RESUMEN
In this study we examined whether the maintenance of Trypanosoma cruzi by long-time in axenic culture produces changes in gene expression and antigenic profiles. The studies were made with a Dm30L-clone from a low-virulent strain and a non-cloned virulent EP-strain of T. cruzi. Both parasites were maintained, for at least seven years, by successive alternate passage triatomine/mouse (triatomine condition), or by serial passage in axenic medium (culture condition). The comparison of the [35S]methionine metabolic labeling products of virulent and non-virulent parasites by 2D-SDS-PAGE, clearly indicates that the expression of metacyclic trypomastigotes (but not of epimastigotes) proteins have been altered by laboratory maintenance conditions. Western blot analysis of EP and Dm30L-epimastigotes using a serum anti-epimastigotes revealed that although most of antigens are conserved, four antigens are characteristics of triatomine condition parasites and three other are characteristics of culture condition parasites. Anti-metacyclics serum revealed significative differences in EP- and Dm30L-metacyclic trypomastigotes from triatomine condition. However, avirulent metacyclic forms were antigenically very similar. These results suggest that besides a possible selection of avirulent subpopulation from T. cruzi strains genetically heterogeneous when maintained by long time in axenic culture, changes in virulence might be due to post-translational modifications of the antigens induced by the absence of the natural alternability (vertebrate-invertebrate) in the life-cycle of T. cruzi.
Asunto(s)
Medios de Cultivo , Expresión Génica , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/genética , Animales , Ratones , Biosíntesis de Proteínas , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidad , Virulencia/efectos de los fármacosRESUMEN
This study reports the results of a vaccine trial established to study the cellular immune responses in vivo (skin-test reactivity) and in vitro (T-cell proliferation and interferon-gamma production) to both leishmanial and mycobacterial antigens following vaccination of healthy volunteers from a leishmaniasis-endemic area with killed leishmanial promastigotes, with or without BCG (Bacille Calmètte-Guerin). Skin tests were performed using purified protein derivative of tuberculin (PPD) and leishmanial antigen in 692 volunteers, and 208 doubly negative subjects (< or = 7 mm induration) were selected to participate in the trial. The study subjects were divided into four vaccine groups: (A) killed promastigotes plus BCG, (B) BCG alone, (C) killed promastigotes alone, and (D) placebo. Three vaccine doses were administered at 6-10-week intervals. The skin-test responses to PPD and leishmanial antigen were reassessed at 4-6- and 12-18-month follow-ups. The results of this trial demonstrated that the combined vaccine, i.e. killed promastigotes of Leishmania plus BCG, results in the stimulation of an immune response to both leishmania and mycobacterial antigens in a high percentage of vaccines (> 85%), manifested either by skin-test conversion, lymphocyte proliferation and/or interferon-gamma production. This was evident after the first dose of vaccine for lymphocyte proliferation and interferon-gamma production and was maintained for a year after the three doses of vaccine. Group B (which received BCG alone), responded as well as group A to PPD but not as well to leishmanial antigen. The reverse was true for group C which received promastigotes alone. Group A attained a 38% leishmanin skin-test conversion at the 4-6-month follow-up, which was associated with double PPD/leishmanial antigen responder status. In contrast, a 35% skin-test conversion was found at the 12-18-month follow-up in group C (promastigotes alone), but this was not associated with responses to PPD. A significant percentage of conversion was observed in the placebo group at the 12-18-month follow-up, both to PPD (58%) and leishmanial (21%) antigens, which suggests either environmental exposure to mycobacterial or leishmanial antigens during the vaccine trial or, more probably, a response to the repeated leishmanial skin tests. Further studies are required to determine whether the presence of proliferative and/or interferon-gamma responses in the absence of a skin-test response are sufficient indicators of potential vaccine success.