Leishmania(Viannia)shawipurified antigens confer protection against ...

Inflamm. Res. (2012) 61:255–263 DOI 10.1007/s00011-011-0407-5

Inflammation Research

ORIGINAL RESEARCH PAPER

Leishmania (Viannia) shawi purified antigens confer protection against murine cutaneous leishmaniasis Luiz Felipe Domingues Passero • Ana Kely Carvalho • Maria Luiza A. C. Bordon • Alexis Bonfim-Melo • Marcos Hikari Toyama Carlos Eduardo Pereira Corbett • Ma´rcia Dalastra Laurenti

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Received: 20 June 2011 / Revised: 21 November 2011 / Accepted: 28 November 2011 / Published online: 14 December 2011 Ó Springer Basel AG 2011

Abstract Objective Leishmania (Viannia) shawi was characterized only recently, and few studies concerning the immunogenic and protective properties of its antigens have been performed. The present study aimed to evaluate the protective potential of the five antigenic fractions isolated from L. (V.) shawi promastigotes in experimental cutaneous leishmaniasis. Materials and methods Soluble antigen from L. (V.) shawi promastigotes was submitted to reverse phase HPLC to purify F1, F2, F3, F4 and F5 antigens. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 lg protein. After 1 week, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 8 weeks, those same mice were sacrificed and parasite burden as well as the cellular and humoral immune responses were evaluated. Results F1 and F5-immunized mice restrained lesion progression and parasite load in the skin. However, only the F1 group was able to control the parasitism in lymph nodes, which was associated with low IL-4 and high IFN-c production; IgG2a isotype was increased in this group. Responsible Editor: Artur Bauhofer. L. F. D. Passero (&) A. K. Carvalho M. L. A. C. Bordon A. Bonfim-Melo C. E. P. Corbett M. D. Laurenti Laborato´rio de Patologia de Mole´stias Infecciosas (LIM-50), Departmento de Patologia, Faculdade de Medicina da Universidade de Saõ Paulo, Av. Dr. Arnaldo, 455, Saõ Paulo, SP 01246-903, Brazil e-mail: [email protected] M. H. Toyama Campus Experimental do Litoral Paulista, Universidade Estadual Paulista, Saõ Vicente, SP, Brazil

Immunizations with F2, F3 and F4 antigens did not protect mice. Conclusion The capability of antigens to restrain IL-4 levels and increase IFN-c was associated with protection, such as in immunization using F1 antigen. Keywords Leishmania (Viannia) shawi Immunization Cellular immune response Humoral immune response

Introduction Leishmaniasis is an infectious disease caused by protozoa belonging to the genus Leishmania. Leishmaniasis occurs worldwide, being endemic in 88 countries with a prevalence of 12 million cases per year [1, 2]. In Brazil, human cases of American cutaneous leishmaniasis (ACL) can be found almost in all territories [3]. In some clinical forms, such as mucosal or anergic diffuse leishmaniasis, the lesions can lead to disfigurement, loss of function in affected areas, and, consequently, social stigmatization of the infected individual [4, 5]. The infection starts after the contact of the promastigote forms with antigen-presenting cells (APC), such as dendritic cells and macrophages. Infected APCs can produce IL-12, which stimulates IFN-c production by NK cells [6]. This cytokine activate macrophages, favoring parasite elimination into host cells [7]; in addition, NK cells can eliminate infected host cells by direct cytotoxicity [8]. APC carrying surviving parasites migrate to regional lymph nodes, where they interact with naı¨ve lymphocytes, generating an acquired immune response. In the context of antigen presentation through MHCII, high amounts of IL-4 in the presentation microenvironment then direct the induction and proliferation of the subset of CD4?Th2 cells

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[9], which is associated with susceptibility to infection [10]. On the other hand, high amounts of IL-12 can lead to differentiation and expansion of the CD4?Th1 cell subset [11], and thus resistance to infection. Parasite antigens presented through MHCI stimulate expansion of CD8?T cells, which can eliminate infected cells by cytotoxicity [12], and can also serve as an additional source of IFN-c [13, 14] by aiding the activation of macrophages in response to the leishmanicidal state. Moreover, stimulated B cells can produce specific antibodies, which, in experimental leishmaniasis have been proposed as interesting markers of susceptibility and resistance [15, 16]; however, immunoglobulins seem not to have a direct impact on parasite clearance [17]. Some recent reports state that patients infected with parasites from the Viannia subgenus present a high cellular immune response, as measured by the Montenegro test, and increased levels of Th1 cytokines produced in the lesion by CD4?T lymphocytes [18]. On the contrary, patients infected with parasites belonging to the Leishmania subgenus present either small or no cellular immune response with elevation of Th2 cytokines [18]. In fact, in human and murine infections by L. (V.) braziliensis few parasites can be detected in lesions, which could be a direct result of exacerbated production of proinflammatory cytokines, such as TNF-a and IFN-c, which favor parasite elimination [19, 20]. On the other hand, the elevated number of parasites in lesions in ATL caused by L. (L.) amazonensis is associated with IL-4 and IL-10 cytokines [21]. These reports suggest that antigens derived from parasites of the Viannia subgenus could be an interesting target for the development of candidate vaccines due to the high immunogenicity of their antigens. In fact, some of these reports indicate that L. (V.) braziliensis antigens are highly immunogenic in relation to L. (L.) amazonensis antigens [22]. Moreover, peripheral blood mononuclear cells (PBMC) from infected patients present a higher index of cell proliferation and IFN-c after stimulation with whole parasite lysate of L. (V.) braziliensis when compared to PBMC incubated with antigens from L. (L.) amazonensis [23], demonstrating elevated antigenicity of L. (V.) braziliensis antigens and the ability to induce a Th1 immune response in vitro and in vivo. In the New World, there are several different parasites belonging to the Viannia subgenus [24–26], but unfortunately few of them have been studied in relation to their immunogenicity and protective potential in experimental immunizations. Concerning L. (V.) shawi, the first human case of infection was recorded in northern Brazil in 1991 [27], and this parasite species has been found recently in the northeast of Brazil suggesting parasite spread within Brazilian territory [28], which justifies the development of prophylactic measures against this parasite. Recently, our

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group showed that soluble antigen of L. (V.) shawi promastigote has immunogenic properties, and that the upregulation of IL-12 and IFN-c in immunized mice could confer partial protection in the skin of immunized mice [29], suggesting the presence of protective antigen in this species. Thus, the present study aimed to evaluate the degree of protection induced by antigens derived from promastigote forms of L. (V.) shawi and to evaluate aspects of the cellular and humoral immune response induced by antigenic fractions in mice—a susceptible model of L. (V.) shawi infection.

Materials and methods Experimental animals Eight-week-old male BALB/c mice obtained from the Animal Facility of the School of Medicine of Saõ Paulo University, Brazil, were maintained in our laboratory during the experiments, in accordance with the institutional guidelines regarding the welfare of experimental animals and with the approval of the Animal Ethics Committee of Saõ Paulo University (0280/07). Parasite The parasite L. (V.) shawi (MHOM/BR/96/M15789) was isolated from a patient with ACL in Buriticupu County, State of Maranhaõ, Brazil, and identified using monoclonal antibodies and multilocus enzyme electrophoresis at the Evandro Chagas Institute, Bele´m, State of Para´, Brazil. The parasites maintained in BALB/c mice footpad were isolated and grown in RPMI-1640 medium (Gibco Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FCS, 0.25 mM HEPES, 10 lg/ml gentamicin and 100 IU/ml penicillin. On day 6 of culture, promastigote forms were centrifuged (1,200 g, 10 min) with phosphate buffered saline solution (PBS, pH 7.4) and used for mice infection. Production of antigens from L. (V.) shawi promastigotes Promastigote forms (*109 promastigotes) in stationary phase were recovered by centrifugation at 1,200 g for 10 min at 4°C, followed by three washes with PBS at 1,200 g for 10 min at 4°C. Lyses buffer (20 mM Tris–HCl; 40 mM NaCl; 10 mM EDTA) was added to the promastigote pellet and the material was frozen in liquid nitrogen and thawed at room temperature three times, then centrifuged at 10,000 g for 1 h at 4°C. The supernatant was collected, lyophilized and applied onto a reverse phase HPLC to purify the antigens, according to the methodology

Leishmania (Viannia) shawi purified antigens confer protection

described by Toyama and collaborators [30]. Briefly, lyophilized whole antigen (10 mg) was dissolved in 250 ll buffer A [0.1% trifluoroacetic acetic acid (TFA)] and the supernatant was then applied to an analytical reverse phase HPLC column, previously equilibrated with buffer A for 15 min. The elution of antigens was conducted using a linear gradient of buffer B (66.6% acetonitrile in buffer A) and the chromatographic run was monitored at 280 nm absorbance for 55 min. The antigenic fractions were collected based on their sharpness and their hydrophilic profile in the elution buffer. The samples were lyophilized and further solubilized with PBS, sterilized in 0.22 lm (Eppendorf, San Diego, CA) and the protein amounts were estimated. The methodology allowed the purification of five major fractions, named F1, F2, F3, F4 and F5. Immunization scheme and challenge Male BALB/c mice, eight per group, were immunized once a week for two consecutive weeks by the subcutaneous route in the rump with 25 lg protein of each antigen. Control mice were injected with 50 ll PBS by the same route. One week after the last immunization, both groups were infected with 106 promastigote forms of L. (V.) shawi into the hind footpad and were identified as the immunized and non-immunized groups, respectively. Another group of healthy mice was injected with PBS into the hind footpad (healthy group). The infection was monitored weekly for eight consecutive weeks by measuring the lesion size using a dial micrometer and expressed as the difference in size between the infected and the contra lateral uninfected footpad. The animals were sacrificed in a CO2 chamber and skin and lymph node fragments were collected to determine parasite burden by the limiting-dilution assay [13]. Evaluation of cellular immune responses To evaluate the cytokine profile in draining lymph nodes, popliteal lymph nodes from all experimental groups were collected at 8 weeks post-infection and macerated under sterile conditions in RPMI 1640 medium. Cells were centrifuged at 900 g, 4°C, 10 min, and suspended in 1 ml culture medium and further counted using Trypan blue (Sigma, St. Louis, MO). Cell concentrations were adjusted to 2 9 106 cell/ml and 100 ll cell suspension plus 10 lg total promastigote antigens (AG) were added to sterile 96-well plate. After 72 h of incubation in humidified atmosphere with 5% CO2, at 37°C, the supernatants were collected and the amounts of IL-2, IL-4, IL-12, and IFN-c were quantified by sandwich ELISA, in accordance with the manufacturer’s recommendations, using recombinant cytokines as standard (BD, San Jose, CA). After supernatant

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collection, the plates were washed three times with PBS and 50 ll 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazole (MTT) at 5 mg/ml in PBS was added to each well to analyze cell proliferation, since only viable cells can reduce MTT to purple formazan. The amount of formazan generated by cells has been claimed to be proportional to cell densities [32]. After 4 h of MTT incubation, 50 ll 10% sodium dodecyl sulfate (SDS) was added to the wells. After 18 h, the plate was read in ELISA reader at 595 nm and cell proliferation was estimated as described previously [32, 33]. Analysis of the humoral immune response The humoral immune response was evaluated by enzymelinked immunosorbent assay (ELISA). High-binding plates (Costar, Cambridge, MA) were coated with 1 lg/well of L. (V.) shawi soluble antigen overnight at 4°C. Next, the plates were blocked with 10% nonfat milk in PBS (2 h at 37°C) to prevent nonspecific binding. Mouse sera (1:50) were added and the plates were incubated for 1 h at 35°C. HPR goat anti-mouse IgG1 and IgG2a (SouthernBiotech, Birmingham, AL) (1:2,000) were added for 1 h at 35°C. After washing, TMB was added to each well for 15 min. The reaction was stopped with 50 ll 2 N sulfuric acid and the plates were read at 450 nm in an ELISA Reader. Sera from healthy and L. (V.) shawi chronically infected mice were also used as negative and positive controls, respectively (data not shown). PBS with 0.05% Tween 20 was used in all washing steps. Statistical analysis The results were expressed as the mean ± standard deviation of three independent experiments and the nonparametric Mann–Whitney U test was used to compare lesion size, parasite load, and cytokine expressions between the groups. Differences were considered statistically significant at a 5% significance level (P \ 0.05). Statistical analysis was performed using SPSS 17.0 for Windows software (SPSS, Chicago, IL).

Results Purification of antigen fractions by reverse phase HPLC The whole antigen was submitted to reverse phase HPLC and five main antigenic fractions were collected. The F1 antigen presented sharpness and homogeneity, indicating the presence of a single molecule. In contrast, fractions F2, F3 and F4 presented multiple peaks, indicating the presence of multiple antigens. F5 antigen presented one

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single peak; however, SDS-PAGE electrophoresis analysis showed the presence of three different molecules (data not shown). Figure 1 shows the chromatographic profile of whole antigen of promastigote forms from L. (V.) shawi parasites. F1- and F5-immunized BALB/c mice inhibited lesion progression and parasitism Lesion size is an important parameter with which to evaluate the efficacy of antigens in experimental immunization as well as in drug trials during disease evolution. In this regard, F1- and F5-immunized mice showed inhibition of lesion progression compared to the non-immunized group from 2 to 8 weeks postchallenge (pc) (P \ 0.05), whereas F3 and F4 antigens induced lesion progression in immunized compared to non-immunized mice from 5 to 8 weeks pc (P \ 0.05). The F2 fraction did not alter the course of infection (Fig. 2). Beside lesion size data, parasite burden analysis, as measured by limiting dilution assay, offers the possibility to enumerate live parasites in tissues, as only viable amastigotes are capable to differentiate in motile promastigotes [31], giving a more realistic profile of parasite burden in the experiments shown herein. Using this methodology, we verified that F1- and F5-immunized groups (Fig. 3) were able to control the multiplication of amastigote forms in the skin (87 and 91% of parasite inhibition, respectively) compared to non-immunized mice (P \ 0.05), although only F1-immunized mice showed a reduction in parasite load in the lymph nodes (92%) compared to non-immunized mice (P \ 0.05). In contrast, F3- and F4-immunized mice (Fig. 3) presented elevated skin parasitism compared to non-immunized mice (260 and 429%, respectively) (P \ 0.05). F2 did not alter cutaneous parasitism in immunized mice in relation to the nonimmunized group. The parasite load in the lymph nodes of

Fig. 2 Size of lesions in immunized and non-immunized BALB/c mice. Purified antigens from L. (V.) shawi promastigotes induced protection and exacerbation of lesions in immunized BALB/c mice as measured by lesion size during 8 weeks. *P \ 0.05 between immunized and non-immunized groups

Fig. 3 Analysis of parasite burden. Parasitism in skin and lymph nodes was quantified in both immunized and non-immunized groups by limiting dilution assay. F1- and F5-immunized mice were able to control parasitism in the skin; on the contrary, F3- and F4-immunized mice presented uncontrolled parasite replication in skin. *P \ 0.05 between parasite load into skin of immunized and non-immunized groups. **P \ 0.05 between parasite load into lymph nodes of immunized and non-immunized groups

the F2-, F3- and F4-immunized groups was similar to that of non-immunized mice (P [ 0.05). Low IL-4, high IFN-c and cell proliferation are linked to protection against L. (V.) shawi infection

Fig. 1 Chromatographic profile of soluble antigen from Leishmania (Viannia) shawi promastigotes. The soluble antigen from L. (V.) shawi promastigotes was submitted to reverse phase chromatography to purify antigens F1, F2, F3, F4 and F5

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After antigen encounter with specific lymphocyte populations, cytokine secretion and proliferation can be triggered, suggesting activation of AG-sensitized lymphocytes. On the other hand, naı¨ve as well as suppressed lymphocytes frequently present low or absent cytokine production and proliferation after antigen encounter.


Upon specific stimulus, cells from the F2, F3 and F4immunized group produced significantly lower levels of IL-2 (Fig. 4a) compared to the non-immunized group (P \ 0.05). Stimulated cells from F1 and F2-immunized mice produced low levels of IL-4 (P \ 0.05), while cells from F3, F4 and F5 groups produced high amounts of this cytokine compared to non-immunized group (Fig. 4b). IL-12 was not detected. Cells from F1-, F3-, F4- and F5-immunized groups produced significantly (P \ 0.05) higher levels of IFN-c compared to culture of lymph node cells from non-immunized mice (Figs. 4c), but cells from F2 group produced low levels of this cytokine (P \ 0.05).

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Cells from healthy mice did not produce detectable cytokines. Cells from all groups without specific stimulation did not produce IL-2, IL-4 and IFN-c. Cells from F1, F2, F3, F4 and F5-immunized groups showed increased proliferation (Fig. 5) compared to nonimmunized mice (P \ 0.05). On the other hand, nonimmunized mice presented similar levels of proliferation compared to healthy mice, indicating the suppressed state of non-immunized mice. IgG2a is associated with protection, and IgG1 with disease progression The levels of specific antibodies can be an important feature that can be used to evaluate the dichotomy of the cellular immune response, since IgG2a frequently can be linked to high IFN-c, and thus resistance to infection, and IgG1 to IL-4, and consequently to disease progression [34]. Compared to the non-immunized control, the F3, F4 and F5-immunized groups showed elevation in the levels of IgG1 (P \ 0.05); F1 and F2-immunized groups showed similar production of IgG1 compared to the non-immunized group (Fig. 6). F1-immunized mice showed elevation in IgG2a compared to the non-immunized group (P \ 0.05).

Discussion In the New World, different vaccine candidates have been characterized and formulated based on L. (L.) amazonensis antigens, but few reports have been able to demonstrate the immunogenicity and protective potential of antigens from species belonging to the Viannia subgenus. L. (V.) shawi—

Fig. 4 Analysis of cellular immune response. The amount of cytokines in lymph nodes cells from immunized and non-immunized mice were measured under specific stimulation using antigens (AG). a IL-2, b IL-4, c IFN-c. *P \ 0.05 between cytokine production of cells from immunized and non-immunized groups

Fig. 5 Analysis of cell proliferation. After specific stimulation using AG, cell proliferation was measured through colorimetric assay based on the conversion of MTT to formazan. In this case, immunized groups presented a different degree of proliferation, suggesting cell activation. *P \ 0.05 between immunized and non-immunized groups

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Fig. 6 Analysis of humoral immune response. Seric IgG1 and IgG2a isotypes were analyzed in all experimental groups. In this case, F3-, F4- and F5-immunized group exhibited elevation in IgG1 isotypes; on the other hand, only F1-immunized mice showed increased IgG2a isotype. *P \ 0.05 between IgG1 of immunized and non-immunized groups. **P \ 0.05 between IgG2a of F1-immunized and nonimmunized mice

a parasite belonging to the Viannia subgenus that seems to have immunogenic antigens capable of polarizing the immune response of BALB/c mice to a Th1 immune phenotype after challenge [29], suggesting the presence of protective antigens in whole intracellular antigen from L. (V.) shawi. In fact, immunization of BALB/c mice with F1 and F5 antigens was able to reduce lesion size and skin parasitism, the main target organs of this parasite in ATL [35]. However, patterns of immunological response differed between groups. Cells from F1-immunized mice produced high IFN-c and low IL-4 concentrations, while cells from F5 exhibited an increase in both cytokines. Control of infection caused by Leishmania parasites can be achieved by IFN-c cytokine, which enhances the microbicidal activity of infected macrophages and also induces differentiation and proliferation of T cells [36, 37]. Moreover, low levels of IL-4 are required to achieve protection, since this cytokine plays an important role in differentiation of naı¨ve CD4?T cells towards the CD4?Th2 T subset of lymphocytes, which in turn can suppress the production and bioactivity of IFN-c [38–40]. Therefore, antigens capable of inducing this immunological profile will induce strong protection in mice. Indeed, the F1-immunized group controlled skin and lymph node parasitism in the presence of high levels of IFN-c and low IL-4. However, the increase in IL-4 level in the supernatant of popliteal lymph node cells from F5-immunized mice could abrogate the protection in lymph nodes. In addition, the results presented herein indicate that both immunizations favored cell proliferation in immunized compared to non-immunized mice, which presented a low cellular proliferation upon specific stimulus, suggesting that the non-immunized group showed

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a suppressive profile such as that occurring in infections caused by other Leishmania species [41, 42]. Concerning the humoral immune response, only F1-immunized animals exhibited an increase in the production of IgG2a compared to non-immunized mice, which could have a direct association with high IFN-c levels [34]. F5-immunized mice exhibited an increase in IgG1 isotype, which can be associated directly with high levels of IL-4 and parasitism in lymph nodes [43]. Therefore, immunization with F1 confers protection on skin and popliteal lymph nodes, associated with an increasing Th1 immune response; however, F5-immunized animals could limit parasite infection of the skin and were associated with a mixed immune response. In contrast, immunization with F3 and F4 antigens favored the development of lesions compared to nonimmunized mice, since an increase in skin parasitism was observed. Functionally, lymph node cells from these groups presented a strong potential to proliferate upon specific antigen stimulation, suggesting activation of T lymphocytes. In addition, these groups showed higher production of IFN-c and IL-4, together with a significant reduction in IL-2 concentration. As stated above, high production of IL-4 can inhibit and regulate the bioactivity of IFN-c; IL-2 alone can activate natural killer cells [44], which can eliminate infected cells through cytotoxicity in vivo and in vitro [8, 45, 46]. Therefore, the low levels of IL-2 in the F3 and F4-immunized groups could be responsible for the decrease in the number of IL2-activated NK cells, thus directing these mice to a susceptibility pole. In addition, F3 and F4-immunization induced mainly IgG1 isotype production. Thus, the F3 and F4-immunized groups showed exacerbated lesions associated with cell proliferation, an increase in the levels of IFN-c, IL-4, IgG1 and a reduction in IL-2. Immunization with F2 antigen did not alter the course of infection; however, this group exhibited changes in immunological patterns. In this case, proliferation also was detected; stimulation with a specific antigen triggered cells to produce low levels of IL-2, IL-4 and IFN-c. IL-4 is able to control a series of immunological mechanisms associated to susceptibility [47], thus low IL-4 levels concomitant with small concentrations of IL-2 and IFN-c could be associated with skin parasitism similar to non-immunized mice. Thus, this immunization induced no exacerbated lesions. IL-12 cytokine can drive the immune response towards an efficient cellular immunity pole. Therefore, in inflammatory processes, the absence of this cytokine can depress cellular immunity, favoring intracellular establishment of the pathogen [7]. In experimental infections by L. major and L. (V.) braziliensis, the pivotal role of IL-12 demonstrates a direct relationship to the immunity of BALB/c mice and resistance profiles [48, 49]. On the other hand, experiments have demonstrated that IL-12-treated C57BL/6 and


C3H mice could not control infections caused by L. (L.) mexicana and L. (L.) amazonensis, respectively [48, 50]. These studies have indicated that some parasite species are able to modulate IL-12 production in the host. On the other hand, other factors, such as IFN-c, iNOS, and STAT4, acting independently of IL-12, seem to be essential to eliminate intracellular amastigotes [50]. In L. (V.) shawi infection, the immunological response can be similar to L. (L.) amazonensis and L. (L.) mexicana; therefore IL-12 was not a requirement to induce protection in F1- and F5immunized groups. Possibly, other cytokines, such as IL-23 and IL-27, should stimulate IFN-c production [51, 52], leading to parasite destruction. The present work shows that antigens derived from L. (V.) shawi promastigotes can interfere with the course of infection through modulation of the immunological response in mice, similarly with antigens derived from L. (V.) braziliensis. In addition, these effects ranged from protection to disease exacerbation, associated with an increase in both the Th1 and Th2 immune responses. Analysis of the data indicated that, even in the presence of high levels of IFN-c, a diminished Th2 response is crucial to inducing protection; moreover, the results strongly suggest the participation of other components of innate immunity in protection, such as NK cells. Besides the demonstration of protective antigens in L. (V.) shawi promastigotes, the present study further discusses the idea that immunological factors associated with resistance and cure in Old World Leishmania sp. can be different in New World species, as observed in mice that suffered exacerbated lesions with high levels of IFN-c—a cytokine responsible for activating infected cells. This work reinforces the view point that disease manifestations caused by Leishmania sp. are generated by a complex network of immunological cells and mediators, and the modulation of specific components of parasites can be useful to develop important target molecules to achieve cure or, in some cases, disease progression. The present work thus described the isolation of a protective antigen of L. (V.) shawi promastigotes and its potential for the development of candidate vaccines. Acknowledgments This work was supported by the Fundaçaõ de Amparo a Pesquisa do Estado de Saõ Paulo (FAPESP) and HCFMUSPLIM50. It is part of the doctoral thesis of L.F.D.P. under the FAPESP fellowship. The authors of the present work would like to thank Prof. Dr. Fernando T. Silveira for kindly donating and identifying the L. (V.) shawi parasite (strain MHOM/BR/96/M15789) and, more importantly, encouraging studies involving the present parasite strain.

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