Charlotte R. Kensil

Structure/function studies of QS-21 adjuvant: assessment of triterpene aldehyde and glucuronic acid roles in adjuvant function.

QS-21, a purified Quillaja saponaria saponin immunologic adjuvant, contains two functional groups that we hypothesized to be involved in the adjuvant mechanism of action through charge or Schiff base interaction with a cellular target. Derivatives, prepared by modification of these sites, were prepared and tested for their ability to augment the immunogenicity of the antigen ovalbumin (OVA) in C57BL/6 mice. QS-21 derivatives that were modified at the carboxyl group on an anionic sugar, glucuronic acid, retained adjuvant activity for antibody stimulation, inducing relative increases in antibody titers similar to those induced by QS-21, although the minimum adjuvant dose required for this stimulation was increased several fold relative to the dose of unmodified QS-21. One of these derivatives also retained significant activity for induction of OVA-specific cytotoxic T-lymphocytes. In contrast, QS-21 derivatives modified at an aldehyde on the triterpene did not show adjuvant activity for antibody stimulation or for induction of cytotoxic T-lymphocytes, suggesting that this functional group may be involved in the adjuvant mechanism.

Structural and Immunological Characterization of the Vaccine Adjuvant QS-21

QS-21 is a triterpene glycoside “saponin” isolated from the bark of the Quillaja saponaria Molina tree, a species native to South America. The bark of this tree, particularly the saponin fraction present in the bark, has long been known as a source of immune stimulators that can be used as vaccine adjuvants. Espinet (1951) noted the adjuvant activity of plant saponins to enhance the potency of foot-and-mouth disease vaccines. A number of commercially available complex saponin extracts were developed for adjuvant use. Not all of these extracts were effective as adjuvants. Dalsgaard (1970) showed that a correlation exists between adjuvant activity and the source of the saponin, and that the most adjuvantactive extracts were derived from the tree Quillaja saponaria. The use of Quillaja saponins as adjuvants has been reviewed by Campbell and Peerbaye (1992).

Three double-blind, randomized trials evaluating the safety and tolerance of different formulations of the saponin adjuvant QS-21

The effects of the adjuvant QS-21 in various formulations on immediate pain on injection after intramuscular injection were evaluated in three Phase I clinical trials in healthy adults. Each trial was designed as a double-blind, randomized, four-way or five-way cross-over study with each subject acting as his/her own control. In the first trial, four formulations designed to evaluate the effect of QS-21 or pH (over a range of 6--7.2) were evaluated: phosphate-buffered saline at pH 6.0 or 7.2, and 50 microg of QS-21 in phosphate-buffered saline at pH 6.0 or 7.2. Thirty-three volunteers received each of the four intramuscular injections in random order separated by approximately 1 week. The volunteers assessed the immediate injection pain from 0 to 10 (none to most pain). The data indicate that the presence of QS-21, but not pH, is associated with transient injection site pain. The second trial, which utilized the same design as the first trial, evaluated formulations of QS-21 in various excipients. Fifteen volunteers received phosphate-buffered saline, QS-21/PBS, QS-21/aluminum hydroxide, and QS-21/4 mg/ml of polysorbate 80. Polysorbate 80, but not aluminum hydroxide, reduced the mean pain score compared to QS-21/PBS. The third trial evaluated formulations of QS-21 in additional excipients. Fifteen volunteers received aluminum hydroxide (without QS-21), QS-21/PBS, QS-21/0.72% benzyl alcohol, QS-21/30 mg/ml of hydroxypropyl-beta-cyclodextrin, and QS-21/8-mg/ml of polysorbate 80. Benzyl alcohol, cyclodextrin, and the higher concentration of polysorbate 80 reduced the pain scores associated with QS-21. Hence, QS-21 is associated with injection pain in simple buffer formulations, but it is possible to improve the acceptability of QS-21-containing formulations through reformulation with certain excipients.

QS-21 structure/function studies: effect of acylation on adjuvant activity

QS-21 is a natural saponin adjuvant derived from the tree Quillaja saponaria Molina. Previous studies over a limited dose range suggested the acylation is critical to adjuvant activity. In this study, we prepared DS-1 (deacylated QS-21) and RDS-1 (reacylated DS-1 with dodecylamine at a different site than QS-21) to determine the effect on a dose-response curve over a wider range in mice. DS-1 and RDS-1 induced IgG1 responses at higher doses compared to that induced by QS-21. DS-1 was inactive for inducing IgG2a or CTL responses at any doses. RDS-1 showed moderate IgG2a response at 240 microg, but did not show CTL response at any dose evaluated.

Structure of the saponin adjuvant QS-21 and its base-catalyzed isomerization product by 1H and natural abundance 13C NMR spectroscopy

The saponin QS-21, derived from the bark of the Quillaja saponaria Molina tree, has shown great potential as an adjuvant with a number of vaccines. Kinetic studies carried out to establish the stability of vaccine formulations show that commercially supplied QS-21 (primarily QS-21A) is converted slowly at pH 5.5, and rapidly at higher pH, to an equilibrium mixture of two regioisomers, QS-21A and QS-21B, in a ratio of 20:1. NMR studies show that QS-21A and QS-21B differ only in the point of attachment of the fatty acyl moiety to the fucose sugar ring. The major isomer, QS-21A, has the fatty acyl portion attached at the 4-hydroxyl group whereas the minor isomer, QS-21B, has the fatty acyl portion attached at the 3-hydroxyl group. The isomerization most likely involves ionization of the 3-hydroxy group and intramolecular acyl transfer from the 4-hydroxy group. The relative stereochemistry of the triterpene and the sugar anomeric centers is also established by NMR methods.

Structure/Function Studies on QS-21, A Unique Immunological Adjuvant from Quillaja saponaria

QS-21 is an acylated triterpene glycoside isolated from the bark of the South American tree Quillaja saponaria Molina’. It is a potent immunological adjuvant. It has been shown to enhance antigen-specific antibody titers to a wide variety of T-dependent and T-independent antigens, including hen egg albumin (ovalbumin)2, recombinant Borrelia burgdorferi OspA and OspB3, recombinant human cytomegalovirus envelope protein gB4, and E. coli 018 polysaccharides’. Further, QS-21-adjuvanted subunit antigen vaccines stimulate a cytotoxic T lymphocyte (CTL) response6,7,8. This response is characterized by the induction of CD8+ T cells that kill target cells expressing specific antigen. Recently, QS-21 was shown to enhance antigen-specific IgM and IgG responses in a Phase I clinical study of a melanoma immunotherapeutic vaccine9.

A Semisynthetic Quillaja Saponin as a Drug Delivery Agent for Aminoglycoside Antibiotics

AbstractPurpose. The purpose of this study was to investigate the utility of a purified, semisynthetic saponin, DS-1, prepared by deacylation of a naturally occurring saponin from the bark of the Quillaja saponaria Molina tree, as a permeation enhancer for mucosal delivery of the aminoglycosides, gentamicin and tobramycin. Methods. Gentamicin or tobramycin formulations, with and without DS-1, were administered to rats nasally, ocularly, and rectally. Serum aminoglycoside levels following mucosal application were compared with those administered intramuscularly. Gentamicin formulations, with and without DS-1, were administered intranasally to mice 60 minutes after a lethal bacterial challenge. To ascertain nasal irritation potential, DS-1 nosedrops were administered to rats twice daily for 7 days in the right nostril only. Comparison of the left (internal control) and right nostril was made with a control group that received only buffer. Results. Significant transport across mucous membranes was only observed in formulations containing DS-1. This effect on drug delivery was transient. Administration of an intranasal gentamicin/ DS-1 formulation reversed the lethal bacterial challenge in mice, demonstrating that biological activity was retained after absorption. Nasal irritation was not observed in groups receiving DS-1 nose-drops, which were identical to control groups. Conclusions. DS-1 has potential as a transmucosal delivery agent for the aminoglycoside antibiotics.

Effects of QS-21 on Innate and Adaptive Immune Responses

Immunological adjuvants typically fall within two classifications: (a) “vehicle” adjuvants that transport antigen from injection sites to lymphoid tissues (generally particulate adjuvants such as emulsions, liposomes, or mineral salts) and (b) “immunomodulators” that act on antigen-presenting cells (APCs) or other cells to induce a cytokine response that influences the immune response to a co-adminstered antigen (1). Most immunomodulators are derived from bacterial sources (2). These include lipopolysaccharides and derivatives such as lipid A or unmethylated deoxyribonucleic acid (DNA) or oligonucleotide sequences, such as CpG enriched sequences from bacteria. However, natural products from other sources can also act as immunomodulators. One such product is the plant saponin QS-21.

QS-21 Augments the Antibody Response to a Synthetic Peptide Vaccine Compared to Alum

The immunomodulating ability of adjuvants has been shown to enhance immune responses directed both specifically and non-specifically against tumor development and growth and to increase immune response to weak antigens. This study compared two adjuvants, alum and QS-21, given separately and together in a candidate p17-based subunit AIDS vaccine, HGP-30-KLH. Alum, the only adjuvant approved for human use, confers a particulate appearance to the immunogen by adsorbing it and retains the immunogen in the body.1 QS-21 is a pure triterpene glycoside saponin adjuvant,2 and is purified from the total saponin fraction of Quillaja saponaria Molina. Crude mixtures of Quillaja saponaria saponins serve as the matrix component of the immunostimulating complex (ISCOM).3 The study’s hypothesis was that these two adjuvants function by different means to increase antibody titer to a synthetic peptide immunogen. QS-21, with its biochemical as well as physical properties, was expected to generate a greater antibody response than alum, and the two combined may be additive or synergistic.