RESUMEN
The CP05 saponin from Calliandra pulcherrima Benth, shows remarkable similarities to the QS21 saponin of Quillaja saponaria Molina. Both shared a monoterpene hydrophobic moiety, a glycidic chain attached to the triterpene C28, and three sugars attached to C3. Different from QS21, the CP05 does not show the aldehyde group in triterpene C4 involved in TH1 response. Balb/c mice were immunized either intact saponin (CP05), the monoterpene-deprived (BS), the C28 carbohydrate-deprived (HS) or the sapogenin fraction, in formulation with the FML antigen of Leishmania donovani and challenged with 2 x 10(8) amastigotes of L. chagasi. While the CP05 induced 90% survival and 92.1% parasite reduction, a 100% survival and 94.1% protection were detected after the BS-vaccine treatment, indicating that the monoterpene acylated moiety, absent in the BS vaccine, is not necessary for the induction of a protective global TH1 response. Only the DTH response of BS vaccines was mildly lower than that of CP05 vaccinees. Maximal anti-FML antibody, CD4(+) and CD8(+) Leishmania specific lymphocytes, IFN-gamma splenocyte secretion, reduction in parasite load and survival was also detected for the BS vaccine. The HSFML vaccine showed diminished responses in all tested variables, except for IFN-gamma secretion, indicating that the integrity of the carbohydrate moiety attached to C28 is mandatory for the these functions. No protection was induced by the sapogenin-FML indicating that the CP05 triterpene which lacks the C4 aldehyde group, is not a immunostimulating compound. No contribution to protection was detected in the CP05 saponin treated control group supporting the specificity of the FML antigenic preparation.
Asunto(s)
Fabaceae/química , Leishmaniasis Visceral/prevención & control , Saponinas/química , Saponinas/farmacología , Terpenos/química , Adyuvantes Inmunológicos , Animales , Femenino , Inmunoglobulinas/sangre , Ratones , Estructura Molecular , Relación Estructura-Actividad , VacunaciónRESUMEN
The adjuvant of the FML-vaccine against murine and canine visceral leishmaniasis, the Riedel de Haen saponin mixture, was fractionated by ion exchange chromatography on DEAE-cellulose to afford one TLC homogeneous Quillaja saponaria Molina QS21 saponin fraction (18.0%), a mixture of two deacylsaponins (19.4%), sucrose (39.9%), sucrose and glucose (19.7%), rutin (0.8%) and quercetin (2.2%), that were identified by comparison of 1H and 13C NMR spectroscopy. The QS21 shows the typical aldehyde group in C-23 (65% equatorial) and a normonoterpene moiety acylated in C-28. The deacylsaponins show the aldehyde group but do not have the normonoterpene moiety. Balb/c mice were vaccinated with 150 microg of FML antigen of Leishmania donovani and 100 microg of each obtained fraction and further challenged by infection with 10(8) amastigotes of Leishmania chagasi. The safety analysis and the effect on humoral and cellular immune responses and in clinical signs showed that the QS21 saponin and the deacylsaponins are the most active adjuvant compounds of the Riedel the Haen saponin mixture. Both induced the highest and non-significantly different increases in DTH, CD4+ T lymphocytes in spleen, IFN-gamma in vitro, body weight gain and the most pronounced reduction of parasite burden in liver (95% for QS21 and 86% for deacylsaponins; p>0.05). While the QS21 showed mild toxicity, significant adjuvant effect on the anti-FML humoral response before and after infection, and decrease in liver relative weight, the deacylsaponins showed no toxicity, less haemolysis and antibody and DTH responses increased mainly after infection, still inducing a stronger Leishmania-specific in vitro splenocyte proliferation. Our results confirm in the Riedel de Haen saponin extract the presence of deacylsaponins normonoterpene-deprivated which are non-toxic and capable of inducing a specific and strong immunoprotective response in vaccination against murine visceral leishmaniasis.