Saponins from Quillaja saponaria Molina: Isolation, Characterization and Ability to Form Immuno Stimulatory Complexes (ISCOMs)

Abstract

ISCOMs have received much attention as vaccine adjuvants due to their immunostimulatory effects. They are colloidal particles typically comprised of phospholipids, cholesterol and Quil A, a crude mixture of saponins extracted from the bark of Quillaja saponaria Molina. We have previously shown that ISCOMs can be prepared by ether injection wherein an ether solution of phospholipids and cholesterol in a mass ratio of 5:2 is injected into a solution of Quil A at a mass ratio of 7 lipids: 3 Quil A. The aim of this study was firstly to isolate and characterise discrete fractions of Quil A and secondly to investigate which of these fractions were able to form ISCOMs by the method of ether injection. Six fractions of Quil A were isolated by semi-preparative reverse phase high performance liquid chromatography (RP-HPLC) and characterised by analytical HPLC, liquid chromatography tandem mass spectrometry (LC-MS) and the qualitative Liebermann-Burchard and Molisch tests for triterpenoids and carbohydrates respectively. ISCOMs were subsequently prepared from the isolated fractions by the method of ether injection and the resulting preparations characterized by photon correlation spectroscopy (PCS) and negative stain transmission electron microscopy (TEM). The molecular weights of the major compounds in the fractions ranged from approximately 1200 to approximately 2300 Da; all fractions tested positive for triterpenoids and saccharides and four of the fractions were identified as QS-7, QS-17, QS-18 and QS-21 by analysis (LC-MS and analytical HPLC). Injection of ether solutions of lipids into aqueous solutions of QS-17, QS-18 or QS-21 all resulted in homogeneous ISCOM dispersions. The combination of lipids and QS-7 by ether injection produced lamellae and liposomes as the prominent structures and a minor amount of ISCOMs. The remaining two hydrophilic, low molecular weight fractions of Quil A did not produce ISCOMs, instead liposomes and helical structures predominated in the samples.

Characterization of in vivo anti-rotavirus activities of saponin extracts from Quillaja saponaria Molina

“…Different types of saponins have different levels of oxidation around the quillaic acid skeleton based on the types, location, and number of sugars and acyl moieties, respectively (Apers et al, 2001; van Setten and van de Werken, 1996) It is likely that the true number of saponin variants present in these extracts would exceed 100 if all conformational isomers are considered (Cox et al, 1998). The saponins content and identification can be determined by RP-HPLC (Fleck et al, 2006; Guo and Kenne, 2000a; Kensil et al, 1991b; Marciani et al, 2003; Pham et al, 2006). At least 22 peaks (designated QS-1 to QS-22) can be distinguished.…”

Carbohydrate-based immune adjuvants

“…In vivo , QS-21 induces inflammatory cytokines, IgG2a antibodies and CD8 T cells in mice, consistent with a Th1 bias [105,107]. Numerous clinical trials have been conducted using QS-21 alone as a vaccine adjuvant and it has also been used in more complex adjuvant formulations such as complexed with cholesterol as immune-stimulating complexes (ISCOMs) [108] or mixed with MPL in an oil-in-water emulsion such as in the proprietary AS02 adjuvant. Because of its potent ability to lyse cell membranes, adverse reactions, including severe injection site pain and hemolysis, are major limiting factors in QS-21 use.…”

Vaccine-induced antibodies linked to bovine neonatal pancytopenia (BNP) recognize cattle major histocompatibility complex class I (MHC I)

“…Quil A is a mixture of saponins, which are extracted from the bark of the tree Quillaja saponaria Molina. Quil A forms immune-stimulatory complexes (ISCOMS) together with the other components of the adjuvant and antigens [16,17]. ISCOMS efficiently induce both, antigen-specific antibodies (IgG1 and IgG2) as well as T-cell response (Th1 and Th2) [18,19].…”