The relative contribution of the NH36-specific-CD4+ and CD8+ T cell producing cells was evaluated in an in vivo depletion assay with monoclonal antibodies ( Fig. 8). In correlation to what was detected for the specific increase of the CD4+ T cells ( Fig. 5), the TNF-α–CD4+ producing T cells ( Fig. 6), only the treatment with anti-CD4+ monoclonal antibody induced a 66% increase in the total LDU counts of mice vaccinated with CA4 saponin, indicating a main contribution of CD4+ T cells ( Fig. 8; p > 0.05) to the vaccine induced protection. On the other hand, the protective effect of the CA3-vaccine is mediated by both CD4+ and the CD8+ T cell
contributions check details since the anti-CD4+ antibody treatment induced a 43% and the anti-CD8+ antibody induced a 16% increase of the total LDU counts of CA3 vaccinated mice, respectively ( Fig. 8). This is in agreement with the increase of the percent of CD8+ NH36-specific T cells by the CA3 vaccine
( Fig. 5) and of the IFN-γ-CD4+ producing T cells ( Fig. 6). The increases in IDR, CD4–TNF-α, CD8–IFN-γ and CD8–TNF-α by the CA4 vaccine were strong correlates of protection and TGF-beta inhibitor were significantly correlated to the decrease of parasite load (p = −0.007). To confirm the relevance of TNF-α in the protection induced by C. alba we vaccinated C57BL6 wild-type and TNF-α-receptor knock-out mice and challenged them with L. chagasi amastigotes. The IDR response against Leishmania lysate was significantly increased (81%) only by the CA4 saponin vaccine in wild type
mice above their respective saline control ( Fig. 9). No increases in IDR were observed however in vaccinated TNF-α-knock-out mice ( Fig. 9). Different from what was detected in Balb/c mice treated with saline (mean = 415 LDU units) ( Fig. 7) the C57Bl6 strain was more sensitive (mean = 1200 LDU units). Confirming the role of IDR as a correlate of protection in visceral leishmaniasis, mafosfamide only the CA4-saponin vaccine (mean = 596 LDU units) induced a significant reduction of 50% of the parasite load ( Fig. 9). The TNF-α-receptor deficient mice lost the ability to clear amastigotes from the liver and showed a mean control value (2185 LDU) 56% greater than the control wild type group (1200 LDU). Protection due to the CA4 saponin was not observed in the TNF-α-receptor deficient mice. To confirm that the presence of an extra-apiose in CA4 is responsible for its increased adjuvant potential, we compared the protective efficacy of the CA3 and CA4-vaccines to the one of vaccines formulated with the CA3X and CA2 saponins of C. alba ( Fig. 1). All these saponins are naturally produced through a glycosylation series by the C. alba plant. The shorter chain is present in CA2 which has only an arabinose and a rhamnose unit attached to C-28 ( Fig. 1) and is followed by the CA3X and CA3 saponins, both with three sugars attached to the C-28 chain. The third sugar is xylose for CA3X and apiose for CA3.