The adjuvanticity of Chiococca alba saponins increases with the length and hydrophilicity of their sugar chains.

The saponins of Chiococca alba are triterpene bidesmosides that contain glycidic moieties attached to the C-3 and C-28 carbon of their aglycone. We describe that their adjuvant potential increases in direct relationship to the length and hydrophilicity of the C-28 attached sugar chain which contains: arabinose-rhamnose in the CA2, arabinose-rhamnose-xylose in the CA3X; arabinose-rhamnose-apiose in the CA3 and arabinose-rhamnose-apiose-apiose in the CA4 saponin. The hydrophile/lipophile balance calculated for CA2 was 12.7, for CA3 and CA3X was 15.8 and for CA4 19.9. All saponins were formulated with the FML antigen for mice prophylaxis against visceral leishmaniasis. The immune response was studied using an ELISA-antibody assay and monitoring of the intradermal response (IDR) to Leishmania antigens, the cytokine expression in supernatants and the intracellular staining of in vitro cultured splenocytes. After challenge, significant increases of IgG and IgG2a antibodies were noted only in the CA4 vaccinated mice that showed extended IDR, higher IFN-γ production by CD8+ and TNF-α production by CD4+ T cells, higher TNF-α secretion and the highest reduction of the parasite load (78%). The increases in IDR, CD4-TNF-α, CD8-IFN-γ and CD8-TNF-α by the CA4 vaccine were strong correlates of protection and were significantly correlated to the decrease of parasite load (p=-0.007). Protection generated by the CA4 vaccine was mainly mediated by a CD4+ T cell and a TNF-α driven response with a lower contribution of CD8+ T cells, as confirmed by an in vivo depletion with monoclonal antibodies and by vaccination assays in TNF-α-receptor knock-out mice. Our results confirm that the superiority of the CA4 saponin is related to the higher hydrophilicity of its longer carbohydrate chain. C. alba saponins were non-toxic and only the xylose-containing saponin CA3X was hemolytic (HD(50)=87 μg/ml). The increase in sugar units of the saponins is positively correlated to the increase of IDR and to the decrease of parasite load.