RESUMO
The golden hamster (Mesocricetus auratus) is a susceptible model to Leishmania (Viannia) spp.; however, available studies employ different infection protocols, which account for clinical and pathological presentation differences. Herein, L. (V.) braziliensis preparations were standardized to contain 10(4), 10(5), or 10(6) parasites to determine an optimal inoculum that ensured cutaneous lesions without causing a disseminated infection in hamsters. Lesion development was followed for 105 days by size measurements, and skin, draining lymph node, spleen, and sera were investigated to check parasite load, spleen visceralization, cytokine expression, histopathological changes, and anti-Leishmania IgG levels. The lesion emergence time was inversely proportional to the parasite concentration in the inocula. Animals infected by 10(4) parasites presented nodular lesions, while those infected with 10(6) parasites often exhibited ulcerated lesions. The differences in the final lesion sizes were observed between 10(4) and 10(5) inocula or 10(4) and 10(6) inocula. High IFNG expression, anti-Leishmania IgG levels, and parasite load occurred independently of the inoculum used. A mild inflammatory skin involvement was observed in animals infected with 10(4) parasites, while extensive tissue damage and parasite spleen visceralization occurred with 10(5) and 10(6) parasites. These results indicate that inocula with different concentrations of parasites generate differences in the time of lesion emergence, clinical presentation, and systemic commitment, despite high and similar IFNG expression and parasite load. This suggests that a modulation in the immune response to different parasite numbers occurs in an early phase of the infection, which could dictate the establishment and magnitude of the chronic phase of the disease.
Assuntos
Citocinas/análise , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/patologia , Carga Parasitária , Pele/patologia , Estruturas Animais/parasitologia , Estruturas Animais/patologia , Animais , Anticorpos Antiprotozoários/sangue , Modelos Animais de Doenças , Feminino , Histocitoquímica , Imunoglobulina G/sangue , Leishmaniose Cutânea/parasitologia , Linfonodos/parasitologia , Linfonodos/patologia , Mesocricetus , Pele/parasitologia , Baço/parasitologia , Baço/patologia , Fatores de TempoRESUMO
Leishmaniasis is a wide-spectrum disease caused by parasites from Leishmania genus. A well-modulated immune response that is established after the long-lasting clinical cure of leishmaniasis can represent a standard requirement for a vaccine. Previous studies demonstrated that Leishmania (Viannia) naiffi causes benign disease and its antigens induce well-modulated immune responses in vitro. In this work we aimed to identify the immunodominant proteins present in the soluble extract of L. naiffi (sLnAg) as candidates for composing a pan-specific anti-leishmaniasis vaccine. After immunoblotting using cured patients of cutaneous leishmaniasis sera and proteomics approaches, we identified a group of antigenic proteins from the sLnAg. In silico analyses allowed us to select mildly similar proteins to the host; in addition, we evaluated the binding potential and degree of promiscuity of the protein epitopes to HLA molecules and to B-cell receptors. We selected 24 immunodominant proteins from a sub-proteome with 328 proteins. Homology analysis allowed the identification of 13 proteins with the most orthologues among seven Leishmania species. This work demonstrated the potential of these proteins as promising vaccine targets capable of inducing humoral and cellular pan-specific immune responses in humans, which may in the future contribute to the control of leishmaniasis.
RESUMO
The golden hamster is a suitable model for studying cutaneous leishmaniasis (CL) due to Leishmania (Viannia) braziliensis. Immunopathological mechanisms are well established in the L. (L.) major-mouse model, in which IL-4 instructs a Th2 response towards progressive infection. In the present study, we evaluated the natural history of L. braziliensis infection from its first stages up to lesion establishment, with the aim of identifying immunological parameters associated with the disease outcome and parasitism fate. To this end, hamsters infected with 104, 105, or 106 promastigotes were monitored during the first hours (4h, 24h), early (15 days, 30 days) and late (50 days) post-infection (pi) phases. Cytokines, iNOS and arginase gene expression were quantified in the established lesions by reverse transcription-quantitative PCR. Compared to the 105 or 106 groups, 104 animals presented lower lesions sizes, less tissue damage, and lower IgG levels. Basal gene expression in normal skin was high for TGF-ß, and intermediary for TNF, IL-6, and IL-4. At 4hpi, no cytokine induction was observed in the 104 group, while an upregulation of IL-6, IL-10, and IL-4 was observed in the 106 group. At 15dpi, lesion appearance was accompanied by an increased expression of all assessed cytokines, markedly in the 105 and 106 groups. Upregulation of all investigated cytokines was observed in the late phase, although less expressive in the 104 group. IFN-γ was the depending variable influencing tissue damage, while IL-6 was associated to parasite load. The network correlating gene expression and clinical and laboratorial parameters indicated inoculum-independent associations at 15 and 30dpi. A strong positive network correlation was observed in the 104 group, but not in the 105 or 106 groups. In conclusion, IL-4, IL-6, IL-10, and TGF-ß are linked o L. braziliensis progression. However, a balanced cytokine network is the key for an immune response able to reduce the ongoing infection and reduce pathological damage.
Assuntos
Citocinas/metabolismo , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Transdução de Sinais , Animais , Biomarcadores , Biologia Computacional/métodos , Cricetinae , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Expressão Gênica , Interações Hospedeiro-Parasita/imunologia , Imunomodulação , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Carga ParasitáriaRESUMO
Leishmania (Viannia) braziliensis is one of the main etiological agents of tegumentary leishmaniasis in Latin America. The establishment of a successful infection in host cells requires several key events including phagocytosis, phagolysosomal maturation impairment, and parasite replication. Autophagy is accountable for the physiological turnover of cellular organelles, degradation of macromolecular structures, and pathogen elimination. In many cases, autophagy control leads to a successful infection, both impairing pathogen elimination or providing nutrients. Here, we have investigated the relationship between autophagy and L. braziliensis infection. We observed that BECLIN1 expression was upregulated early on infection in both in vitro macrophage cultures and biopsies of cutaneous lesions from L. braziliensis infected patients. On the other hand, LC3B expression was downregulated in cutaneous lesions biopsies. A transient pattern of LC3+ cells was observed along L. braziliensis infection, but the number of LC3 puncta did not vary. Additionally, autophagy induction, with rapamycin treatment or through starvation, reduced infection. As expected, rapamycin increased the percentage of LC3+ cells and the number of puncta, but the presence of parasite restricted this effect, indicating LC3-associated autophagy impairment by L. braziliensis. Finally, silencing LC3B but not BECLIN1 promoted infection, confirming BECLIN1 independent and LC3B-related control by the parasite. Taken together, these data indicate macrophage autophagic machinery manipulation by L. braziliensis, resulting in successful establishment and survival into the host cell.
Assuntos
Autofagia , Leishmania braziliensis/fisiologia , Leishmaniose Cutânea/imunologia , Macrófagos/citologia , Macrófagos/parasitologia , Animais , Proteína Beclina-1/metabolismo , Feminino , Humanos , Leishmaniose Cutânea/metabolismo , Macrófagos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , FagocitoseRESUMO
In this study we determined whether exposing mice to hyperbaric oxygen (HBO) would alter various disease parameters of a susceptible mouse strain infected with Leishmania amazonensis. BALB/c mice exposed to HBO (100% O2 at a pressure of 2.5 ATA, 1h before parasite inoculation and subsequently for 20 days) showed significant delay in lesion development and reduction in lesion parasite burdens compared with HBO-unexposed mice. Circulating levels of interferon gamma (IFN-gamma) and tumor necrosis factor (TNF-alpha) were significantly elevated in HBO-exposed as compared to HBO-unexposed mice. Concanavalin A-stimulated lymph nodes cultures from HBO-exposed mice released significantly more IFN-gamma and less interleukin 10 (IL-10) than cultures from HBO-unexposed mice, consistent with a skewed Th1 response. These results demonstrate, for the first time, that HBO can play a pathogen control role during leishmaniasis. Further studies are needed to elucidate whether hyperoxia alone or increased atmospheric pressure alone can exert a similar effect.
Assuntos
Oxigenoterapia Hiperbárica/métodos , Leishmania/crescimento & desenvolvimento , Leishmaniose/terapia , Infecções dos Tecidos Moles/terapia , Animais , Feminino , Histocitoquímica , Interferon gama/sangue , Interleucina-10/imunologia , Leishmaniose/imunologia , Leishmaniose/parasitologia , Leishmaniose/patologia , Linfonodos/imunologia , Linfonodos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções dos Tecidos Moles/imunologia , Infecções dos Tecidos Moles/parasitologia , Infecções dos Tecidos Moles/patologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: Cutaneous leishmaniasis (CL) is a neglected disease with a broad spectrum of clinical manifestations, ranging from small cutaneous nodules to severe mucosal tissue destruction. Leishmania (Viannia) braziliensis is the main species attributed to CL in the Americas. However, studies of experimental infection are limited in the murine model due to the self-resolutive pattern of the disease. Previously, our group demonstrated that the hamster model reproduces many of the clinical and histopathological features observed in humans. Herein, we standardized a RT-qPCR gene expression assay to evaluate a panel of immunological markers and a qPCR assay in order to quantify with high sensitivity and reproducibility the parasite load in skin lesions. METHODS: Hamsters were intradermally infected in the footpad with 10(5) promastigotes of L. (V.) braziliensis and 110 days post-infection skin lesions and popliteal lymph nodes were removed for RNA and DNA extraction, both from the same tissue fragment. Gene expression of IFN-É£, IL-10, TGF-ß TNF, IL-4, IL-6, iNOS and arginase were measured using non-infected animal tissue as a calibrator. Parasite load was quantified from DNA extracted from lesions by qPCR targeting Leishmania kDNA and normalized by hamster GAPDH, using a SYBR Green-based absolute quantification methodology. RESULTS: A relative quantification RT-qPCR assay was standardized for the evaluation of mRNA levels from skin and lymph node samples of golden hamsters, with PCR efficiencies ranging from 92.3 to 116.4 %. In uninfected animals, higher basal mRNA levels in lymph nodes were observed for IFN-É£, TGF-ß, TNF and IL-4 (111.4 ± 92.2; 5.6 ± 1.2; 5.3 ± 1.7; and 60.3 ± 26.8, respectively) in comparison to skin. In golden hamsters infected with L. (V.) braziliensis, an increase in the expression of all immunological markers evaluated was observed, ranging from 2.7 ± 0.2 for TGF-ß to 1018.5 ± 809.0 for iNOS in skin lesions, and 2.4 ± 1.6 for TGF-ß to 600.2 ± 666.4 for iNOS in popliteal lymph nodes. Interestingly, significantly higher levels of IFN-É£, TNF and IL-10 mRNA were observed in skin in comparison to lymph nodes, while a lower significant level of arginase mRNA was observed in skin. In parallel, parasite loads were quantified by qPCR from the skin lesions of infected animals, ranging from 27.0 to 6647.0, with a median of 553.4 (416.7-1504.0) parasites/mg skin equivalents, whereas lesion size varied from 0.3 to 3.1 mm. Despite the tendency of larger lesions to present higher parasite load, the correlation observed was not statistically significant. CONCLUSIONS: In this study, we describe for the first time a sensitive, reproducible and cheaper molecular assay to quantify from the same tissue fragment the gene expression of immunological markers and the parasite load in skin lesions, observing a mixed profile of immune response in the hamster model infected by L. (V.) braziliensis.
Assuntos
Leishmania braziliensis , Leishmaniose Cutânea/imunologia , Carga Parasitária , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Cricetinae , Citocinas/genética , Citocinas/metabolismo , DNA de Protozoário/análise , DNA de Protozoário/genética , Feminino , Regulação da Expressão Gênica , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/parasitologia , Mesocricetus , RNA Mensageiro , RNA de Protozoário , Sensibilidade e Especificidade , Pele/parasitologiaRESUMO
Leishmania amazonensis and Leishmania braziliensis are the main causal agents of anergic diffuse cutaneous leishmaniasis and hyperergic mucosal leishmaniasis in man, respectively. In this work we demonstrate that intramuscular vaccination of BALB/c mice with whole antigens of L. amazonensis (LaAg) but not L. braziliensis (LbAg) results in increased susceptibility to cutaneous leishmaniasis. LaAg vaccination resulted in an increased capacity of the draining lymph nodes to produce IL-10 and TGF-beta during antigen recall responses. In vitro cultivation with LaAg but not LbAg induced increased apoptosis of CD8+ T cells. Following infection with L. amazonensis, LaAg-vaccinated mice produced significantly more TGF-beta and a higher serum IgG1/IgG2a antibody ratio compared with LbAg-vaccinated and non-vaccinated animals. The association of TGF-beta with enhanced susceptibility to infection was confirmed in mice co-vaccinated with LaAg and neutralizing anti-TGF-beta antibodies. Upon parasite challenge, these animals developed much smaller lesion sizes and parasite burdens, comparable with non-vaccinated controls. The disease-promoting effect of LaAg vaccination is not a general event, as in contrast to BALB/c, the disease outcome in C57Bl/6 mice was unaltered. Together, these findings indicate that species-specific components of L. amazonensis activate overt TGF-beta production that predisposes more susceptible individuals to aggravated disease following vaccination.
Assuntos
Antígenos de Protozoários/administração & dosagem , Apoptose/imunologia , Leishmania/imunologia , Leishmaniose/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Antígenos de Protozoários/imunologia , Suscetibilidade a Doenças , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos BALB C , VacinaçãoRESUMO
BACKGROUND: Previous results have shown that oral and intranasal administration of particulate Leishmania (Leishmania) amazonensis antigens (LaAg) partially protects mice against L. amazonensis infection. However, vaccination studies on species of the subgenus Viannia, the main causative agent of cutaneous and mucosal leishmaniasis in the Americas, have been hampered by the lack of easy-to-handle bio-models that accurately mimic the human disease. Recently, we demonstrated that the golden hamster is an appropriate model for studying the immunopathogenesis of cutaneous leishmaniasis caused by L. (Viannia) braziliensis. Using the golden hamster model, our current study investigated whether the protective effect of intranasal immunisation with LaAg can be extended to L. braziliensis infection. METHODOLOGY/PRINCIPAL FINDINGS: Golden hamsters vaccinated with either two intranasal (IN) doses of LaAg (10 µg) or two intramuscular doses of LaAg (20 µg) were challenged 2 weeks post-vaccination with L. braziliensis. The results showed that IN immunisation with LaAg significantly reduced lesion growth and parasitic load as well as serum IgG and IgG2 levels. At the experimental endpoint on day 114 post-infection, IN-immunised hamsters that were considered protected expressed IFN-γ and IL10 mRNA levels that returned to uninfected skin levels. In contrast to the nasal route, intramuscular (IM) immunisation failed to provide protection. CONCLUSIONS/SIGNIFICANCE: These results demonstrate for the first time that the nasal route of immunisation can induce cross protection against L. braziliensis infection.
Assuntos
Antígenos de Protozoários/imunologia , Leishmania braziliensis/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Cutânea/prevenção & controle , Administração Intranasal , Animais , Anticorpos Antiprotozoários , Cricetinae , Imunoglobulina G/sangue , Interferon gama/metabolismo , Vacinas contra Leishmaniose/administração & dosagem , Carga Parasitária , Pele/metabolismo , Pele/parasitologiaRESUMO
The objective of this work was to verify if hydrophilic gels containing benzophenone-3 loaded nanocapsules (HG-NCBZ3) could improve the sunscreen in vitro effectiveness against UVA radiation and its photostability compared to a conventional hydrogel containing the free sunscreen (HG-BZ3). In parallel, the immune response of the nanostructured system was evaluated by mouse ear swelling test and the local lymph node assay. The nanocapsules were prepared by interfacial deposition of poly(epsilon-caprolactone) and characterized in terms of particle size, polydispersity index, zeta potential, drug content and encapsulation efficiency. HG-NCBZ3 UV scans showed higher absorption intensity values than HG-NCplacebo, prepared using unloaded nanocapsules. Films of the gels were irradiated with UVA light and the BZ3 recovery was evaluated by HPLC. BZ3 recovery decreased from 100% to 29% for HG-BZ3 and to 56% for HG-NCBZ3 after 13 h. After wavelength scans within 13 h, the relative areas under the curves (AUC) decreased from 1.00 to 0.62 for HG-BZ3 and remained constant for HG-NCBZ3. Sensitization assay showed that stimulation indexes lower than 3 for all the hydrogel samples. Formulations did not induce increases higher than 10% in ear swelling, indicating that the hydrogels did not cause cutaneous sensitization in mice. The nanoencapsulation improved both the photostability and the effectiveness of BZ3 compared to the non-encapsulated sunscreen and the topical application of free and nanoencapsulated BZ3 did not produce significant allergy response in mice.
Assuntos
Portadores de Fármacos/química , Toxidermias/imunologia , Nanoestruturas/química , Protetores Solares/administração & dosagem , Animais , Portadores de Fármacos/efeitos adversos , Portadores de Fármacos/efeitos da radiação , Toxidermias/diagnóstico , Luz , Teste de Materiais , Camundongos , Nanoestruturas/efeitos adversos , Nanoestruturas/efeitos da radiação , Pós , Protetores Solares/efeitos adversosRESUMO
We previously showed that intranasal (i.n.) vaccination with pCIneo plasmid encoding the leishmanial LACK gene (pCIneo-LACK) induces long-lasting protective immunity against cutaneous leishmaniasis in mice. In this work, we proposed to investigate whether the efficacy of i.n. pCIneo-LACK is extensive to visceral leishmaniasis. BALB/c mice received two i.n. doses of 30 microg pCIneo-LACK prior to intravenous (i.v.) infection with Leishmania chagasi. Vaccinated mice developed significantly lower parasite burden in the liver and spleen than control mice receiving empty pCIneo or saline. The spleen cells of vaccinated mice produced significantly increased IFN-gamma and IL-4 concomitant with decreased IL-10 production during infection. Serum levels of specific IgG were elevated whereas TNF-alpha were decreased as compared with controls. These results show that the practical needle-free i.n. pCIneo-LACK vaccine displays potential broad-spectrum activity against leishmaniasis.
Assuntos
Antígenos de Protozoários/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Visceral/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Administração Intranasal , Animais , Antígenos de Protozoários/genética , DNA de Protozoário/genética , Interferon gama/metabolismo , Interleucina-4/metabolismo , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/prevenção & controle , Leishmaniose Visceral/genética , Fígado/efeitos dos fármacos , Fígado/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/administração & dosagem , Proteínas de Protozoários/genética , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/genética , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/parasitologia , Resultado do TratamentoRESUMO
We previously showed the opposing effect of systemic and mucosal vaccination with whole Leishmania amazonensis antigen (LaAg). Here, the role played by lipophosphoglycan (LPG) as the key disease-promoting component of intramuscular (i.m.) LaAg and its usefulness as a defined intranasal vaccine was investigated in murine cutaneous leishmaniasis. BALB/c mice were twice vaccinated by the i.m. route with 25mug of intact LaAg or with LaAg that was pretreated with anti-LPG 3A1-La monoclonal antibody, prior to infection with L. amazonensis. LPG neutralization rendered the otherwise disease-promoting LaAg antigen protective, as observed by the smaller lesion sizes and reduced parasite burden. The increased resistance was accompanied by a markedly lower antigen-driven TGF-beta and IL-10 responses in the lesion-draining lymph nodes, concomitant with significantly higher IFN-gamma production. To test for intranasal efficacy, 10 microg of affinity-purified LPG and its parental LaAg were twice instilled in the nostrils prior to L. amazonensis infection. In both cases, similarly slower lesion growth and lower parasite burden were found that was associated with increased IFN-gamma and IL-10 responses in the lesion-draining lymph nodes. These results support a role for LPG in the dual route-related effect of LaAg and shows its strong potential as a defined needle-free and adjuvant-free vaccine for cutaneous leishmaniasis.
Assuntos
Antígenos de Protozoários/imunologia , Glicoesfingolipídeos/imunologia , Leishmania mexicana/imunologia , Leishmaniose Cutânea/prevenção & controle , Vacinas Protozoárias/administração & dosagem , Administração Intranasal , Animais , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Protozoárias/imunologia , VacinaçãoRESUMO
The induction of oral tolerance against disease-inducing antigens has emerged as a feasible strategy to prevent immunopathologies. In this study, we investigated the effect of oral immunization with whole antigens of Leishmania amazonensis promastigotes (LaAg) on murine cutaneous leishmaniasis. We found that two oral doses with 100 microg LaAg rendered BALB/c and C57BL/6 mice more resistant against subsequent infection with L. amazonensis. The oral vaccine also partially protected BALB/c mice against Leishmania major infection. Unlike the oral route, hepatic immunization was without effect, indicating a requirement for antigen passage through the gut mucosa. Oral LaAg significantly impaired the capacity of infected BALB/c mice to mount a disease-associated hypersensitivity response, compatible with peripheral tolerization. Both IFN-gamma and IL-10, but not IL-4 were greatly elevated in the mesenteric lymph nodes whereas only IFN-gamma was increased in the peripheral lymph nodes, compatible with a TH1 cytokine response. Gamma delta TCR+ T cells may be an important component in antigenic sensitization of the gut mucosa since their depletion during oral immunization reverted protection. These results demonstrate for the first time the feasibility of using the oral route of immunization to induce protection against cutaneous leishmaniasis using a crude parasite antigen.
Assuntos
Indutores de Interferon/farmacologia , Interferon gama/biossíntese , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/prevenção & controle , Vacinas Protozoárias/imunologia , Vacinação , Administração Oral , Animais , Antígenos de Protozoários/imunologia , Citocinas/biossíntese , Hipersensibilidade a Drogas/imunologia , Injeções , Injeções Subcutâneas , Leishmaniose Cutânea/patologia , Fígado/fisiologia , Linfonodos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/efeitos adversos , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Pele/patologia , Linfócitos T/imunologiaRESUMO
We have previously demonstrated that oral delivery of a disease-promoting particulated antigen of Leishmania amazonensis (LaAg) partially protects mice against cutaneous leishmaniasis. In the present work, we sought to optimize a mucosal vaccine by using the intranasal route for delivery of different antigen preparations, including (i) LaAg, (ii) soluble recombinant p36/LACK leishmanial antigen (LACK), and (iii) plasmid DNA encoding LACK (LACK DNA). BALB/c mice that received two intranasal doses of 10 microg of LaAg and were challenged 1 week postvaccination with L. amazonensis developed delayed but effective control of lesion growth. A diminished parasite burden was accompanied by enhancement of both gamma interferon (IFN-gamma) and interleukin-10 levels in the lesion-draining lymph nodes. The vaccine efficacy improved with time. At 4 months postvaccination, when a strong parasite-specific TH1-type response was present in vivo, the infection was controlled for at least 5 months after challenge. In contrast to the particulated LaAg, soluble LACK (10 microg/dose) had no effect. Interestingly, LACK DNA (30 microg/dose), but not empty DNA, promoted rapid and durable protective immunity. Parasite growth was effectively controlled, and at 5 months after challenge LACK-reactive cells in both the mucosal and lesion-draining lymph nodes produced high levels of IFN-gamma. These results demonstrate for the first time the feasibility of using the intranasal route for long-lived memory vaccination against cutaneous leishmaniasis with adjuvant-free crude antigens or DNA.