RESUMO
In the present study, two proteins cloned from Leishmania braziliensis species, a hypothetical protein (LbHyp) and the eukaryotic initiation factor 5a (EiF5a), were evaluated to protect BALB/c mice against L. amazonensis infection. The animals were immunized with the antigens, either separately or in combination, using saponin as an immune adjuvant in both cases. Spleen cells from vaccinated and later infected mice produced significantly higher levels of protein and parasite-specific IFN-γ, IL-12, and GM-CSF, in addition to low levels of IL-4 and IL-10. Evaluating the parasite load by means of a limiting dilution technique and quantitative Real-Time PCR, vaccinated animals presented significant reductions in the parasite load in both infected tissues and organs, as well as lower footpad swelling, when compared to the control (saline and saponin) groups. The best results regarding the protection of the animals were achieved when the combined vaccine was administered into the animals. Protection was associated with an IFN-γ production against parasite antigens, which was mediated by both CD4+ and CD8+ T cells and correlated with antileishmanial nitrite production. In conclusion, data from the present study show that this polyprotein vaccine, which combines two L. braziliensis proteins, can induce protection against L. amazonensis infection.
Assuntos
Antígenos de Protozoários/imunologia , Reações Cruzadas/imunologia , Leishmania braziliensis/imunologia , Leishmania mexicana/imunologia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Cutânea/prevenção & controle , Fatores de Iniciação de Peptídeos/imunologia , Proteínas de Ligação a RNA/imunologia , Animais , Antígenos de Protozoários/química , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Parasita/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Camundongos , Carga Parasitária , Fatores de Iniciação de Peptídeos/química , Proteínas de Ligação a RNA/química , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
We evaluated whether four recombinant antigens previously used for vaccination against experimental infection with Leishmania (Leishmania) major could also induce protective immunity against a challenge with Leishmania (Viannia) braziliensis, the species responsible for 90% of the 28,712 annual cases of cutaneous and mucocutaneous leishmaniasis recorded in Brazil during the year of 2004. Initially, we isolated the homolog genes encoding four L. (V.) braziliensis antigens: (i) homologue of receptor for activated C kinase, (ii) thiol-specific antioxidant, (iii) Leishmania elongation and initiation factor, and (iv) L. (L.) major stress-inducible protein 1. At the deduced amino acid level, all four open reading frames had a high degree of identity with the previously described genes of L. (L.) major being expressed on promastigotes and amastigotes of L. (V.) braziliensis. These genes were inserted into the vector pcDNA3 or expressed as bacterial recombinant proteins. After immunization with recombinant plasmids or proteins, BALB/c mice generated specific antibody or cell-mediated immune responses (gamma interferon production). After an intradermal challenge with L. (V.) braziliensis infective promastigotes, no significant reduction on the lesions was detected. We conclude that the protective immunity afforded by these four vaccine candidates against experimental cutaneous leishmaniasis caused by L. (L.) major could not be reproduced against a challenge with L. (V.) braziliensis. Although negative, we consider our results important since they suggest that studies aimed at the development of an effective vaccine against L. (V.) braziliensis, the main causative agent of cutaneous leishmaniasis in the New World, should be redirected toward distinct antigens or different vaccination strategies.
Assuntos
Antígenos de Protozoários/imunologia , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Mucocutânea/imunologia , Vacinas Protozoárias/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/genética , Modelos Animais de Doenças , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Humanos , Imunoensaio/métodos , Leishmania braziliensis/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/prevenção & controle , Leishmaniose Mucocutânea/parasitologia , Leishmaniose Mucocutânea/prevenção & controle , Estágios do Ciclo de Vida , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fases de Leitura Aberta , Fatores de Iniciação de Peptídeos/biossíntese , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/imunologia , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/genética , Vacinas Protozoárias/farmacologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/farmacologiaRESUMO
The interaction of the innate immune system with the microbial world involves primarily two sets of molecules generally known as microbial pattern recognition receptors and microbial pattern recognition molecules, respectively. Examples of the former are the Toll receptors present particularly in macrophages and dendritic cells. Conversely, the microbial pattern recognition molecules are conserved protist homopolymers, such as bacterial lipopolysaccharides, lipoteichoic acids, peptidoglycans, glucans, mannans, unmethylated bacterial DNA, and double-strand viral RNA. However, for protists that lack most of these molecules, such as protozoans, the innate immune system must have evolved receptors that recognize other groups of microbial molecules. Here we present evidence that a highly purified protein encoded by a Leishmania brasiliensis gene may be one such molecule. This recombinant leishmanial molecule, a homologue of eukaryotic ribosomal elongation and initiation factor 4a (LeIF), strongly stimulates spleen cells from severe combined immunodeficient (SCID) mice to produce interleukin-12 (IL-12), IL-18, and high levels of gamma interferon. In addition, LeIF potentiates the cytotoxic activity of the NK cells of these animals. Because LeIF is a conserved molecule and because SCID mice lack T and B lymphocytes but have a normal innate immune system (normal reticuloendothelial system and NK cells), these results suggest that proteins may also be included as microbial pattern recognition molecules. The nature of the receptor involved in this innate recognition is unknown. However, it is possible to exclude the Toll receptor Tlr4 as a putative LeIF receptor because the gene encoding this receptor is defective in C3H/HeJ mice, the mouse strain used in the present studies.