RESUMO
In order to test recombinant Toxoplasma as adjuvant and live vaccine carrier in the infectious disease model of murine experimental leishmaniasis, we engineered the attenuated, temperature-sensitive Toxoplasma gondii strain ts-4 to express the heterologous Leishmania antigen kinetoplastid membrane protein-11 (KMP-11). Transgenic ts-4 clones were obtained which express KMP-11 as cytoplasmatic protein or target it to the secretory pathway of the tachyzoites. Immunization of BALB/c mice with these stably transformed parasites elicited proliferative responses to both T. gondii antigen and recombinant KMP-11. When challenged with Leishmania major, we observed significant protection in animals that had been vaccinated with the KMP-11-expressing ts-4 mutants. The adjuvant attenuated only the onset of the Leishmania infection, but animals were ultimately not able to control the disease. Thus, our findings demonstrate that recombinant Toxoplasma has the potential to serve as an efficient vaccine carrier for cutaneous leishmaniasis. Furthermore, they establish a protective role for the antigen KMP-11 when given in such a vaccine formulation.
Assuntos
Leishmania/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Toxoplasma/genética , Vacinas Sintéticas/imunologia , Animais , Feminino , Leishmaniose/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologia , Vacinação , Vacinas Atenuadas/imunologiaRESUMO
Parasitologic confirmation of cutaneous leishmaniasis is obligatory before chemotherapy can be considered. Direct microscopic examination of scrapings taken from indurated borders of ulcers has been routinely used as primary method of diagnosis. In this report we compared the sensitivity of examination of dermal scrapings taken from the bottoms of ulcers (BDS) with that of dermal scrapings taken from indurated active margins of lesions (MDS) in a total of 115 patients. The sensitivities of the microscopic examination were 90.4 and 78.3% for BDS and MDS samples, respectively. When the PCR method was used with a group of 40 patients, we also observed a higher sensitivity when BDS samples were examined (80.8% in BDS samples versus 57.7% in MDS samples). The improvement of the diagnostic sensitivity in the BDS samples appears to be related to the higher parasite load and more easily detectable morphology of amastigotes in the centers of the ulcers. Other parasitologic diagnostic methods, such as culture and histopathologic examination of biopsies, are less sensitive (67.5 and 64.3%, respectively). Aspirate culture, however, was shown to be the most sensitive method for the diagnosis of patients with chronic ulcers. When microscopic examinations of both MDS and BDS samples are combined, the sensitivity of diagnosis may rise up to 94%. We therefore recommend this method as a primary routine procedure for diagnosis of cutaneous leishmaniasis.
Assuntos
Leishmaniose Cutânea/diagnóstico , Úlcera Cutânea/patologia , Animais , Biópsia , Colômbia , Humanos , Leishmania/classificação , Leishmania/isolamento & purificação , Leishmaniose Cutânea/patologia , Reação em Cadeia da Polimerase/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Pele/patologia , Testes Cutâneos , Úlcera Cutânea/parasitologiaRESUMO
The Leishmania infantum Mat-1 gene--recently described in L. major as a highly stage-specific, metacyclogenesis-associated transcript--has been cloned. The 420-bp Mat-1 coding region is conserved with respect to the L. major gene (82% sequence homology). Analysis of the predicted amino-acid sequence reveals structural motifs showing homology with the class of leucine-zipper transcription factors. Southern-blot hybridization analysis suggests that Mat-1 is a low-copy-number gene, probably consisting of two gene copies. The recombinant Mat-1 protein expressed in fusion with the Escherichia coli maltose-binding protein shows a tendency to form dimers in the presence of the leucine-rich C-terminal domain. Bacteria expressing the Mat-1 open reading frame are highly growth-attenuated and tend to delete or modify the insert, which suggests that expression of Mat-1 is toxic for the bacteria.
Assuntos
Leishmania/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Dimerização , Leishmania/crescimento & desenvolvimento , Leishmania/metabolismo , Leishmania infantum/genética , Leishmania infantum/metabolismo , Leishmania major/genética , Leishmania major/metabolismo , Dados de Sequência Molecular , Proteínas de Protozoários/químicaRESUMO
The kinetoplastid membrane protein-11 (KMP-11) is a major target of the humoral immune response during Leishmania-infections. The majority of sera from visceral leishmaniasis, mucocutaneous leishmaniasis and even some cutaneous leishmaniasis patients contain detectable IgG antibodies against KMP-11. We also provide evidence that this protein may act as a potent antigen in T. cruzi infections, since most Chagas sera show immunological cross-reactivity. Therefore, KMP-11 cannot be used as a specific diagnostical tool for the serodiagnosis of leishmaniasis in those regions where both, Leishmania and T. cruzi infections overlap geographically. When analyzing the subclass specificity of the antibody response to KMP-11 we observed the following order of reactivity: IgG1 > > IgG3 > IgG2 > IgG4, which is similiar to that seen in crude parasite extract. The mapping of antigenic determinants by using synthetic 20-mer peptides revealed the existence of predominantly conformational epitopes in leishmaniasis, while 50% of sera from Chagas patients reacted with a particular KMP-11 peptide. These results therefore suggest the presence of disease-specific B-cell epitopes.
Assuntos
Anticorpos Antiprotozoários/sangue , Doença de Chagas/imunologia , Imunoglobulina G/sangue , Leishmaniose Cutânea/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas de Protozoários/imunologia , Sequência de Aminoácidos , Especificidade de Anticorpos , Linfócitos B/imunologia , Brasil , Surtos de Doenças , Mapeamento de Epitopos , Humanos , Imunoglobulina G/classificação , Isotipos de Imunoglobulinas/sangue , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Peptídeos/imunologia , Proteínas de Protozoários/genética , Proteínas Recombinantes/imunologiaRESUMO
The kinetoplastid membrane protein 11 (KMP-11) has been recently described in Leishmania (Leishmania) donovani as a major component of the promastigote membrane. Two oligonucleotide primers were synthesized to PCR-amplify the entire encoding region of New World Leishmania species. The Leishmania (Viannia) panamensis amplification product was clone, sequenced and the putative amino acid sequence determined. A remarkably high degree of sequence homology was observed with the corresponding molecule of L. (L) donovani and L. (L) infantum (97% and 96%, respectively). Southern blot analysis showed that the KMP-11 locus is conformed by three copies of the gene. the L. (V) panamensis ORF was subsequently clone in a high expression vector and the recombinant protein was induced and purified from Escherichia coli cultures. Immunoblot analysis showed that 80%, 77% and 100% sera from cutaneous, mucocutaneous and visceral leishmaniasis patients, respectively, recognized the recombinant KMP-11 protein. In a similar assay, 86% of asymptomatic Leishmania-infected individuals showed IgG antibodies against the rKMP-11. We proposed that KMP-11 could be used as a serologic marker for infection and disease caused by Leishmania in America.