β production, which act in an autocrine manner to further downregulate parasite killing. DC: Dendritic cell; MQ: Macrophage; NK: Natural killer; NO: Nitric oxide; ROI: Reactive oxygen intermediate. Data taken from [16,32–39]. 

Innate immune response Following the deposition of infective metacyclic promastigotes into the dermis, the skin innate immune system detects invading promastigotes,

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recruits inflammatory cells to sites of invasion within minutes and promotes the induction of adaptive immunity [40] . Initial sensing of the parasite involves pattern recognition receptors

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Review  Khadem & Uzonna and complement receptors present on different cell types including neutrophils, macrophages, dendritic cells (DCs) and natural killer (NK) cells. Several Toll-like receptors (TLRs) such as TLR2, TLR3 [41] , TLR4 [42] , TLR7 [43] and TLR9 [42] have been shown to contribute to innate sensing and recognition of Leishmania by various innate immune cells. This recognition leads to activation of intracellular signaling pathways that are necessary for the initiation of inflammatory responses and control of parasite proliferation by the innate immune response [44] . Neutrophils are essential cells involved in inflammatory response and contribute to phagocytosis and killing of microbial pathogens. However, the precise role of these cells in VL remains to be addressed. It has been previously shown that the turnover and activity of granulocytes including neutrophil and eosinophil are increased in human cases of VL [45] . Despite their efficient leishmaniacidal machinery, L. donovani has developed some strategies to transiently survive inside neutrophils. For instance, L. donovani promastigotes inhibit activation of oxidative burst, avoid being targeted to lytic compartments such as lysosome via their surface lipophosphoglycans (LPG) and induce rapid release of neutrophil extracellular traps, which leads to containment of parasite at the site of inoculation thereby facilitating their uptake by mononuclear phagocytes [46] . Neutrophils also play an important role in early control of parasite growth in the spleen but not in the liver. Neutrophil depletion at the beginning of L. donovani infection leads to increase in parasite burden in the spleen and bone marrow but not in the liver, enhanced splenomegaly, a delay in the maturation of hepatic granulomas and a decrease in inducible nitric oxide synthase (iNOS) expression within granulomas [47] . It appears that the absence of neutrophils provided a perfect milieu for the development of nonprotective Th2 responses as evident by a dramatic increase in serum levels of IL-4 and IL-10 and a significant increase in the ratio of L. donovani-specific serum IgG1/IgG2a levels [47] . The importance of neutrophils in resistance to VL is also supported by observations in dogs, which suggests that there might be neutrophil dysfunction depending on the different disease stages. In moderate stage of canine VL, superoxide production is decreased without any alteration in neutrophil viability. In contrast, dogs in very severe stage of the disease show increased neutrophil apoptosis

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and decreased superoxide production, which was associated with uremia [48] . The early interaction of Leishmania with macrophages and DCs and its influence on the host immune response have been recently reviewed [40] . Therefore, only very salient points will be emphasized in the present review, with special emphasis on aspects that could influence immunotherapy. Both macrophages and DCs phagocytose Leishmania and contribute to the initial decision processes that regulate resistance and/or susceptibility. For example, infected DCs produce IL-12, which is critical for the development of IFN-γ-producing CD4 + Th1 cells. Failure to produce functional IL-12 by DCs leads to progressive expansion of Th2 cells, upregulation of arginase activity in macrophages and parasite proliferation. In contrast to DCs, Leishmania-infected macrophages are unable to produce IL-12 and are therefore believed to play no significant role in the initiation of protective adaptive immunity [49] . Although promastigotes are capable of directly invading DCs and macrophages following their deposition by infected sandflies, several TLRs have been shown to contribute to this process and play a vital role in the production of proinflammatory cytokines that are critical for immunity [31] . In particular, TLR2 and TLR3 participate in the phagocytosis of L. donovani promastigotes by macrophages [41] . Interestingly, during L. donovani infection, the parasite modulates TLR2 expression and signaling to suppress IL-12 production [50] . TLR3 is involved in the leishmaniacidal activity of IFN-γ-primed macrophages [41] . A recent report showed that TLR7-mediated activation of interferon regulatory factor 5 is essential for the development of Th1 responses to L. donovani in the spleen during chronic infection [43] . Interferon regulatory factor 5 also indirectly regulates iNOS e­xpression in infected cells [43] . In addition to the TLRs, complement receptors also contribute to detection of Leishmania infection and the initiation of inflammatory responses. Following injection of promastigotes into the skin, both the classical and alternative pathways of the complement system are activated [51] and within a few minutes after serum contact, more than 90% of all inoculated parasites are lysed via the membrane attack complex [32] . Interestingly, certain species of the parasite have evolved to exploit this pathway to enhance their infectivity and survival in the host. For example,

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Immunity to visceral leishmaniasis: implications for immunotherapy  metacyclic promastigotes of L. infantum chagasi enter macrophages via the CR3 and this t­ransiently subverts macrophage activities [40] . During the initial uptake and phagocytosis of Leishmania promastigotes, macrophage produces superoxide anions. Following macrophage activation by IFN-γ, the second and most important anti-leishmania event occurs via nitric oxide (NO) generation. Failure to activate macrophages, resulting from the absence of a proinflammatory response and NO production, leads to invasion of L. donovani promastigotes within the mammalian host and their successful establishment [52] . Infection with Leishmania also stimulates the production of IFN-γ from NK cells via IL-2-dependent and -independent pathways. Since DCs (and not macrophages) are the critical source of early IL-12 production following Leishmania infection, DC–T-cell inter­action is thought to provide the ­microenvironment for initial NK-cell activation [49] . Adaptive immune response The activation of the adaptive immune system during active VL in humans leads to the production of both macrophage-activating (IFN-γ and TNF-α) and deactivating (IL-10 and TGF-β) cytokines mainly in the spleen and liver [53] . The balance between these activating and deactivating factors ultimately detects the outcome of infection, pathology and the ensuing disease. Unlike murine models of most Leishmania infections, there is no clear dichotomy in Th1 and Th2 cytokines in human VL and their impacts on resistance and susceptibility is not clearly defined. However, patients with active VL have higher plasma levels of IL-10, IL-4, IFNγ, TNF-α and IL-12 relative to asymptomatic and cured subjects [54] . L. donovani derivedexosomes have been recently implicated in this immunomodulatory process by promoting IL-10 production and inhibiting TNF-α production by human monocytes [55] . Experimental studies in mice suggest that the control of VL may be associated with the development of parasite-specific cell-mediated immune responses involving both CD4 + and CD8 + T cells [53] . These cells produce IFN-γ, which activates infected macrophages leading to the production of NO and other free radicals that kill the parasites. DCs activate CD8 + T cells through mechanisms that involve antigen cross presentation [56] . In contrast, susceptibility to L. infantum or L. donovani in infected

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mice is associated with the production of high amounts of IL-10, which is a potent immunosuppressive and anti-inflammatory cytokine [57] . Both conventional CD4 + T cells and Tregs have been shown to contribute to IL-10 secretion and immunosuppression in VL [58] . In addition to IL-10, the production of TGF-β1 by Tregs also contributes to disease pathogenesis and susceptibility [59] . In VL, there is a fine line between immune responses that effectively control parasite growth and induce long-term immunity and those that allow parasite persistence and associated disease. Thus, differences in splenic and hepatic tissue microenvironments dictate differences in the ability to generate effective immune responses and parasite control in these organs. As in other forms of leishmaniasis, studies have shown that sandfly salivary proteins play a crucial role in shaping the quality of adaptive immunity in VL. For instance, vaccination with a DNA encoding an 11-kDa salivary protein from Lutzomyia longipalpis called LJM19, protected hamsters against an otherwise fatal challenge with virulent L. chagasi mixed with L. longipalpis saliva [60] . This protection was associated with high IFN-γ/TGF-β ratio, increased iNOS expression in the spleens and livers and a strong DTH response [60] . Interestingly, vaccination with LJM19 induced a rather weak response in the dogs, which is the main reservoir of VL [61] . Reverse antigen screening studies have identified other salivary proteins (LJL143 and LJM17) of sandfly capable of inducing protection against experimental VL [61] . Vaccination with these proteins induces strong systemic and local Th1 cell-mediated immune response in dogs characterized by remarkable IFN-γ and IL-12 expression [61] . Notably, this protective response was recalled by sandfly bites and increased leishmanicidal activity of macrophages in vitro [61] . It has been demonstrated that a proteophosphoglycanrich mucin-like gel is secreted by L. infantum inside the midgut of L. longipalpis and this is regurgitated along with parasites during a blood meal [62] . Interestingly, Rogers et al. have demonstrated that L. infantum–derived proteophosphoglycans enhances parasite establishment at the site of inoculation suggesting that L. longipalpis saliva could synergistically enhance ­establishment of infection in the skin [63] . Liver versus spleen immunity in VL The development of a vaccine against VL is greatly hindered by our limited understanding

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Review  Khadem & Uzonna of the specific immune mechanisms required for controlling parasite growth without inducing pathology in the liver and spleen. In most experimental models of VL, infection in the spleen remains chronic over a prolonged period of time whereas liver infections are self-resolving [43] . Resolution in the liver is associated with the development of controlled but effective granulomas that promote parasite clearance. In contrast, pathology is mediated by changes to the local tissue microenvironments that contribute to an inability to generate effective immune responses. Therefore, understanding the development of immunity in the liver may lead to potential means to improve parasite clearance in the spleen. The development of inflammatory granulomas around infected liver macrophages leading to immunity is a T-cell-driven event. This Th1-dominated response is mediated by TLR7, TLR8, TLR9, IL-1 and IL-18 via the MyD88 signaling pathway [43] . An efficient granuloma formation involves the expression of inducible iNOS by macrophages [64] , which is regulated by several proinflammatory (Th1) cytokines such as IL-12, IFN-γ, TNF-α, lymphotoxin, granulocyte/macrophage colony-stimulating factor and IL-2 [65] as well as intact and functional NK and NKT cells [33] . Interestingly, a report suggests that IL-4 (a prototypic Th2 cytokine known to inhibit granuloma formation) is also required for the resolution of hepatic infection and for priming of CD8 + T cells that are critical for long-term protection [56] . Although IL-10, TGF-β and IL-27 can suppress the control of L. donovani growth, they appear to have little impact on granuloma formation in the liver [66] . In VL, the spleen and bone marrow also become chronically infected by mechanisms that are less well understood. In experimental murine VL, the spleen becomes enlarged (akin to the condition in humans) and splenomegaly can account for up to 15% of the body weight of infected mice in as little as 6–8 weeks postinfection [34] . The persistence of parasites in the spleen is associated with changes in the splenic lymphoid microenvironment, and concomitant increases in the rate of T-cell apoptosis and decreased responsiveness to leishmanial antigens [34] . In addition, elevated levels of TNF-α in the spleen may contribute to this process since members of the TNF superfamily of cytokines are known to promote apoptotic cell death [67] . The production of IL-10 in the spleen contributes

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significantly to the establishment of infection and immunological dysfunction associated with the disease [34] . Subversion of host immune response in VL Several pathogens including Leishmania have evolved mechanisms to subvert the host immune system in order to establish production infection and enhance their survival [68] . In the case of Leishmania that are obligate intracellular organisms, subversive events that enhance intracellular survival, inside the mamalian host cells are important for their survival and disease pathogenesis. This is important given that Leishmania resides and replicates inside macrophages that are equipped with lytic enzymes capable of destroying infecting pathogens once the cell is appropriately activated. Hence, Leishmania parasites have evolved to actively subvert several aspects of the host cell signaling events, ranging from preventing the production of microbicidal molecules and protective cytokines to interferring with effective antigen presentation. A number of virulence factors including surface and secreted molecules have been shown to actively interfere with macrophage activities in vivo and in vitro. These include fructose 1,6 bisphosphate aldolase, secreted acid phosphatise, elongation factor 1α, glycoprotein 63 (GP63), LPG and peroxidoxins. For example, the interaction of L. donovani elongation factor 1α secreted within exosomes and fructose 1,6 bisphosphate aldolase with host SHP-1 has been shown to inactivate macrophage [69] . Similarly, it has been proposed that lipid raft dependent release of GP63 (a protease) by L. donovani promastigotes and its subsequent translocation into the macrophage cytosol leads to cleavage of several macrophage proteins including protein tyrosine phosphatises thereby promoting macrophage deactivation [70] . In addition, L. donovani promastigotes and amastigotes secrete acid phosphatise, which have been shown to d­ephosphorylate host proteins [35] . To prevent activation of an effective immune response, Leishmania directly or indirectly suppresses a number of cytokines involved in host immune response. For example, L. donovani promastigotes have the ability to inhibit IL-12 production in macrophages thereby impairing cell-mediated immune response and IFN-γ production [65] . Similarly, L. chagasi inhibits host immune responses by inducing TGF-β

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Immunity to visceral leishmaniasis: implications for immunotherapy  production in macrophages [71] . Leishmania also induces the production of IL-10 in macrophages, which causes suppression of IL-1, IL-12, TNF-α and NO production and inhibits optimal expression of costimulatory molecules including B7–1/2 [72] . Leishmania donovani inhibits antigen presentation by repressing the MHC class I and II expression on macrophages [73] . Recently, we reported that Leishmania glycophosphate conjugates including phosphoglycans and LPGs, influence the host early immune response by inhibiting antigen presentation in DCs [74] . Another mechanism by which L. donovani limits leishmaniacidal activity of the host is by inhibiting the activities of some important kinases in macrophages. L. donovani LPG strongly inhibits protein kinase C mediated intracellular signaling and its downstream functions [75] . Similarly, L. donovani-infected macrophages are defective in their ability to phosphorylate JAK1, JAK2 and STAT1 protein [35] . Leishmania donovani also inhibits nuclear translocation of STAT1α, AP-1 and NF-κB in macrophages [76] . Leishmania infection also leads to activation of the phosphoinositide 3 kinase pathway macrophages resulting in inhibition of macrophage apoptosis [77] . The enhancement of host cell survival consequently leads to pathogen replication, persistence and spread [77] . Furthermore, L. donovani infection of macrophage leads to alteration of MAPK pathway and promotes parasite survival and propagation within infected cell [77] . Manipulation of host protein tyrosine phosphatise pathway is another mechanism by which Leishmania subverts the host immunity [35] . In L. donovani-infected macrophages, the activation of SHP-1 prevents IFN-γ-dependent NO production via inactivation of JAK2 and ERK1/2 [78] . This results in failure of NF-κB and AP-1 to translocate to the nucleus thereby inhibiting transcription of key genes including those involved in macrophage activation and ­production of ­proinflammatory cytokines and NO [78] . Vaccines & vaccination strategies against VL Despite the global public health importance of leishmaniasis, progress in developing vaccines against the disease has lagged because of some key technical hurdles, including the fact that the disease occurs mostly in the world’s poorest countries and absence of financial incentives to

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pharmaceutical companies. The current status of scientific and technical progress in the development of novel neglected tropical disease vaccines has been reviewed recently [79] . The reader is therefore referred to this review for comprehensive information on the status of vaccination progress against the disease. Many in vivo experimental models have been developed for antileishmanial drug and vaccine screening and their respective advantages and disadvantages have been thoroughly reviewed elsewhere [12,80] and is outside the scope of the present review. Currently, there is no animal model that accurately reproduces all the unique features of human VL. Although the Syrian golden hamster (Mesocricetus auratus) provides a more synchronic infection in the liver and spleen resulting in chronic infection and death if untreated (akin to human VL) [81] , the limited availability of reagents severely hampers its use for immunologic studies. Inbred strains of mice show varying degrees of susceptibility to VL and variable similarities with human disease. While BALB/c mice infected with L. donovani complex do not exhibit high susceptibility and result in a selfhealing chronic infection [82] , it is still among the most commonly used experimental models due to their availability, disease reproducibility, ease of handling and availability of reagents. In general, several vaccination and/or control strategies have been used and/or proposed including leishmanization, vaccination with virulent or genetically modified live-attenuated leishmania parasites, killed parasites, whole-cell lysates/purified fractions, parasite protein components or subunits, whole native/recombinant parasite proteins, plasmid DNA and viral vector based vaccine candidates e­ncoding parasite virulence factors (reviewed in [83]). There are currently only four vaccines against leishmaniasis: a killed vaccine for immunotherapy in Brazil, a live vaccine in Uzbekistan, a recombinant vaccine for prophylaxis in dogs in Brazil [84] and an European canine vaccine (CaniLeish) prepared from purified excretedsecreted proteins of L. infantum [85] . CaniLeish has been shown to induce a strong and effective Th1-dominated anti-L. infantum response in vaccinated dogs, which was associated with significant reduction in parasite burden and disease intensity in vaccinated animals [85] . However, the impact of this vaccine on the overall immune system of the vaccinated dogs is currently unknown [86] . In addition, the efficacy

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Review  Khadem & Uzonna of these vaccines remains controversial [84] . In order to develop a safe, effective and practical vaccine against VL, the identification of protective antigens and improvement of antigen delivery platforms and vaccine formulations that could induce effective T-cell responses are critical and challenging hurdles that must be overcome. In addition, a comprehensive proteomics approach is needed in order to identify cross protective Leishmania antigens that could protect against both cutaneous and visceral forms of the disease. Several vaccination studies (at least in experimental models of the disease) point to the feasibility of developing an effective Leishmania vaccine. Vaccination with the Leishmania histone H1 protein has been reported to induce strong protection against VL caused by L. infantum in mice. The protection was associated with enhancement of IFN-γ production by T cells and a reduction in IL-10 production by DCs from vaccinated mice [87] . Recently, the safety and efficacy of a recombinant Leishmania polyprotein vaccine, Leish-F1 in MPL-SE adjuvant against VL was tested in India. The vaccine was shown to be safe and well tolerated in subjects with and without history of previous Leishmania infection [88] . In addition, the vaccine was also strongly immunogenic and induced IFN-γ production by T cells [88] , suggesting that it may offer some level of protection against the disease. The efficacy of Leish-F1 vaccine has also been tested in CL in Brazil. As in VL, the vaccine was found to be safe and immunogenic in CL patients. Interestingly it shortened the time to cure in patients when combined with meglumine antimoniate [89] . Perhaps, the most encouraging vaccine that has raised high hopes for potential development of a vaccine against human VL is Leishmune, a purified glycoprotein derived from fucose-mannose ligand of L. donovani promastigotes and formulated in a saponin adjuvant [90] . Leishmune is a transmission blocking vaccine that has been demonstrated to be effective against canine VL in both prophylactic and immunotherapeutic manners [91] . One of the major groups of virulence factors in Leishmania belongs to cysteine proteinase (CP) family. Considering the essential role of dogs in the pathogenesis of the VL, Rafati S. et al. have proposed that the combination of DNA and recombinant protein vaccination using CP type I and II of L. infantum can interrupt the domestic cycle of the disease [92] . In addition,

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they have reported that VL prime-boost vaccination based on CP type III induces appropriate humoral and cellular immune responses in vivo that leads to lower parasite load [93] . Because of the importance of live parasites in the maintenance of anti-Leishmania immunity [94] and the proven efficacy of leishmanization against CL [95] and VL [96] , several studies have focused on live-attenuated organisms as viable vaccine candidates against leishmaniasis. Attenuated parasites have the advantage of being infectious but not pathogenic, and as such are able to be taken up by the host antigen-presenting cells in the same compartment as virulent organisms. In addition, these may persist long enough to induce appropriate immune response without causing disease [97] . In line with this, a genetically modified live-attenuated L. donovani lacking the centrin gene (termed LdCen−/−) has been shown to induce protection in BALB/c mice and Syrian hamsters against homologous and heterologous Leishmania challenges [98] . A recent study also showed that LdCen−/− parasites induce strong humoral and cell-mediated immune responses in vaccinated dogs [99] . Although protection against virulent challenge was not assessed in this study, the induction of strong CD4 + and CD8 + responses suggested that this may be a viable vaccine candidate against canine VL. One of the concerns of live-attenuated vaccine is the potential of the parasites to revert to virulence, which could lead to the development of active disease, particularly in immunocompromised individuals. As a result, efforts are being made to utilize nonpathogenic parasites as potential vaccine candidates. The lizard Leishmania specie, L. tarentolae has been shown to activate DC maturation processes and induce strong protective CD4 + Th1-cell-mediated immunity against L. donovani challenge in mice [100] . In addition, recombinant expression of amastigote-specific L. donovani A2 antigen in L. tarentolae elicits protection against L. infantum challenge in mice [100] . Recently, a nanotechnological approach comprising DNA/ live and live/live prime-boost vaccination strategies against L. infantum infection in BALB/c mice was carried out using a recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB) [101] . The results show that recombinant L. tarentolae-expressing tri-fusion proteins elicited a strong protective response and

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Immunity to visceral leishmaniasis: implications for immunotherapy  protected vaccinated mice against VL caused by L. infantum [101] . Immunotherapy of VL Despite significant advances in the treatment of VL, there are still concerted efforts toward developing new therapies and treatment regimens. This is because most conventional antileishmanial therapeutic strategies have several drawbacks such as difficulty in administration, prolonged duration of treatment, toxicity, high cost of treatment, emergence of drug-resistant strains and disease relapse [3,16] . Therefore, immunotherapy, which is the use of biological substances to modulate or modify immune responses in order to achieve a prophylactic and/or therapeutic goal, has been proposed as a rational treatment option for leishmaniasis. The rationale behind immunotherapy against VL stems from the belief that following infection, progressive disease occurs when there is either a failure, suboptimal or excessive host immune response against the parasite. Hence, it is reasoned that the induction of appropriate immune modulation or interventions using immunomodulatory agents or biological response modifiers could reverse or ameliorate disease progression [102] . In addition, immunotherapy prior to or in synergy with conventional therapy, may lower the required drug dose and treatment regimen, reduce drug toxicity, improve drug efficacy, reduce emergence of drug-resistant strains and consequently reduce the chances of disease relapse [16] . Efforts to use immunotherapy as an alternative treatment for leishmaniasis have been mostly focused on human CL in South America. This involves the administration of killed parasites either alone or in combination with adjuvants (such as BCG) or chemotherapeutic agents to patients with chronic cutaneous disease. Such combination treatments were shown to be effective in curing American CL [103] . Because of the similarities in certain aspects of the immunopathogenesis of CL and VL, it is plausible that similar immunotherapeutic endeavors utilizing killed parasites, adjuvants and anti-Leishmania compounds could also be beneficial in the treatment of VL. Indeed, it has been shown that a combination of killed parasites, BCG and sodium stibogluconate was very effective in curing persistent post-kala-azar dermal leishmaniasis in East Africa [104] . The combined therapy successfully

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Review

cured the disease in 87% of patients, which was significantly different from those that received the drug alone [104] . Studies in experimental CL have shown that vaccinating Leishmania-infected mice with DNA expressing immunogenic proteins could ameliorate the disease and in some cases lead to cure [105] . This cure was attributed to the vaccine’s ability to shift existing disease-promoting Th2 cytokine toward a protective Th1 cytokine response [105] . Similarly, vaccination with a recombinant DNA expressing the L. chagasi nucleoside hydrolase protein (NH36-DNA) modulates the outcome of VL in BALB/c mice previously infected with L. chagasi, leading to survival from an otherwise lethal disease [106] . A single dose of a recombinant adenoviral vector containing L. donovani antigens, HASPB and KMP11, significantly reduced liver parasite burden in mice previously infected with L. donovani. This immunotherapeutic effect was associated with increased antigen-specific CD4 + and CD8 + T-cell responses resulting in enhanced DTH [107] . Thus, the inherent adjuvant activity of the adenoviral vector provides a novel vaccination strategy for administering immunotherapeutic candidate genes to hosts with existing VL infection [107] . Collectively, these experimental murine studies suggest that DNA vaccines may induce immunomodulatory activities in human VL patients that could result in disease cure. Experimental studies in mice suggest that the outcome of VL may also be altered by the administration of subunit vaccines. A vaccine containing the fucose mannose ligand protein induced a significant reduction in liver parasite load and decrease in IL-10 level in mice experimentally infected with L. donovani [108] . Similarly, Leishmune®, the first commercial licensed vaccine against canine leishmaniasis that comprise fucose mannose ligand fractions in saponin adjuvant has been shown to lower clinical disease and parasite burden when administered several months after L. donovani infection [109] . This protection was associated with recovery of CD4 + T-cell response and decreased serum antibody levels [109] . These few encouraging reports highlight the potential of DNA or subunit vaccines as immunotherapeutic strategy for the treatment of VL. Another strategy with potential immuno­ therapeutic implication is targeting of cytokines. As with CL, protective immunity against VL is dependent on an IL-12-driven

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Review  Khadem & Uzonna IFN-γ production by T cells. By contrast, susceptibility is usually associated with the preferential expansion of T cells that produce immunoregulatory cytokines [110] . Thus, targeting immune imbalances to preferentially expand protective T-cell responses provides a rational strategy for treatment of VL. For example, high levels of TGF-β, a cytokine that suppresses lymphocyte activation, has been shown to be partly responsible for the immunosuppression observed in experimental leishmaniasis by inducing T-cell apoptosis [111] . Therefore, targeting TGF-β may potentially minimize lymphocyte apoptosis and restore the suppressed cell-mediated immunity in the host, which could result in cure. Similarly, VL is associated with high levels of serum IL-10 as well as production of high levels of IL-10 by splenic cells [112] . Treatment of mice infected with L. donovani with anti-IL-10R mAb has been shown to result in significant amelioration of the disease [113] . Thus, targeting this cytokine could be potentially effective in t­reating human VL. Because progression of VL is usually associated with increased Th2 and a concomitant decreased Th1 cytokine response, it is conceivable that a combination of Th1-inducing cytokines or immunostimulants with chemotherapy could enhance Th1 response and possibly lead to cure of clinical disease. For example, while treatment with IFN-γ alone was not beneficial in VL treatment in India [114] , a combination of IFN-γ and meglumine in Guatemala was more effective than meglumine alone in treating patients infected with L. braziliensis [115] . Another mediator that could hold potential immunotherapeutic promise in treatment of leishmaniasis is interferon inducible protein 10 (IP-10), a CXC chemokine. Treatment of infected BALB/c mice with recombinant IP-10 induced a strong protective Th1 immune response that was associated with marked decrease in TFG-β and IL-10-secreting CD4 + T cells. In addition, IP-10-mediated decrease in production of immunosuppressive cytokines was correlated with a marked reduction in the frequency of CD4 + CD25 + Tregs [116] . Thus, a detailed mechanistic insight into IP-10-mediated regulation of Tregs and enhanced immunity during VL might be helpful in designing immunotherapeutic intervention strategies against the disease.

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Conclusion & future perspective Most studies on the immunobiology and vaccinology of leishmaniasis focus on cutaneous leishmaniasis due to the erroneous belief that knowledge gained from experimental CL could also be applicable to VL. However, it should be noted that the regulatory mechanisms governing resistance or susceptibility to both experimental and natural VL and CL are widely distinct. Therefore, vaccine or immunotherapy modalities that work for CL would not necessarily be expected to be effective against VL. Whether a Leishmania infection remains asymptomatic or progresses toward VL in the host depends on several factors such as the interactions between the environment, the parasite and the host genetic makeup that ultimately govern the quality and magnitude of the host immune response. Indeed, designing novel immunotherapeutic strategies against VL requires obtaining in-depth knowledge about basic mechanisms underlying immunity and immunopathology of VL in animal models and assessing their translational relevance and potentials in humans. Despite tremendous efforts in the development of immunotherapeutic strategies against VL, there is still an unmet need to address several issues regarding their mechanism of action as well as safety and efficacy considerations. Importantly, a global standardized protocol should be developed to evaluate and compare immunotherapeutic benefits of all emerging strategies. This protocol should include pharmacokinetic considerations such as assessment of the dose and duration of therapy. Considering the huge variations in readouts of immunologic assays such as classical cytokine measurement and parasite burden, this protocol should provide the field with new technological advances to minimize these variations in both human patients and animal models. More importantly, formulation of efficient drug delivery systems should be considered as an urgent priority for successful design of effective VL i­mmunotherapeutic strategies and agents. As mentioned earlier, VL is a complex heterogeneous disease with various innate and adaptive immune mechanisms involved in the disease pathology. This necessitates designing a unified strategy aimed at targeting different mediators and pathways in a comprehensive manner. In this regard, Novel concepts such as VL genomics and proteomics as well as

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Immunity to visceral leishmaniasis: implications for immunotherapy  computational modeling could provide insightful clues for developing potential VL immunotherapeutics. For example, several efforts are being made to generate a computational pipeline to aid the identification, mapping and characterization of conserved intercoding sequences in the Leishmania genomes that are involved in gene regulation [117] . Furthermore, identifying proteomic data from two different life stages of L. donovani (the promastigote and amastigote stages) has helped characterize specific subset of proteins that are expressed in both parasite forms [118] . Computational modeling using these identified proteins could help develop effective immunotherapy treatment regimens. Recently, it was reported that Leishmania organisms use an exosome-based secretion mechanism to export and deliver effector molecules into host

Review

cells. Therefore, studies focused on identifying the properties and functional activities of Leishmania exosomes and their cargo contents could generate important new knowledge that are likely to identify novel targets for rational immunotherapy and candidates for vaccine development. Financial & competing interests disclosure Funding for this study was provided by the Canadian Institutes of Health Research and the Manitoba Health Research Council. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

EXECUTIVE SUMMARY Visceral leishmaniasis ●●

Visceral leishmaniasis (VL) is primarily caused by Leishmania donovani in India and East Africa, L. infantum in the Mediterranean countries and L. chagasi in the New World.

●●

Over 90% of VL cases occur in seven countries: Bangladesh, Brazil, Ethiopia, India, Nepal, South Sudan and Sudan.

●●

Due to relatively incomplete epidemiological data, official figures are likely to underestimate the real prevalence of the disease.

●●

The Leishmania infected female Phlebotomus genus sand fly is the vector in the Old World and the Lutzomyia genus sand fly serves as the vector in the New World.

Treatment of visceral leishmaniasis ●●

Several drugs including sodium stibogluconate, amphotericin B and miltefosine, among others, along with combination therapy are used in VL chemotherapy.

●●

Various factors such as endemicity, infecting species, disease severity, drug dose and frequency and host immunocompetence have significant impact on VL cure rate.

Host immune response to visceral leishmaniasis ●●

The early interaction of Leishmania with macrophages and dendritic cells influences the host immune response and guides it toward appropriate or malfunctional adaptive immune response.

●●

Control of Leishmania infection in mouse liver requires a coordinated host response resulting in productive granuloma formation.

●●

In VL, the spleen and bone marrow become chronically infected by mechanisms that are less understood.

Vaccines & vaccination strategies against visceral leishmaniasis ●●

Vaccine development for VL has lagged because of some key hurdles: not adequate knowledge/information on the host immune mechanism and absence of financial incentives.

Immunotherapy of visceral leishmaniasis ●●

Immunotherapeutic strategies prior to or in synergy with VL conventional therapy, may lower the required dose and

treatment regimen or toxicity, improve drug efficacy, reduce emergence of drug-resistant strains and circumvent the problems of treatment in immunocompromised hosts.

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Review  Khadem & Uzonna 15 Tunccan OG, Tufan A, Telli G et al. Visceral

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