**1. Introduction**

The principle of antimicrobial vaccines is to increase immunity against a specific infectious agent so when the individual is challenged by that agent the appropriate immune response is mounted rapidly and efficiently. Vaccines for infectious agents have historically developed from whole live or dead microorganisms to more defined native or recombinant pure fractions, following antigen-coding DNA and the latest approaches of antigen-pulsed dendritic cells. Although bacterial and viral infections have a quite long list of effective vaccines, parasitic infections – from worms to protozoa – have been a hard challenge for researchers to be able to develop proper vaccines. Currently, the most advanced anti-parasitic vaccine is the RTS,S/ AS01 for malaria with a protection that covers 30-40% [1]. Despite several attempts during seven decades of research with some promising approaches, so far there is no vaccine available for human leishmaniasis and the options available for veterinary use have zone-restricted market authorization, being inaccessible to many endemic countries.

Traditionally, live vaccines incorporate attenuated strains that after entering the host cause a non-pathological short-lived infection, being rapidly controlled by the innate and adaptive immune systems. In few words, the microorganism is taken up and processed by antigen presenting cells (APCs) that efficiently expose the microbial antigens *via* MHC class I or MHC class II molecules, activating the cognate T cell receptors (TCRs) on the surface of CD8+ or CD4+ T cells, respectively. From here, the effector cellular and humoral machinery develop a specific response aiming to eliminate the aggressor. When a sterile cure (*i.e.* complete elimi‐ nation of the microorganisms) is achieved, a contraction in all the effectors takes place, though specific central memory T cells and antibodies endure [2], being ready to initiate a stronger response upon a second encounter with a similar microorganism. However, the abidance time

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of memory is highly dependent on the strength of the primary response. Studies using mice models have shown that small numbers of parasites restricted to the inoculation site, without causing clinical manifestations, are essential for protection from a virulent challenge [3], indicating that antigen persistence is of major importance in a vaccination protocol for leishmaniasis. In fact, this is the concept behind the leishmanization strategy applied in humans.

In this chapter we address some general aspects of the epidemiology of human and canine leishmaniasis to introduce the needs for a vaccine and the desirable immune response to be generated upon vaccination. We present the animal models most commonly used in leishma‐ niasis vaccine research, the road so far travelled by the scientific community attempting to discover the vaccine for leishmaniasis and its current status. Finally, we show our experimental study in BALB/c mice about the influence of a primary infection of *Leishmania infantum* on the outcome of a *de novo* infection with a homologous or heterologous strain with distinct infec‐ tivity and immunomodulation.

#### **1.1. Human leishmaniasis**

Leishmaniasis is endemic in 98 countries and 3 territories ranging the Mediterranean Basin, the Middle East, the Indian sub-continent, and the tropical regions from America and Africa [4]. The last WHO report on the epidemiology of leishmaniasis estimates that every year 0.7 to 1.2 million new cases of cutaneous leishmaniasis (CL) are mounted and 0.2 to 0.4 million people develop visceral leishmaniasis (VL) which, in turn, is responsible for 20000 to 40000 deaths [4]. Nevertheless, in endemic countries most of the *L. infantum*- or *L. donovani*- infected people are asymptomatic carriers or self-healers [5, 6].

The relation of leishmaniasis with poverty catalogues it as a neglected tropical disease. In fact, 72 of the endemic countries are developing nations with a burden of 90% of the VL, CL and mucocutaneous leishmaniasis (MCL) [7]. In these regions, the majority of the population lives in rural areas, where higher densities of sand flies are found, and is malnourished, a condition that leads to immunosuppression. In addition, HIV concomitant infection is frequent, contri‐ buting to a severe state of immunodeficiency [8]. The close geographical overlap of *Leishma‐ nia* and HIV promote the concomitant infection of both pathogens. In fact, HIV infection increases in 100-2320 times the risk of developing VL in the endemic regions. HIV/*Leishma‐ nia* coinfections correspond to 2-9% of all the VL cases in endemic countries [9]. Furthermore, leishmaniasis is nowadays an important issue in developed countries due to coinfection cases with HIV where *Leishmania* arises as an opportunistic infectious agent, the third of the parasitic infections after *Toxoplasma gondii* and *Cryptosporidium* spp. [10]. Indeed, 90% of the reported HIV/*Leishmania* cases are from Southern European countries, namely Spain, Portugal, Italy and France [8]. The routine use of highly active antiretroviral therapy (HAART) by the end of 1990's produced a clear decrease of HIV/*Leishmania* coinfection cases in southern Europe, but it is now a growing concern in those major *foci* of leishmaniasis in developing countries like Ethiopia, where the incidence of HIV is still high [9]. In addition, following the climatic changes that currently allow the presence of the vector in higher latitudes and the constant circulation of people and animals crossing frontiers and oceans, nowadays leishmaniasis cannot anymore be considered restricted to the endemic countries but is otherwise spread in the world.

of memory is highly dependent on the strength of the primary response. Studies using mice models have shown that small numbers of parasites restricted to the inoculation site, without causing clinical manifestations, are essential for protection from a virulent challenge [3], indicating that antigen persistence is of major importance in a vaccination protocol for leishmaniasis. In fact, this is the concept behind the leishmanization strategy applied in

In this chapter we address some general aspects of the epidemiology of human and canine leishmaniasis to introduce the needs for a vaccine and the desirable immune response to be generated upon vaccination. We present the animal models most commonly used in leishma‐ niasis vaccine research, the road so far travelled by the scientific community attempting to discover the vaccine for leishmaniasis and its current status. Finally, we show our experimental study in BALB/c mice about the influence of a primary infection of *Leishmania infantum* on the outcome of a *de novo* infection with a homologous or heterologous strain with distinct infec‐

Leishmaniasis is endemic in 98 countries and 3 territories ranging the Mediterranean Basin, the Middle East, the Indian sub-continent, and the tropical regions from America and Africa [4]. The last WHO report on the epidemiology of leishmaniasis estimates that every year 0.7 to 1.2 million new cases of cutaneous leishmaniasis (CL) are mounted and 0.2 to 0.4 million people develop visceral leishmaniasis (VL) which, in turn, is responsible for 20000 to 40000 deaths [4]. Nevertheless, in endemic countries most of the *L. infantum*- or *L. donovani*- infected

The relation of leishmaniasis with poverty catalogues it as a neglected tropical disease. In fact, 72 of the endemic countries are developing nations with a burden of 90% of the VL, CL and mucocutaneous leishmaniasis (MCL) [7]. In these regions, the majority of the population lives in rural areas, where higher densities of sand flies are found, and is malnourished, a condition that leads to immunosuppression. In addition, HIV concomitant infection is frequent, contri‐ buting to a severe state of immunodeficiency [8]. The close geographical overlap of *Leishma‐ nia* and HIV promote the concomitant infection of both pathogens. In fact, HIV infection increases in 100-2320 times the risk of developing VL in the endemic regions. HIV/*Leishma‐ nia* coinfections correspond to 2-9% of all the VL cases in endemic countries [9]. Furthermore, leishmaniasis is nowadays an important issue in developed countries due to coinfection cases with HIV where *Leishmania* arises as an opportunistic infectious agent, the third of the parasitic infections after *Toxoplasma gondii* and *Cryptosporidium* spp. [10]. Indeed, 90% of the reported HIV/*Leishmania* cases are from Southern European countries, namely Spain, Portugal, Italy and France [8]. The routine use of highly active antiretroviral therapy (HAART) by the end of 1990's produced a clear decrease of HIV/*Leishmania* coinfection cases in southern Europe, but it is now a growing concern in those major *foci* of leishmaniasis in developing countries like Ethiopia, where the incidence of HIV is still high [9]. In addition, following the climatic changes that currently allow the presence of the vector in higher latitudes and the constant circulation

humans.

tivity and immunomodulation.

278 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

people are asymptomatic carriers or self-healers [5, 6].

**1.1. Human leishmaniasis**

The progression of a *Leishmania* infection to clinical disease comprises multifactorial phenom‐ ena, including the tropism of the species and strains, the genetic background of the host and the efficiency of the immune response developed against the parasite [11]. Studies using mice models have helped the scientific community to better understand the host-parasite relation‐ ship in leishmaniasis. Interleukin (IL) -12 is considered a key cytokine in the early development of the effective immune response due to its requirement for the activation of natural killer cells and T lymphocytes [12]. Activation of these cells leads to the secretion of interferon-γ (IFNγ), another commander cytokine.

Both in mice as in humans, macrophages are classically activated by IFNγ. This leads to the transcription of inducible nitric oxide synthase (iNOS) and phagocyte NADPH oxidase (phox) that produce nitric oxide (NO) and reactive oxygen species, respectively, specimens generally considered indispensable for macrophage-direct killing of *Leishmania* [13]. Macrophages activated by IL-12-driven IFNγ secretion by Th1 lymphocytes – named M1 macrophages – also produce TNFα, IL-1β and IL-6, pro-inflammatory cytokines that favor the protective response against *Leishmania* infection. These macrophages are, then, both effectors and inducers of the Th1 polarized immune response [14]. Nevertheless, the strong Th1 pro-inflammatory response must be balanced with the secretion of IL-10 and transforming growth factor-β (TGFβ) to avoid immunopathology through excessive tissue damage [15].

Effector CD4<sup>+</sup> and CD8<sup>+</sup> T cells that were activated by the recognition of *Leishmania* antigens on the cognate TCR and expanded to respond to infection will face a massive contraction on their numbers of about 90% after the elimination of the parasite, leaving a subset of experienced cells that constitute the memory pool. Memory cells are long-lived cells that rapidly expand in response to a secondary challenge with the priming antigen [16]. They form a heterogeneous pool with distinct abilities in proliferation, migration and cytokine production, which allow their classification in central memory (TCM) or effector memory T cells (TEM).

Memory cells were demonstrated to have great importance in the control of leishmaniasis, with distinct roles described for TCM and TEM cells. Zaph *et al*. have shown that in mice both TCM and TEM CD4<sup>+</sup> cells require parasite presence to be developed, though maintenance of TCM is independent of antigen persistence [2]. This achievement, however, seems highly dependent on the initial overall T cell response, since in some immunization experiments that used low dose of parasites protection was lost after the elimination of the parasites, possibly due to insufficient expansion of the TCM pool [3]. Adoptive transfer of TCM from *L. major*infected mice to naïve animals conferred protection upon a challenge. When facing the antigen, TCM expanded in the lymph nodes, acquired effector functions, including CD62L downre‐ gulation which allowed their migration to the infection site and effective protection [2]. In accordance, analysis of CD4<sup>+</sup> memory T cells from patients with CL stimulated *ex-vivo* with soluble *Leishmania* antigen (SLA) revealed the high proliferative ability and IL-2 production of TCM and high percentage of IFNγ-secreting TEM [17].

Nevertheless, concomitant immunity, *i.e.* efficient protection upon a challenge due to the longtermandsimultaneouspersistenceofthepathogen, seems tobe ahallmarkinleishmaniasis [18].

#### **1.2. Canine leishmaniasis**

Dogs are primary reservoir hosts of zoonotic visceral leishmaniasis (ZVL) caused by *Leishmania infantum* and play a key role in the long-term maintenance of the parasite in the endemic areas of Mediterranean countries, the Middle East, Asia and Latin America. Epidemiological surveys estimate that, for example in western Mediterranean countries, seroprevalence ranges from 5 to 37%, varying from region to region depending on ecological aspects. Nevertheless, surveys based on PCR diagnosis demonstrated high infection rates in endemic areas, for example 80% in Marseille, France [19], and 67% in Majorca, Spain [20]. Longitudinal studies in Italy have also shown high incidences (40-92%) during the season of transmission [21]. Importantly, not all infected dogs develop canine leishmaniasis; more than 50% of infected dogs remain asymptomatic after infection, though it has been shown that these asymptomatic carriers are also infective to sandflies [22].

The high prevalence of infected dogs in endemic areas, their common presence in the domestic surroundings where ZVL transmission occurs, and the high infectiousness of both sympto‐ matic and asymptomatic animals makes that *Leishmania*-infected dogs represent not only a serious veterinary but also an important public health problem. Infected dogs have been associated with the emergence of new *foci* of ZVL, like those in the North of Argentina, where the appearance of human cases is preceded by those of canine leishmaniasis [23], and also with the spread of VL observed in large Brazilian cities [24] and the northward spread of the disease reported in Italy [25]. Therefore, the control of parasite-infected dogs is of prime urgency to reduce the number of cases of human VL by decreasing prevalence in dogs [26].

The outcome of *Leishmania* infection in dogs is variable and depends on the persistence and multiplication of the parasite and the immune response of the animal. Not all the infected dogs will develop clinical disease, part of them can control the expansion of the parasite and spontaneously cure the infection; in others, the infection is subclinical for an undefined time (years or even the whole life) during which the animal remains asymptomatic. Few than 50% of infected animals do not have (or have lost) the capacity to control the parasite, in these cases being distributed extensively throughout the organism: spleen, liver, lymph nodes, bone marrow, kidney, skin, etc., (as opposed to what occurs in humans, where the parasite is normally limited to bone marrow, spleen and liver) [26]. In these dogs the disease progresses, the parasite burden and the *Leishmania*-specific antibody levels increase, and after two to four months of incubation the symptoms of canine leishmaniasis appear [27].

The natural history of canine leishmaniasis mostly depends on the efficacy of the dog´s immune response to *L. infantum* infection which determines the development of resistance or susceptibility to the disease. In general, resistance is associated with low levels of specific antibodies and presence of a predominant Th1 cell-mediated response against the parasite, with the production of IFNγ that is able to stimulate, in collaboration with TNFα, the leish‐ manicidal activity of macrophages mediated by the induction of iNOS. Absence of symptoms is related with high levels of IFNγ expression in the peripheral blood as detected by quanti‐ tative real-time PCR [28]. When dogs develop such parasite-specific cell-mediated immunity, they are able to control parasite dissemination and present an overall low tissue parasitism. This status of resistance is reflected in the development of a positive leishmanin skin test or/and an *in vitro* lymphoproliferative response after stimulation of peripheral blood mono‐ nuclear cells (PBMCs) with leishmanial antigens. In these animals, it has been observed that *in vitro* stimulation of PBMCs with *L. infantum* SLA induces the expression of IL-2, IFNγ, TNFα, IL-4 and IL-10, confirming the existence of both *Leishmania*-specific Th1 and Th2 clones [29]. Also, quantification of the cytokine expression by real-time PCR allowed to establish that PBMCs from resistant dogs expressed high levels of IFNγ and TNFα after *in vitro* stimulation with purified parasite antigens [30, 31]. Therefore, the evaluation of IFNγ expression level from PBMCs constitutes a good approach to evaluate the *in vitro* immunogenicity of leishmanial molecules to identify vaccine candidates able to induce the protective cellular immune response to canine leishmaniasis [30, 32].

Different attempts have been made to confirm a correlation between the classes and subclasses of immunoglobulins and the type of response against *Leishmania* infection in dogs. Early studies associated the appearance of specific IgG2 antibodies against *Leishmania* with the asymptomatic state of the dogs, and the preponderance of IgG1 with progression of the disease [33]. However, other studies have failed to show this [34, 35]. Recent reports have proposed the analysis of IgG, IgG1 and IgG2 isotypes as immune biomarkers for the assessment of the immunogenicity of vaccines against canine leishmaniasis. Since IgG1 and IgG2 responses are largely T cell dependent, the evaluation of the specific isotypes has been considered an important aspect to evaluate the overall immunity induced by a specific vaccine. It has been seen that IgG2 induced by vaccination with *L. infantum* excreted/secreted proteins (*Li*ESP) had a potent inhibitory effect on the *in vitro* growth of both amastigotes and promastigotes and that the pre-treatment of amastigotes with this serum reduced significantly their *in vitro* infectivity in canine macrophages [36].

It is important to remark that the lack of *Leishmania*-specific cell mediated immunity constitutes a key aspect in the pathogenesis of canine leishmaniasis and also in the recovery of the animal after treatment. It has been confirmed that successful chemotherapy of the animals correlates not only with the disappearance of external signs of leishmaniasis but also with a significant increment in the percentage of CD4+ T cells and the appearance of a parasite-specific prolifer‐ ative response of PBMCs [37].
