RESUMO
We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79% in attenuated parasite-vaccinated monkeys, versus 75% in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-gamma) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application.
Assuntos
Modelos Animais de Doenças , Interferon gama/biossíntese , Leishmania major/imunologia , Vacinas Protozoárias/imunologia , Animais , Antígenos de Protozoários/imunologia , Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Hipersensibilidade Tardia/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/prevenção & controle , Macaca mulatta , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/efeitos adversos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/imunologiaRESUMO
We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79 percent in attenuated parasite-vaccinated monkeys, versus 75 percent in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-g) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application
Assuntos
Animais , Interferon gama , Leishmania major , Vacinas Protozoárias , Vacinas Atenuadas , Vacinas de Produtos Inativados , Antígenos de Protozoários , Vacina BCG , Hipersensibilidade Tardia , Leishmaniose Cutânea , Macaca mulatta , Vacinas Protozoárias , Vacinas Atenuadas , Vacinas de Produtos InativadosRESUMO
In this study we have compared the immune response of normal human cells cultured in vitro to two virulent strains of Leishmania major (CC1 and LV39), and to an avirulent vaccine strain (dhfr-ts-) made by targeted deletion of the essential gene DHFR-TS. We utilized an in vitro system in which naive T cells from normal human donors were primed with autologous Leishmania-infected macrophages. All three parasites infected macrophages and transformed into amastigotes within the cells. However, whereas LV39 and CC1 replicated in macrophages, dhfr-ts- did not. When peripheral blood lymphocytes (PBL) were stimulated with autologous macrophages infected with any of the three parasites, the lymphocytes produced a type-1-biased cytokine response. Finally, addition of IL-12 during the first stimulation period increased the production of interferon-gamma but decreased IL-5 secretion. On the other hand, anti-IL-12 resulted in the opposite effect.
Assuntos
Citocinas/biossíntese , Leishmania major/imunologia , Leishmania major/patogenicidade , Células Th1/imunologia , Animais , Células Cultivadas , Humanos , Interferon gama/biossíntese , Interleucina-12/fisiologia , Interleucina-5/biossíntese , Leishmania major/enzimologia , Leishmania major/genética , Tetra-Hidrofolato Desidrogenase/deficiência , Tetra-Hidrofolato Desidrogenase/genética , Células Th1/metabolismo , Células Th1/parasitologia , Células Th2/imunologia , Células Th2/metabolismo , Células Th2/parasitologia , Timidilato Sintase/deficiência , Timidilato Sintase/genética , VirulênciaRESUMO
E10-5A3 is a dhfr-ts- Leishmania major double knockout auxotrophic shown previously to induce substantial protection against virulent L. major infection in both genetically susceptible and resistant mice. We investigated the capacity of dhfr-ts- to protect against heterologous infection by L. amazonensis. The degree of protection was evaluated by immunization of BALB/c or C57BL/6 mice with E10-5A3, followed by L. amazonensis challenge. Whether immunized by subcutaneous (SC) or intravenous (IV) inoculation, susceptible and resistant mice displayed a partial degree of protection against challenge with virulent L. amazonensis. SC-immunized BALB/c mice developed lesions 40 to 65 percent smaller than non immunized mice, while IV immunization led to protection ranging from 40 to 75 percent in four out of six experiments compared to non immunized animals. The resistant C57BL/6 mice displayed comparable degrees of protection, 57 percent by SC and 49 percent by IV immunization. Results are encouraging as it has been previously difficult to obtain protection by SC vaccination against Leishmania, the preferred route for human immunization
Assuntos
Camundongos , Animais , Antígenos de Protozoários/administração & dosagem , Leishmania major/imunologia , Leishmaniose Cutânea/prevenção & controle , Vacinas Protozoárias/imunologia , Timidilato Sintase/imunologia , Leishmaniose Cutânea/imunologia , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos MutantesRESUMO
We have used a chromosome-specific approach to generate a 300 kb long 'contig' across Leishmania major 500 kb chromosome. Clones from a 13-hit genomic library served as templates to generate end-specific probes that were used in hybridization to a high density array of the library. The 'contig' generated contained 12 markers uniformly spaced. Three restriction endonucleases were mapped within the map extending its resolution. Map extension indicated a peculiar feature of sequence organization in subtelomeric regions where chromosome-specificity of mapping is lost. End-probes generated from clones mapping to the extremes of a 300 kb 'contig' rescued a high percentage of 2 types of clones from the genomic library, 1 of which showed positive hybridization to the hexameric telomere repeat. Fine mapping at these regions revealed that these 2 clones contained elements common to all chromosomes of the parasite. The physical map generated constitutes ready-to-use data for the study of many aspects of genome organization. Being cloned in a shuttle vector, the genomic sequences reordered in the map can be used to generate genetic information by transfection into the parasite.
Assuntos
Mapeamento Cromossômico , Leishmania major/enzimologia , Complexos Multienzimáticos/genética , Proteínas de Protozoários/genética , Tetra-Hidrofolato Desidrogenase/genética , Timidilato Sintase/genética , Animais , Leishmania major/genéticaRESUMO
A method for discriminating among Leishmania is described, based upon small subunit ribosomal DNA sequence differences. The method was to amplify the entire 2.2 kb small subunit rDNA by polymerase chain reaction using conserved primers specific for the 5' and 3' termini of the small subunit ribosomal RNA, and then hybridize the product dotted onto nylon membranes with labeled oligonucleotides. The design of the hybridization probes was based upon complete small subunit rDNA sequences from L. amazonensis, L. major and L. guyanensis and partial sequences of L. mexicana, L. braziliensis, L. tropica and L. chagasi. A high degree of sequence similarity (> 99%) among species was found. However, sufficient sequence divergence occurred to permit the design of internal oligonucleotide probes specific for species complexes. This procedure successfully discriminated amongst a wide range of Leishmania isolates. The method detected as few as 10 cultured organisms and detected parasites in tissue samples from experimentally infected animals. Non-radioactive labeling showed the same specificity and sensitivity as radioactive probes.
Assuntos
DNA de Protozoário/análise , Leishmania/isolamento & purificação , Leishmaniose/diagnóstico , Reação em Cadeia da Polimerase/métodos , Animais , Sequência de Bases , Clonagem Molecular , Cricetinae , DNA Ribossômico/análise , DNA Ribossômico/genética , Eletroforese em Gel de Ágar , Humanos , Leishmania/classificação , Leishmania/genética , Leishmaniose/parasitologia , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sondas de OligonucleotídeosRESUMO
The question is addressed whether antigens of Leishmania, a parasite residing in the endosomal compartment of macrophages, can be presented in the context of major histocompatibility complex class I molecules. We used E. coli beta-galactosidase as a model antigen which can be expressed in high levels in L. mexicana promastigotes (L. mexicana-gal). Infection of BALB/c mice with L. mexicana-gal induces beta-galactosidase-specific cytotoxic T cells (CTL), which can be isolated using a beta-galactosidase-expressing mastocytoma line as an antigen-presenting cell. These CTL recognize epitopes of beta-galactosidase in the context of H-2Kd; however, they do not recognize L. mexicana-gal-infected macrophages even after killing of the intracellular amastigotes by drug treatment or macrophage activation by lymphokines, although class I-peptide interaction and the presentation of endogenously produced antigens is normal. It is concluded that parasite antigens can induce a CTL response in vivo but that these CTL cannot recognize infected macrophages because the relevant epitopes cannot gain access to class I molecules. The effect of priming in vivo may be explained by the well-known but ill-understood phenomenon of cross-priming.
Assuntos
Leishmania mexicana/imunologia , Macrófagos/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Células Cultivadas , Citotoxicidade Imunológica , Feminino , Antígenos de Histocompatibilidade Classe I/fisiologia , Leishmaniose Cutânea/imunologia , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos , beta-Galactosidase/análise , beta-Galactosidase/genética , beta-Galactosidase/imunologiaRESUMO
The sequence of the Leishmania tarentolae SSU rRNA (small subunit or 18S rRNA) gene was completely determined from 2 different strains and used to determine phylogenetic relationships between this organism and other trypanosomatids. Extensive structural similarities were observed between L. tarentolae and mammalian leishmanias the SSU rRNA. Phylogenetic reconstructions, using distance matrix or parsimony methods, showed large evolutionary distances between trypanosomes, either African and American, and L. tarentolae. Further analysis using intergenic rDNA spacer (IGS) sequences as probes in dot blot experiments confirmed the results obtained with the SSU rDNA comparisons. The data presented here clearly indicate that L. tarentolae is closely related to the mammalian parasite Leishmania donovani and highly divergent from trypanosomes.
Assuntos
Leishmania/classificação , Leishmania/genética , RNA de Protozoário/genética , RNA Ribossômico 18S/genética , Animais , Sequência de Bases , DNA Ribossômico/genética , Leishmania donovani/classificação , Leishmania donovani/genética , Dados de Sequência Molecular , Filogenia , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Immunization with the GP46/M-2 membrane glycoprotein of Leishmania amazonensis has been shown to induce a protective immune response against infection. We have surveyed a variety of trypanosomatid species and genera for the presence and expression of this gene family, information that will be relevant to future vaccine studies against leishmaniasis. Molecular karyotype analysis revealed the presence of GP46/M-2 genes in all members of the Leishmania mexicana complex, Leishmania major, Leishmania donovani, Leishmania tarentolae, and Crithidia fasciculata. In contrast, DNAs from species of the Leishmania braziliensis complex (L. braziliensis, Leishmania guyanensis, and Leishmania panamensis) failed to hybridize to GP46/M-2 probes. Western blot analyses with several polyclonal antisera against the GP46/M-2 protein revealed protein expression in L. major and L. donovani, but not L. panamensis or L. braziliensis. Phylogenetic analysis suggests that a loss of the GP46A gene family occurred following separation of the L. braziliensis complex, prior to speciation events within this complex. These data indicate that GP46/M-2 membrane glycoprotein may not be critical to parasite survival, but may play an ancillary role during the developmental cycle.
Assuntos
Leishmania braziliensis/genética , Glicoproteínas de Membrana/genética , Família Multigênica , Proteínas de Protozoários/genética , Animais , Evolução Biológica , Southern Blotting , Western Blotting , Cariotipagem , Leishmania/genéticaRESUMO
A new DNA amplification is described from an isolate of the lizard parasite Leishmania tarentolae. This DNA is present in up to 50 copies in the Trager line of this species and present but not amplified in all other lines tested. This amplification has been named the T amplification (for Tarentolae/Trager). Restriction enzyme digestion and electrophoresis of total DNA reveal amplified fragments totalling 19 kb following staining with ethidium bromide, a finding confirmed by the use of specific hybridization probes. Much of the amplified T DNA occurs as extra-chromosomal circular molecules. No cross-hybridization was observed between the T region and other amplified DNA of Leishmania, or the maxicircle of L. tarentolae, nor was resistance to methotrexate, chloroquine or primaquine detected in the T-amplified line. Combined with our previous results showing H region amplification in 2 other unselected lab stocks, these data demonstrate the prevalence of apparently spontaneous gene amplifications in L. tarentolae.