Q. brasiliensis (A. St.-Hil. Et Tul) Mart. leaves were collected in Canguçu, RS, Brazil (31°23′42″ S-52°40′32″ W) (voucher ICN 142953, deposited at the Herbarium of the Federal University of Rio Grande do Sul). The extraction and purification of saponins were carried out as previously described (22 (link)).
The nanoadjuvanted formulation with the zEDIII antigen was prepared by the modified ethanol injection technique (17 (link), 23 (link)). Briefly, the zEDIII protein solution (1 mg/mL or 0.2 mg/mL) was added to a mixture of Q. brasiliensis saponins fraction (QB-80, 1 mg/mL). Ethanol-dissolved cholesterol (Sigma-Aldrich, USA) and di-palmitoylphosphatidyl-choline (Avanti Polar Lipids, USA) were immediately injected into the mixture, which was then stirred for 48 h at 4°C.
This procedure resulted in the nanoadjuvant derived from QB-80, which was named IQB80. In order to check the toxicity of the nanoadjuvant, hemolysis tests were performed, as previously described (17 (link), 18 (link), 24 (link)), and none of the concentrations tested (ranging from 100 to 10 μg) have been shown to cause membrane damage (data not shown). The experimental vaccine formulations were prepared under aseptic conditions, filtered through 0.22 μm filters and kept at 4°C until use.
The nanoadjuvanted formulation with the zEDIII antigen was prepared by the modified ethanol injection technique (17 (link), 23 (link)). Briefly, the zEDIII protein solution (1 mg/mL or 0.2 mg/mL) was added to a mixture of Q. brasiliensis saponins fraction (QB-80, 1 mg/mL). Ethanol-dissolved cholesterol (Sigma-Aldrich, USA) and di-palmitoylphosphatidyl-choline (Avanti Polar Lipids, USA) were immediately injected into the mixture, which was then stirred for 48 h at 4°C.
This procedure resulted in the nanoadjuvant derived from QB-80, which was named IQB80. In order to check the toxicity of the nanoadjuvant, hemolysis tests were performed, as previously described (17 (link), 18 (link), 24 (link)), and none of the concentrations tested (ranging from 100 to 10 μg) have been shown to cause membrane damage (data not shown). The experimental vaccine formulations were prepared under aseptic conditions, filtered through 0.22 μm filters and kept at 4°C until use.