RESUMO
Leishmaniasis is a vector-borne infectious disease caused by multiple Leishmania (L.) species with diverse clinical manifestations. There is currently no vaccine against any form of the disease approved in humans, and chemotherapy is the sole approach for treatment. Unfortunately, treatment options are limited to a small number of drugs, partly due to high cost and significant adverse effects. The other obstacle in leishmaniasis treatment is the potential for drug resistance, which has been observed in multiple endemic countries. Immunotherapy maybe another important avenue for controlling leishmaniasis and could help patients control the disease. There are different approaches for immunotherapy in different infectious diseases, generally with low-cost, limited side-effects and no possibility to developing resistance. In this paper, different immunotherapy approaches as alternatives to routine drug treatment will be reviewed against leishmaniasis.
Assuntos
Imunoterapia/métodos , Leishmania/imunologia , Leishmaniose/imunologia , Leishmaniose/terapia , Animais , Antiprotozoários/administração & dosagem , Antiprotozoários/efeitos adversos , Antiprotozoários/economia , Antiprotozoários/uso terapêutico , Ensaios Clínicos como Assunto , Citocinas/uso terapêutico , Modelos Animais de Doenças , Resistência a Medicamentos , Humanos , Fatores Imunológicos/uso terapêutico , Imunoterapia/efeitos adversos , Imunoterapia/economia , Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Vacinas contra Leishmaniose/imunologia , CamundongosRESUMO
Recent findings have demonstrated the suitability of interferon-gamma-induced protein 10 (IP-10) or CXCL-10 as an immunotherapy tool in treatment of leishmaniasis. This chemokine can overcome Leishmania (L.) infection through inducing nitric oxide (NO) production for parasite elimination. This study was undertaken to investigate the therapeutic effects of recombinant Leishmania tarentolae expressing CXCL-10 and an expression vector encoding CXCL-10 (pcDNA-CXCL-10-EGFP) in a model of BALB/c mice susceptible to infection by Leishmania major. The outcome of intervention was examined at 3 weeks post-treatment by evaluating the parameters of parasite burden (PB), arginase activity, NO and various cytokines such as IFN-γ, IL-4, IL-6 and IL-10. The results have shown that despite the efficacy of CXCL-10 expression vector as gene therapy, the live therapy strategy using L. tarentolae expressing CXCL-10 was more effective in terms of decreasing PB. Nitric oxide production increased, especially in the live therapy approaches. Arginase activity also decreased in all regimens, which demonstrates the potency of the treatment. The overall cytokine production shifted in favour of Th1 responses in the treated mice. Altogether, recombinant L. tarentolae expressing CXCL-10 represents a promising therapeutic strategy to improve treatment of cutaneous leishmaniasis.
Assuntos
Quimiocina CXCL10/genética , Terapia Genética/métodos , Imunoterapia/métodos , Leishmania major/imunologia , Leishmaniose Cutânea/terapia , Óxido Nítrico/metabolismo , Animais , Arginase/metabolismo , Quimiocina CXCL10/biossíntese , Interferon gama/imunologia , Interleucina-10/imunologia , Interleucina-4/imunologia , Interleucina-6/imunologia , Leishmania major/genética , Leishmania major/metabolismo , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Células Th1/imunologiaRESUMO
Cutaneous leishmaniasis (CL) is one of the most important vector-borne parasitic diseases, highly endemic in Iran, and its prevalence is increasing all over the country. Arginase (ARG) activity in isolated Leishmania parasites from CL patients is yet to be explored. This study aimed to compare the ARG activity of isolated Leishmania promastigotes from CL patients with a standard strain of Leishmania major and its influences on the disease pathogenesis. We recruited 16 confirmed CL patients from Qom Province, in central Iran; after detection of Leishmania species using PCR-RFLP, we assessed the levels of ARG in the isolated promastigotes and determined the parasites' growth rate. Only L. major was identified from CL patients. The level of ARG activity in the isolated Leishmania promastigotes from CL patients was significantly higher than that obtained from the standard strain of L. major. No significant correlations between ARG activity and lesion size, number or duration were observed; in contrast, a significant negative correlation was seen between ARG level and Leishmania' growth rate. The obtained results suggest that increased ARG expression and activity in the isolated Leishmania promastigotes might contribute to the higher parasite infectivity and play a major role in the pathogenicity of the CL.
Assuntos
Arginase/metabolismo , Leishmania/enzimologia , Leishmaniose Cutânea/parasitologia , Adulto , Arginase/genética , Feminino , Humanos , Irã (Geográfico)/epidemiologia , Leishmania/crescimento & desenvolvimento , Leishmania/isolamento & purificação , Leishmania/patogenicidade , Leishmania major/enzimologia , Leishmania major/crescimento & desenvolvimento , Leishmania major/isolamento & purificação , Leishmania major/patogenicidade , Leishmaniose Cutânea/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Adulto JovemRESUMO
Leishmaniasis caused by protozoan parasites of the genus Leishmania. Intracellular infections treatment such as leishmaniasis is frequently hampered by limited access of drugs to infected cells. Moreover, most of the current drugs are confined to some toxic compounds, and there are increasing incidences of development of drug resistance. Hence, production of a new antileishmanial compound is crucial. Paromomycin sulphate (PM) is a promising antileishmanial drug. One strategy to improve drug effectiveness is to use appropriate delivery systems. Solid lipid nanoparticle (SLN) is as an excellent substitute delivery system to other colloidal carrier. In the present study, PM was loaded in solid lipid nanoparticles (PM-SLN) and the in vivo efficacy was studied against Leishmania (L.) major-infected BALB/c mice. For this reason, the footpad swelling was measured and real-time PCR was performed to quantify the parasite load after infectious challenge. The level of cytokines including interleukin-4 (IL-4) and gamma interferon (IFN-γ) and nitric oxide was evaluated. Altogether, this study showed that the PM-SLN formulation is a safe compound and SLN in PM-SLN compound is effective for treatment of leishmaniasis by improving the effectiveness of PM in killing the parasite and switching towards Th1 response.
Assuntos
Antiprotozoários/farmacologia , Composição de Medicamentos , Leishmania/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Nanopartículas , Paromomicina/farmacologia , Animais , Antiprotozoários/administração & dosagem , Citocinas/metabolismo , Modelos Animais de Doenças , Portadores de Fármacos , Feminino , Interferon gama/metabolismo , Interleucina-4/metabolismo , Leishmaniose/parasitologia , Lipídeos , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Paromomicina/administração & dosagemRESUMO
BACKGROUND: Cutaneous leishmaniasis (CL) in Iran is mainly caused by Leishmania major (L. major) and L. tropica. Arginase mediated L-arginine metabolism is an important issue in Leishmania parasite propagation. Arginase activity in human CL due to L. major and L. tropica have not been studied up to now. OBJECTIVES: We aimed to compare the clinical and laboratory aspects of acute and chronic CL, focussing on arginase activity. METHODS: In this case-control study, 30 patients with acute CL (duration ≤ 1 year), 13 patients with chronic CL (duration ≥ 2 year) and 11 healthy controls were recruited. Arginase activity was measured in skin biopsies of lesions, peripheral blood polymorphonuclear cells (PMNs), peripheral blood mononuclear cells (PBMCs) and plasma by standard methods. RESULTS: The median of arginase activity in the acute lesions was higher than in chronic samples and significantly higher than in healthy controls (P = 0.008). PMNs of both acute and chronic patients showed higher levels of arginase activity as compared to the levels in PBMCs and plasma. The median of arginase activity in the PMNs of patients with chronic CL was higher than that of patients with acute CL and significantly higher than that of the healthy controls (P = 0.010). CONCLUSION: The level of arginase activity in lesions of patients with acute and chronic CL was higher than the skin of healthy controls. The highest level of arginase activity was observed in PMNs from patients with chronic CL. This suggests that the high level of arginase activity in PMNs of patients with chronic CL may contribute to the chronicity.
Assuntos
Arginase/metabolismo , Leishmania major/patogenicidade , Leishmania tropica/patogenicidade , Leishmaniose Cutânea/metabolismo , Doença Aguda , Estudos de Casos e Controles , Doença Crônica , Humanos , Leishmaniose Cutânea/psicologiaRESUMO
The use of an appropriate delivery system has recently emerged as a promising approach for the development of effective vaccination against visceral leishmaniasis (VL). Here, we compare two vaccine delivery systems, namely electroporation and cationic solid-lipid nanoparticle (cSLN) formulation, to administer a DNA vaccine harbouring the L. donovani A2 antigen along with L. infantum cysteine proteinases [CPA and CPB without its unusual C-terminal extension (CPB(-CTE) )] and evaluate their potential against L. infantum challenge. Prime-boost administration of the pcDNA-A2-CPA-CPB(-CTE) delivered by either electroporation or cSLN formulation protects BALB/c mice against L. infantum challenge and that protective immunity is associated with high levels of IFN-γ and lower levels of IL-10 production, leading to a strong Th1 immune response. At all time points, the ratio of IFN-γ: IL-10 induced upon restimulation with rA2-rCPA-rCPB and F/T antigens was significantly higher in vaccinated animals. Moreover, Th2-efficient protection was elicited through a high humoral immune response. Nitric oxide production, parasite burden and histopathological analysis were also in concordance with other findings. Overall, these data indicate that similar to the electroporation delivery system, cSLNs as a nanoscale vehicle of Leishmania antigens could improve immune response, hence indicating the promise of these strategies against visceral leishmaniasis.
Assuntos
Eletroporação , Vacinas contra Leishmaniose/administração & dosagem , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/prevenção & controle , Nanopartículas , Vacinas de DNA/administração & dosagem , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Cisteína Proteases/genética , Cisteína Proteases/imunologia , Feminino , Imunidade Humoral , Imunização Secundária , Imunoglobulina G/sangue , Interferon gama/biossíntese , Interleucina-10/biossíntese , Leishmania donovani/imunologia , Leishmania donovani/fisiologia , Leishmania infantum/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/parasitologia , Lipídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Vacinação , Vacinas de DNA/imunologiaRESUMO
To control cervical cancer, efficient vaccination against human papillomavirus (HPV) is highly required. Despite the advantages and safety of the protein vaccines, additional strategies to enhance their immunogenicity are needed. E7 is a transforming protein which represents a perfect target antigen for vaccines or immunotherapies. Heat shock proteins (HSPs) facilitate cellular immune responses to antigenic peptides or proteins bound to them. Regarding to previous studies, vaccination with purified HSP/antigen complexes efficiently elicit antigen-specific immune responses in mice model. The N-terminal of glycoprotein 96 (NT-gp96) has adjuvant effect and can induce effective cumulative immune response against clinical disorders, especially cancers. In this study, the recombinant HPV16 E7 and E7 linked to NT-gp96 (E7-NT-gp96) proteins were generated in prokaryotic expression system. Mice were vaccinated twice with this recombinant proteins and the immunogenicity of the fusion protein was determined. The preventive efficacy of E7-NT-gp96 fusion protein was also evaluated and compared to E7 protein after challenging with cancerous TC-1 cell line. In vitro re-stimulated splenocytes of mice vaccinated with rE7-NT-gp96 protein induced higher IFN-γ response in comparison with E7 protein immunization. Moreover, immunization with E7-NT-gp96 protein displayed low but stable humoral responses at post-challenge time. The data showed that vaccination with fused E7-NT-gp96 protein delayed the tumour occurrence and growth as compared to protein E7 alone. These results suggest that fused adjuvant-free E7-NT-gp96 protein vaccination could direct the immune responses towards Th1 immunity. Furthermore, the linkage of NT-gp96 to E7 could enhance protective anti-tumour immunity.
Assuntos
Glicoproteínas/imunologia , Papillomavirus Humano 16/imunologia , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/imunologia , Neoplasias do Colo do Útero/virologia , Vacinas Virais/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Feminino , Glicoproteínas/genética , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Imunização/métodos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/prevenção & controle , Infecções por Papillomavirus/virologia , Estatísticas não Paramétricas , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/prevenção & controle , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/farmacologia , Vacinas Virais/genética , Vacinas Virais/farmacologiaRESUMO
Stimulation of neutrophils may potentiate immunity to Leishmania major. CpG-containing oligodeoxynucleotide (ODN) has immune stimulatory effects and has been suggested as adjuvants and therapeutics to potentiate efficacy of vaccines and treatments against leishmaniasis. Here, we examined the stimulatory effect of synthetic ODN containing CpG motifs class A and B on cytokine production by neutrophils. Neutrophils from healthy donors responded to CpG-ODN type A, but not to class B, with secretion of IL-8 and following GM-CSF pretreatment with TNF-α production. To test whether neutrophil responses were altered in cutaneous leishmaniasis (CL) and to better understand the role of neutrophils in susceptibility and resistance to disease, we evaluated cytokine responses in GM-CSF preconditioned neutrophils from asymptomatic (Leishmanin skin test positive, LST+) and nonhealing CL individuals to CpG-ODN class A and assessed the expression levels of toll-like receptors (TLR2), 4 and 9. LST+ and healthy donor, but not nonhealing CL neutrophils, responded with TNF-α secretion. Neutrophils from nonhealing CL displayed increased mRNA expression levels of TLR2, 4 and 9 compared to neutrophils from LST+ or healthy donors. Therefore, failure to cure CL is associated with reduced ability of neutrophils to secrete TNF-α and correlates with high TLR 2, 4 and 9 expressions.
Assuntos
Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Ativação de Neutrófilo , Neutrófilos/imunologia , Oligodesoxirribonucleotídeos/imunologia , Adolescente , Adulto , Animais , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Interleucina-8/biossíntese , Interleucina-8/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Oligodesoxirribonucleotídeos/administração & dosagem , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor 2 Toll-Like/biossíntese , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/biossíntese , Receptor 4 Toll-Like/imunologia , Receptor Toll-Like 9/biossíntese , Receptor Toll-Like 9/imunologia , Fator de Crescimento Transformador beta/biossíntese , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Appropriate adjuvant, proper antigen(s) and a suitable formulation are required to develop stable, safe and immunogenic vaccines. Leishmanial cysteine proteinase type I (CPB) is a promising vaccine candidate; nevertheless, it requires a delivery system to induce a potent immune response. Herein, solid lipid nanoparticles (SLN) have been applied for CPB [with and without C-terminal extension (CTE)] formulation to utilize as a vaccine against Leishmania major infection in C57BL/6 mice. Therefore, SLN-CPB and SLN-CPB(-CTE) formulations were prepared from cetyl palmitate and cholesterol, using melt emulsification method. After intraperitoneal vaccination and subsequent L. major challenge, a strong antigen-specific T-helper type 1 (Th1) immune response was induced compared to control groups. Lymph node cells from immunized mice displayed lower parasite burden, higher IFN-γ, IgG2a and lower IL-4 production, indicating that robust Th1 immune response had been induced. Our results revealed that CTE is not necessary for inducing protective responses against L. major infection as the IFN-γ/IL-4 ratio was significantly higher, whereas IgG1 responses were lower in the SLN-CPB(-CTE) vaccinated group, post-challenge. Thus, SLN-CPB(-CTE) was shown to induce specific Th1 immune responses to control L. major infection, through effective antigen delivery to the peritoneal antigen presenting cells.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Cisteína Proteases/imunologia , Leishmania major/imunologia , Leishmaniose Cutânea/prevenção & controle , Lipossomos/administração & dosagem , Nanopartículas/administração & dosagem , Vacinas Protozoárias/imunologia , Adjuvantes Imunológicos/química , Animais , Cisteína Proteases/administração & dosagem , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Feminino , Imunoglobulina G/imunologia , Interferon gama/metabolismo , Interleucina-4/metabolismo , Leucócitos Mononucleares/imunologia , Lipossomos/química , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Vacinas Protozoárias/administração & dosagemRESUMO
Carboxy-terminus of merozoite surface protein-1 (MSP-1(19) ) is the major protein on the surface of the plasmodial merozoite that acts as one of the most important blood-stage vaccine candidates. The present investigation was designed to evaluate the immune responses when either two recombinant antigens (rPvMSP-1(19) + rPfMSP-1(19)) or two plasmid constructs (pcDNA3.1 hygro-PvMSP-1(19) + pcDNA3.1 hygro-PfMSP-1(19)) were administered in combination at a single site in mice by using different immunization strategies (protein/protein, DNA/DNA and DNA/protein) at weeks 0, 5 and 8. All mice were monitored for the level of MSP-1(19) -specific antibody for up to 40 weeks. The inclusion of both recombinant antigens in a vaccine mixture could not inhibit induction of antibodies to the other antigen when the two recombinant antigens were combined in immunization formulation. Interestingly, antisera from immunized mice with either recombinant antigen failed to cross-react with heterologous antigen. Moreover, the results of this study showed that co-immunization with both antigens at a single site generated a substantial PvMSP-1(19) - and PfMSP-1(19) -specific antibody responses and also IFN-γ cytokine production (Th1 response) in DNA/protein prime-boost immunization strategies. The increased humoral response to PvMSP-1(19) and PfMSP-1(19) lasted nearly a year after immunization. Therefore, the results of this study are encouraging for the development of multi-species malaria vaccine based on MSP-1(19) antigen.
Assuntos
Imunização/métodos , Vacinas Antimaláricas/imunologia , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium falciparum/imunologia , Plasmodium vivax/imunologia , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Células COS , Chlorocebus aethiops , Citocinas/biossíntese , Citocinas/imunologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Imunização Secundária , Interferon gama/biossíntese , Interferon gama/imunologia , Malária/imunologia , Malária/prevenção & controle , Proteína 1 de Superfície de Merozoito/administração & dosagem , Proteína 1 de Superfície de Merozoito/genética , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Células Th1 , Vacinas Sintéticas/imunologiaRESUMO
Leishmania protozoa are obligate intracellular parasites that reside in the phagolysosome of host macrophages and cause a large spectrum of pathologies to humans known as leishmaniases. The outcome of the disease is highly dependent on the parasite species and on its ascribed virulence factors and the immune status of the host. Characterization of the genome composition of non-pathogenic species could ultimately open new horizons in Leishmania developmental biology and also the disease monitoring. Here, we provide evidence that the lizard non-pathogenic to humans Leishmania tarentolae species expresses an Amastin-like gene, cysteine protease B (CPB), lipophosphoglycan LPG3 and the leishmanolysin GP63, genes well-known for their potential role in the parasite virulence. These genes were expressed at levels comparable to those in L. major and L. infantum both at the level of mRNA and protein. Alignment of the L. tarentolae proteins with their counterparts in the pathogenic species demonstrated that the degree of similarity varied from 59% and 60% for Amastin, 89% for LPG3 and 71% and 68% for CPB, in L. major and L. infantum, respectively. Interestingly, the A2 gene, expressed specifically by the L. donovani complex which promotes visceralization, was absent in L. tarentolae. These findings suggest that the lack of pathogenicity in L. tarentolae is not associated with known virulence genes such as LPG3, CPB, GP63 and Amastin, and that other factors either unique to L. tarentolae or missing from this species may be responsible for the non-pathogenic potential of this lizard parasite.
Assuntos
Leishmania/patogenicidade , Lagartos/parasitologia , Fatores de Virulência/genética , Animais , Biologia Computacional/métodos , DNA de Protozoário/análise , DNA de Protozoário/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Leishmania/classificação , Leishmania/genética , Leishmania major/genética , Leishmania major/patogenicidade , Macrófagos Peritoneais/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade da EspécieRESUMO
There are little information about Th17 cells and cutaneous Leishmaniasis (CL), due to an important effect of Th17 cells on immune response, it is worth to explore the role of Th17 on CL. The purpose of this study was to assess Th17 population in patients with acute vs. chronic CL lesions in comparison with skin samples collected from healthy volunteers in an endemic region of Old World CL. A total of 49 patients with clinical manifestations of chronic (n=16) and acute (n=33) CL lesions were recruited. The clinical diagnosis of CL was confirmed by direct smear or PCR. Biopsy specimens from prelesional skin of non-infectious lesions of 30 healthy individuals were used as control. Tissue sections of 3µm thickness were prepared and used for immunohistochemistry (IHC) analysis with primary antibody specific for Th17 associated antigen (CD161). For IHC, Envision+ (DakoCytomation) system was used and developed by using diaminobenzidine (DakoCytomation). The mean age of 33 patients with acute CL and the mean age of 16 patients with chronic CL were accordingly 45.24±16.43 and 33.56±15.87. In acute and chronic CL the mean (±standard deviation) and median (±interquartile range) were accordingly 2.92±2.21, 2.56±2.9 and 2.1±1.99, 1.54±2.81. In healthy controls the mean (±standard deviation) and median (±interquartile range) were 0.72±0.41 and 0.61±0.58 respectively. With pairwise comparison of acute, chronic and control groups, there were significant difference between acute and control (P value < 0.001), chronic and control (P value = 0.043). The results showed that there was an increasing cellular response of Th17 in both acute and chronic CL patients. Th17 was significantly higher in patients with acute and chronic CL lesions in comparison with healthy control group. However, there was no significant difference between acute and chronic infection concerning to Th17 cells.
Assuntos
Leishmaniose Cutânea/imunologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK/análise , Células Th17/imunologia , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Imuno-Histoquímica , Irã (Geográfico) , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Cutaneous leishmaniasis is a zoonotic, vector-borne disease causing a major health problem in several countries. No vaccine is available and there are limitations associated with the current therapeutic regimens. Immune responses to sand fly saliva have been shown to protect against Leishmania infection. A cellular immune response to PpSP15, a protein from the sand fly Phlebotomus papatasi, was sufficient to control Leishmania major infection in mice. This work presents data supporting the vaccine potency of recombinant live non-pathogenic Leishmania (L.) tarentolae secreting PpSP15 in mice and its potential as a new vaccine strategy against L. major. We generated a recombinant L. tarentolae-PpSP15 strain delivered in the presence of CpG ODN and evaluated its immunogenicity and protective immunity against L. major infection in BALB/c mice. In parallel, different vaccination modalities using PpSP15 as the target antigen were compared. Humoral and cellular immune responses were evaluated before and at three and eight weeks after challenge. Footpad swelling and parasite load were assessed at eight and eleven weeks post-challenge. Our results show that vaccination with L. tarentolae-PpSP15 in combination with CpG as a prime-boost modality confers strong protection against L. major infection that was superior to other vaccination modalities used in this study. This approach represents a novel and promising vaccination strategy against Old World cutaneous leishmaniasis.
Assuntos
Proteínas de Insetos/imunologia , Leishmania major/fisiologia , Leishmania/metabolismo , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/prevenção & controle , Animais , Formação de Anticorpos/imunologia , DNA/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Proteínas de Fluorescência Verde/metabolismo , Imunidade Celular/imunologia , Interferon gama/biossíntese , Interleucina-17/biossíntese , Vacinas contra Leishmaniose/imunologia , Leishmaniose Cutânea/parasitologia , Linfonodos/metabolismo , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos/imunologia , Parasitos/imunologia , Proteínas Recombinantes/imunologia , VacinaçãoRESUMO
In this study, we report the identification of two parasite polypeptides recognized by human sera of patients infected with Leishmania major. Isolation and sequencing of the two genes encoding these polypeptides revealed that one of the genes is similar to the L. major cathepsin L-like gene family CPB, whereas the other gene codes for the L. major homologue of the cysteine proteinase a (CPA) of L. mexicana. By restriction enzyme digestion of genomic DNA, we show that the CPB gene is present in multiple copies in contrast to the cysteine proteinase CPA gene which could be unique. Specific antibodies directed against the mature regions of both types expressed in Escherichia coli were used to analyze the expression of these polypeptides in different stages of the parasite's life cycle. Polypeptides of 27 and 40 kDa in size, corresponding to CPA and CPB respectively, were detected at higher level in amastigotes than in stationary phase promastigotes. Purified recombinant CPs were also used to examine the presence of specific antibodies in sera from either recovered or active cases of cutaneous leishmaniasis patients. Unlike sera from healthy uninfected controls, all the sera reacted with recombinant CPA and CPB. This finding indicates that individuals having recovered from cutaneous leishmaniasis or with clinically apparent disease have humoral responses to cysteine proteinases demonstrating the importance of these proteinases as targets of the immune response and also their potential use for serodiagnosis.
Assuntos
Antígenos de Protozoários/imunologia , Cisteína Endopeptidases/imunologia , Leishmania major/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Southern Blotting , Catepsinas/biossíntese , Catepsinas/genética , Catepsinas/imunologia , Clonagem Molecular , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/genética , Genoma de Protozoário , Humanos , Soros Imunes/imunologia , Immunoblotting , Leishmania major/enzimologia , Leishmaniose Cutânea/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Família Multigênica/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologiaRESUMO
This study was evaluated the ability of DNA vaccine encoding L7/L12 protein of Brucella sp. to induce cellular and humoral immune responses in BALB/c mice and the profile of cytokines and IgG sub classes were determined. Intra muscular vaccination of mice using L7/L12 gene. Three vaccinations at 3 week intervals were performed. Cytokines and IgG subclasses were analyzed 3 week after the last DNA vaccination. Splenic lymphocytes from L7/L12pCDNA3-vaccinated mice produced high levels of IFNy (3100 pg mL(-1)) and low levels of IL-5 (300 pg mL(-1)), 3 weeks post-vaccination. The L7/L12pCDNA3 immunizations elicited high IgG2a isotype response in mice immunized. This antigen also induced IgG1 titers which were slightly lower than the IgG2a titers. Immunological analysis shows the appropriate immune response in BALB/c mice model after vaccination with L7/L12 gene. The high level of IFNgamma and low level of IL-5 in combination with high IgG2a/IgG1 ratio show the activation of Th1 cell response. The lower bacterial cfu from vaccinated mice in comparison with control groups show the efficiency of L7/L12 DNA vaccination in mice model.
Assuntos
Brucella abortus/metabolismo , Citocinas/metabolismo , Genes Bacterianos/genética , Imunoglobulina G/metabolismo , Ribossomos/metabolismo , Linfócitos T/imunologia , Animais , Brucella abortus/genética , Proliferação de Células , Sistema Imunitário , Imunização , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Tempo , Vacinas de DNARESUMO
Cellular immune mechanisms resulting in interferon-gamma (IFN-gamma) production are essential for protection against cutaneous leishmaniasis. Antigens of the intracellular amastigote form of the parasite, found in mammalian hosts, are likely to be good candidates for the induction of T cell response and protection from development of leishmaniasis. We purified a stage-specific antigen from amastigote soluble antigen (A-SLA) of Leishmania major by immunoaffinity chromatography. The purified protein was characterized as a cysteine proteinase with enzymatic activity which is inhibited by E-64, and it was named the amastigote cysteine proteinase (ACP). BALB/c mice were immunized by two intraperitoneal injections, at a month interval, of 5 microg of ACP or A-SLA in Freund's complete adjuvant (FCA). Animals were challenged 4 weeks later with 106 L. major promastigotes and examined 4 months after the last injection. The immunized animals developed significantly smaller or no lesions compared with controls. Spleen cells from immunized mice showed a significant proliferative response and produced a high level of IFN-gamma in response to ACP, suggesting the induction of Th1 cells after immunization. These results make 24-kD ACP a possible component for an eventual cocktail vaccine against L. major infection.
Assuntos
Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/uso terapêutico , Leishmania major/enzimologia , Leishmania major/imunologia , Leishmaniose Cutânea/prevenção & controle , Vacinas Protozoárias/uso terapêutico , Animais , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Antígenos de Protozoários/uso terapêutico , Linfócitos B/imunologia , Divisão Celular/imunologia , Cisteína Endopeptidases/isolamento & purificação , Feminino , Imunidade Celular , Interferon gama/biossíntese , Interferon gama/imunologia , Leishmaniose Cutânea/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Protozoárias/imunologia , Vacinas Protozoárias/isolamento & purificação , Solubilidade , Linfócitos T/imunologia , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/uso terapêuticoRESUMO
Soluble Leishmania antigen (SLA) from both developmental stages of L. major (L. major MRHO/IR/75/ER) were prepared. Three and five subfractions of SLA from amastigote and promastigote were obtained by fast protein liquid chromatography (FPLC), respectively. Biochemical analyses and comparison of amastigote and promastigote SLA were done. The biochemical analyses revealed that the first fraction of L. major amastigote possesses a distinct band on its electrophoretic mobility pattern corresponding to a position of 24 kD, and it has enzymatic activity with characteristics of a cysteine proteinase. The isolated fractions of amastigote were tested for induction of proliferation, interferon-gamma (IFN-gamma) and IL-4 production in cultures of peripheral blood mononuclear cells (PBMC) from individuals who had recovered and also chronic patients of cutaneous leishmaniasis caused by L. major. The cells of recovered individuals compared with chronic cases proliferated profoundly in response to the first fraction of amastigote SLA. In all recovered individuals, the IFN-gamma, but not IL-4, was secreted in response to stimulation with the first fraction of amastigote SLA. In chronic cutaneous leishmaniasis, IFN-gamma was infrequently observed in response to stimulation by all three fractions of amastigote SLA, but secretion of IL-4 was observed. These data indicate that first fraction of amastigote SLA is a strong inducer of primed human immune response to L. major, and may have a protective function.
Assuntos
Antígenos de Protozoários/imunologia , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Animais , Antígenos de Protozoários/análise , Feminino , Humanos , Interferon gama/imunologia , Interleucina-4/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
The protection elicited by the intramuscular injection of two plasmid DNAs encoding Leishmania major cysteine proteinase type I (CPb) and type II (CPa) was evaluated in a murine model of experimental cutaneous leishmaniasis. BALB/c mice were immunized either separately or with a cocktail of the two plasmids expressing CPa or CPb. It was only when the cpa and cpb genes were co-injected that long lasting protection against parasite challenge was achieved. Similar protection was also observed when animals were first immunized with cpa/cpb DNA followed by recombinant CPa/CPb boost. Analysis of the immune response showed that protected animals developed a specific Th1 immune response, which was associated with an increase of IFN-gamma production. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a long lasting protective response, whereas the separate injection of cysteine proteases genes is not protective.
Assuntos
Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Leishmania major/enzimologia , Leishmania major/imunologia , Leishmaniose Cutânea/prevenção & controle , Vacinas Protozoárias/genética , Vacinas Protozoárias/farmacologia , Vacinas de DNA/genética , Vacinas de DNA/farmacologia , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/sangue , Cisteína Endopeptidases/administração & dosagem , Feminino , Genes de Protozoários , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Interferon gama/biossíntese , Leishmania major/genética , Leishmaniose Cutânea/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Vacinas Protozoárias/administração & dosagem , Vacinas Combinadas/administração & dosagem , Vacinas de DNA/administração & dosagem , Vacinas Sintéticas/administração & dosagemRESUMO
The immune responses induced against Leishmania antigens in volunteers who were vaccinated in a double-blind, randomized field efficacy trial of a preparation of autoclaved Leishmania major (ALM) mixed with a low dose of Bacille Calmette-Guerin vaccine (BCG) who developed either a cutaneous leishmaniasis (CL) lesion due to exposure to infected sandfly bite(s) or did not develop a lesion during the course of the trial were studied and compared with those of non-vaccinated controls. Blood samples were also assayed from different groups including volunteers with history of CL and volunteers with previous positive or negative leishmanin skin test (LST) without a history of CL. The vaccinated volunteers had received a single dose of either ALM mixed with a low dose of BCG or the same dose of BCG alone. The LST and in vitro proliferative response (stimulation index, SI), interferon gamma (IFN-gamma) production and, in a few cases, interleukin (IL)-4 production of peripheral blood mononuclear cells to soluble Leishmania antigens were measured. The results indicated that volunteers who developed CL in the vaccine arm showed a slightly higher SI than cases who received BCG alone. Volunteers with history of CL and volunteers with positive LST demonstrated the strongest proliferation indices and IFN-gamma production. The data suggest that a single dose of ALM + BCG induces a weak Th1 response in vaccinated volunteers that is far lower than that in volunteers with prior subclinical infection or volunteers with history of CL, who are presumed to be immune.
Assuntos
Vacina BCG/imunologia , Leishmaniose Cutânea/imunologia , Vacinas Protozoárias/imunologia , Adolescente , Adulto , Animais , Antígenos de Protozoários/imunologia , Divisão Celular/imunologia , Células Cultivadas , Método Duplo-Cego , Humanos , Interferon gama/biossíntese , Interleucina-4/biossíntese , Leishmania major/imunologia , Vacinas contra Leishmaniose , Leishmaniose Cutânea/prevenção & controle , Ativação Linfocitária , Pessoa de Meia-Idade , Células Th1/imunologia , Vacinas Combinadas/imunologiaRESUMO
In a murine model of experimental cutaneous leishmaniasis, we investigated the protection elicited by injection of histone H1 isolated from parasites by perchloric extraction, of a H1 recombinant protein produced in E. coli, and of H1 long and short synthetic peptides, against infection by L. major. Partial protection was achieved in most of the animals as shown by reduction in lesion size, upon immunization with histone H1 or its peptides, provided that the region 1-60 was present in the molecule. These observations argue in favor of a thorough examination of the possibility of including histone H1 described here in a cocktail vaccine against human leishmaniasis.