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.

Plasmodium falciparum: immunogenicity of alum-adsorbed clinical-grade TBV25-28, a yeast-secreted malaria transmission-blocking vaccine candidate.

Gozar, M. M. G., Muratova, O., Keister, D. B., Kensil, C. R., Price, V. L., and Kaslow, D. C. 2001. Plasmodium falciparum: Immunogenicity of alum-adsorbed clinical-grade TBV25-28, a yeast-secreted malaria transmission-blocking vaccine candidate. Experimental Parasitology 97, 61-69. The fusion of Pfs25 and Pfs28, two major surface antigens on zygotes and ookinetes of Plasmodium falciparum, as a single recombinant protein (TBV25-28) was previously shown to elicit potent transmission-blocking antibodies in mice. Clinical-grade TBV25-28 was subsequently manufactured and its potency was evaluated in rabbits. Rabbits received three doses of either clinical-grade TBV25H or clinical-grade TBV25-28 adsorbed to alum with or without QS-21. As measured in a standard membrane-feeding assay, addition of QS-21 to the formulations appeared to enhance transmission-blocking potency of rabbit sera after two vaccinations but not after three vaccinations. Surprisingly, TBV25H elicited more potent transmission-blocking antibodies than did TBV25-28, a result strikingly different from those of previous mouse experiments using research-grade TBV25-28. The apparent decrease in potency of clinical-grade TBV25-28 in rabbits appears to reflect an enhancement in potency of clinical-grade TBV25H in a new formulation rather than simply a species difference in immunogenicity of TBV25-28.

Oral QS-21 requires early IL-4 help for induction of mucosal and systemic immunity.

The highly purified saponin derivative, QS-21, from the Quillaja saponaria Molina tree has been proved to be safe for parenteral administration and represents a potential alternative to bacterial enterotoxin derivatives as a mucosal adjuvant. Here we report that p.o. administration of QS-21 with the vaccine protein tetanus toxoid elicited strong serum IgM and IgG Ab responses, which were only slightly enhanced by further oral immunization. The IgG Ab subclass responses were predominantly IgG1 followed by IgG2b for the 50-microg p.o. dose of QS-21, whereas the 250-microg p.o. dose also induced IgG2a and IgG3 Abs. Low oral QS-21 doses induced transient IgE Ab responses 7 days after the primary immunization, whereas no IgE Ab responses were seen in mice given the higher QS-21 dose. Further, low but not high p.o. QS-21 doses triggered Ag-specific secretory IgA (S-IgA) Ab responses. Th cell responses showed higher IFN-gamma (Th1-type) and lower IL-5, IL-6, and IL-10 (Th2-type) secretion after the high QS-21 p.o. dose than after low doses. Interestingly, the mucosal adjuvant activity of low oral QS-21 doses was diminished in IL-4(-/-) mice, suggesting a role for this cytokine in the initiation of mucosal immunity by oral QS-21. In summary, our results show that oral QS-21 enhances immunity to coadministered Ag and that different doses of QS-21 lead to distinct patterns of cytokine and serum Ab responses. We also show that an early IL-4 response is required for the induction of mucosal immunity by oral QS-21 as adjuvant.

Enhancement of intestinal model compound transport by DS-1, a modified Quillaja saponin.

DS-1, a modified Quillaja saponin, has recently been shown to promote the absorption of insulin and aminoglycoside antibiotics via the ocular and nasal route. The purpose of this study is to investigate the effect of DS-1 on intestinal permeability, the mechanism of its action, and reversibility of the effect. The permeation-enhancing activity of DS-1 was evaluated in cultured monolayers of the Caco-2 intestinal epithelial cells by examining its effect on the transepithelial electric resistance (TEER) and on transport of mannitol and a model D-decapeptide. Mucosal addition of DS-1 promptly reduced the TEER of the Caco-2 monolayers, and a propensity of recovery of the TEER was observed upon its removal. DS-1 added at 0.01-0.1% (w/v) increased the transports of both mannitol and D-decapeptide in a dose-dependent manner; a relatively "flat" concentration-dependence was seen at 0.1-0.2%. Visualization studies conducted by confocal laser scanning microscopy (CLSM) seem to suggest that DS-1 enhances the Caco-2 permeability mainly via a transcellular route. Histological examination failed to reveal noticeable morphological alterations in the cell monolayers pretreated with DS-1. The integrity of the Caco-2 monolayers, as assessed by their permeability to mannitol, was found to be recoverable following the mucosal pretreatment of DS-1. These results suggest that DS-1 is an efficacious intestinal permeation-enhancing agent with low adverse effect on the epithelial viability and barrier function.

Induction of systemic and mucosal immune responses to human immunodeficiency virus type 1 by a DNA vaccine formulated with QS-21 saponin adjuvant via intramuscular and intranasal routes.

Induction of mucosal and cell-mediated immunity is critical for development of an effective vaccine against human immunodeficiency virus (HIV). We compared intramuscular and intranasal immunizations with a DNA vaccine encoding env of HIV-1 and evaluated the QS-21 saponin adjuvant for augmentation of the systemic and mucosal immune responses to HIV-1 in a murine model. Vaccination via the two routes elicited comparable systemic immune responses, and QS-21 consistently enhanced antigen-specific serum immunoglobulin G2a (IgG2a) production, delayed-type hypersensitivity reaction, and cytolytic activity of splenocytes. Intestinal secretory IgA production and cytolytic activity of the mesenteric lymph node cells are preferentially elicited by intranasal immunization, and QS-21 augmented these activities as well. This adjuvant augmented production of interleukin-2 (IL-2) and gamma interferon (IFN-gamma) associated with decrease in IL-4 synthesis by antigen-restimulated splenocytes. The serum immunoglobulin subtype profile showed a dominant IgG2a response and less strong IgG1 and IgE production in a QS-21 dose-dependent manner. As expected, enhancements of humoral and cell-mediated immune responses by QS-21 were abrogated by treatment with anti-IL-2 and anti-IFN-gamma monoclonal antibodies. These results suggest that the intranasal route of DNA immunization is more efficient than the intramuscular route in inducing mucosal immunity mediated by sIgA and mesenteric lymphocytes. Furthermore, QS-21 is able to act as a mucosal adjuvant in DNA vaccination and demonstrates its immunomodulatory property via stimulation of the Th1 subset. This study emphasizes the importance of the route of immunization and the use of an adjuvant for effective DNA vaccination against HIV-1.

QS-21 and QS-7: purified saponin adjuvants.

QS-21 and QS-7 are two adjuvant-active saponins that can be obtained in high purity from Quillaja saponaria Molina extracts. QS-21 is a highly characterized compound and is known to be a potent adjuvant for antibody and CD8+ CTL response to subunit antigens. Less is known about the activity and structure of the hydrophilic saponin QS-7. Hence, we have carried out a detailed structural and immunological characterization. As with QS-21, QS-7 was shown to be a 3,28-O-bisglycoside quillaic acid, with some differences being a higher degree of glycosylation and a considerably shorter fatty acyl unit in QS-7. These differences were correlated to a lower lytic activity against sheep red blood cells. Different doses of QS-7 were evaluated for stimulation of immune response to the antigen ovalbumin, given three times by subcutaneous route to C57BL/6 mice. QS-7 doses of 40 micrograms or higher were shown to induce a strong CD8+ CTL response reproducibly against E. G7-OVA targets (similar to that induced by a 5-10 micrograms dose of QS-21). QS-7 (at doses above 5 micrograms) was also shown to stimulate CTL against peptide 18 of HIV-1IIIB gp120 after three immunizations of Balb/c mice with recombinant gp120 and different doses of QS-7. These data suggest that a hydrophilic saponin with low lytic activity can stimulate MHC Class I CTL responses although a higher minimum dose may be required for some antigens.

QS-21: a water-soluble triterpene glycoside adjuvant.

QS-21 is a purified immunological adjuvant derived from a natural source, the bark of the tree Quillaja saponaria. It is a water soluble triterpene glycoside with amphiphilic character that can be mixed with a soluble antigen in a fully soluble vaccine formulation or combined with emulsion or mineral salt adjuvants. QS-21 has been shown to enhance antibody and cell-mediated immune responses to subunit antigens, as well as DNA vaccines in animal models. It acts as an immunostimulatory adjuvant, eliciting production of immunomodulatory cytokines, and not as an antigen depot. QS-21 is currently under clinical evaluation with various vaccines. This includes a Phase II evaluation of a QS-21 adjuvanted pneumococcal polysaccharide vaccine and a Phase III evaluation of a QS-21 adjuvanted GM2-KLH (ganglioside GM2 vaccine) immunotherapeutic product for melanoma. At present, more than 1600 individuals have received vaccines containing QS-21 adjuvant. In most studies, QS-21-containing vaccines have been well-tolerated. No serious adverse events have occurred.

Induction of cross-reactive cytotoxic T-lymphocyte responses specific for HIV-1 gp120 using saponin adjuvant (QS-21) supplemented subunit vaccine formulations.

The antigenic variation associated with Human Immunodeficiency Virus type-1 (HIV-1) envelope proteins could limit their utility in vaccines if the immune responses induced are specific for immunodominant variable epitopes. We evaluated the ability of experimental subunit vaccines, containing recombinant forms of the envelope glycoprotein (rgp120) from two HIV-1 variants, to induce immune responses capable of recognizing unrelated HIV-1 variants. A vaccine formulation based on HIV-1IIIB/LAI rgp120 and supplemented with saponin adjuvant (QS-21) induced neutralizing antibodies specific for the HIV-1IIIB/LAI variant. This antibody response was presumably specific for the variable principle neutralizing determinant (PND) of the third variable region of gp120, the V-3 region. This formulation induced cytotoxic T-lymphocytes (CTL) specific for the dominant V-3 epitope but also to an additional unidentified epitope outside of this region. The CTL specific for this second epitope also recognized gp120 from the HIV-1MN and HIV-1RF variants in a "cross-reactive" manner. A second vaccine formulation based on HIV-1MN rgp120 and QS-21 adjuvant induced neutralizing antibodies that were again variant-specific but also CTL that recognized all three HIV-1 variants in a cross-reactive manner. These data demonstrate that CTL capable of recognizing different HIV-1 variants, which are presumed to be specific for a conserved HIV-1 gp120 epitope, can be induced using subunit vaccines with the appropriate adjuvant while variant-specific antibody responses are produced. These findings support further evaluation of this vaccine format.

Structure-function relationship among Quillaja saponins serving as excipients for nasal and ocular delivery of insulin.

The purpose of this investigation was to explore the structure-function relationship among naturally occurring Quillaja saponins and derivatives for their ability to stimulate insulin delivery from nosedrops and eyedrops and to test the hypothesis that stimulation of peptide drug delivery was correlated with surfactant strength. Native saponins, including QS-21, were purified from an aqueous extract of Quillaja saponaria bark by adsorption chromatography and HPLC. Native saponins were then deacylated by mild alkaline hydrolysis to form DS-1 and DS-2, derivatives that are smaller and more hydrophilic than their parent compounds. DS-1 was further treated either to reduce an aldehyde residue to form DS-1(R) or to remove the fucose-containing oligosaccharide to form QH-957. Rats receiving eyedrops or nosedrops formulated with insulin, but without any Quillaja saponins, showed no hypoglycemic response. Rats receiving eyedrops or nosedrops formulated with insulin plus saponins showed a dose-dependent hypoglycemic response, with the following rank order: QS-21 > DS-1 > DS-1(R) > DS-2 > QH-957. Surfactant strength was determined by measurement of the critical micellar concentration (cmc) and hemolysis of sheep erythrocytes. The cmc was lowest for the parent saponins QS-21 and QS-18, and increased for the deacylated saponin derivatives DS-1, DS-2, and QH-957; hemolysis of sheep erythrocytes was observed at low concentrations (approximately 0.006 mM) of the parent saponins, QS-21 and QS-18, at intermediate concentrations (0.06-0.08 mM) of DS-1 and DS-2, and at higher concentrations of DS-1(R) (0.45 mM) and QH-957 (1.5 mM). Hence, efficacy as an absorption-enhancing agent was greatest in those saponins with the lowest hemolytic titers and cmc values. However, this relationship was not a strict one, because DS-1, which differs from DS-2 only in the absence of one glucose residue, was significantly more potent than DS-2 in stimulating the absorption of insulin. DS-1 and DS-2 share a similar cmc and hemolytic titer, so this difference in efficacy must be due to some specificity beyond simple surfactant strength. Furthermore, DS-1 does not trigger an immune response when administered to animals, whereas QS-21 is a strong immune system activator. Therefore, DS-1 has emerged as an interesting candidate for inclusion in an eyedrop or nosedrop formulation.

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.

Isomerization and formulation stability of the vaccine adjuvant QS-21.

The stability of the immunologic adjuvant QS-21 (Cambridge Biotech Corp.) was optimized for use in the MN rgp120 HIV-1 subunit vaccine. QS-21, a saponin purified by reversed phase HPLC from an extract of the bark of the Quillaja saponaria Molina tree, consisted initially of one species (QS-21A), but converted to two species, QS-21A and QS-21B, in aqueous solution. NMR studies indicated that the two species are structural isomers and that isomerization occurs by intramolecular trans-esterification of the fatty acid moiety between the 3- and 4-hydroxyl groups of the fucose ring (Jacobsen et al. Carbohydr. Res., in press). Both isomers were adjuvant active. Storage of QS-21 in aqueous solution resulted in the interconversion between these isomer forms, as well as the slow formation of degradation products due to ester hydrolysis. The critical micellar concentration of QS-21 in succinate buffer was measured by a fluorescent probe method to be 51 +/- 9 micrograms/mL. Studies were performed at different concentrations of QS-21 to assess the influence of micelle formation on stability. These experiments indicated that QS-21 is more stable in the micellar form, presumably because the most labile ester bond linking the fatty acid moiety to fucose is constrained or buried in the hydrophobic micellar environment. The pH of maximum stability was pH 5.5, the pH for minimum degradation of most esters. The final formulation, 500 micrograms/mL QS-21 in 20 mM sodium succinate, 150 mM NaCl, pH 5.5, provided a shelf-life of greater than 2 years.

Saponins as vaccine adjuvants.

Naturally occurring triterpene glycosides (saponins) from Quillaja saponaria have considerable adjuvant activity. Adjuvant functions include stimulation of high levels of antibody to T-dependent and T-independent antigens, induction of mouse IgG1, IgG2b, and IgG2a isotypes, and induction of cytotoxic T lymphocyte responses. This article reviews responses due to specific saponins of saponin preparations, effect of formulation, structure/function studies, and use in different preclinical and clinical vaccine applications.

A semisynthetic Quillaja saponin as a drug delivery agent for aminoglycoside antibiotics.

PURPOSE: 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 nosedrops, which were identical to control groups. CONCLUSIONS: DS-1 has potential as a transmucosal delivery agent for the aminoglycoside antibiotics.

DS-1, a modified Quillaja saponin, enhances ocular and nasal absorption of insulin.

The purpose of this study was to test DS-1, a modified Quillaja saponin, for its efficacy as an absorption enhancer. Anesthetized rats receiving eyedrops or nosedrops formulated with regular pork insulin in saline showed no hypoglycemic response, indicating no systemic absorption of insulin. However, rats receiving eyedrops or nosedrops formulated with insulin plus 0.025-0.10% DS-1 showed rapid absorption of insulin and a concomitant decrease in serum D-glucose levels. No response was observed following sublingual or buccal delivery of insulin. In conclusion, the modified saponin DS-1 was efficacious at enhancing nasal or ocular insulin delivery at extremely low concentrations. The mechanism of DS-1 action is not yet known.

Effect of adjuvants on immunogenicity of MN recombinant glycoprotein 120 in guinea pigs.

The immunogenicity of recombinant gp120 from the MN strain of HIV-1, a candidate HIV-1 vaccine, was evaluated in guinea pigs using adjuvant formulations with different physical and chemical properties. The adjuvants tested included Freund's adjuvant (FA), alum, and the novel adjuvant QS-21. These studies demonstrated that QS-21 provides a number of advantages compared to the two other adjuvants tested. QS-21 formulations accelerated the production of antibodies to MN rgp120 and elicited complete seroconversion after a single immunization. QS-21 shifted the antigen dose-response curve for antibody production by as much as three orders of magnitude, enabling a more economical use of antigen. Antibody titers to MN rgp120 and to the principal neutralizing determinant in the V3 domain were higher in animals receiving QS-21 formulations than in animals immunized with the other adjuvants, and correlated well with higher virus neutralization titers in an in vitro assay. These results support the testing of QS-21 in future clinical trials of candidate HIV-1 vaccines.

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.

Phase 1 trial of immunological adjuvant QS-21 with a GM2 ganglioside-keyhole limpet haemocyanin conjugate vaccine in patients with malignant melanoma.

Increasing doses of saponin fraction QS-21 were administered as immunological adjuvant in a Phase 1 trial with a constant dose of the melanoma ganglioside GM2 covalently attached to keyhole limpet haemocyanin (KLH). Twenty-eight patients with AJCC Stage III or IV melanoma who were free from disease after surgery were treated with six vaccinations administered subcutaneously over a 5-month period. Local and systemic reactions were QS-21 dose-related. Doses of < or = 100 micrograms induced mild local tenderness and inflammation at vaccination sites lasting 2-4 days and occasional brief low-grade fever and malaise, but no significant incapacitation. The 200 micrograms dose induced low-grade fever and malaise after 30% of vaccinations and local reactions as large as 20 cm in diameter were seen in all patients, resulting in discomfort with usage of the injected extremity for 5-10 days. The titres of IgM and IgG antibodies against GM2, and IgG antibodies against KLH, were highest at the 100 and 200 micrograms QS-21 doses. No antibodies against QS-21 were detected. This trial identifies the 100 micrograms dose of QS-21 as the optimal well tolerated dose for induction of antibodies against both the melanoma ganglioside/GM2 and the protein KLH in melanoma patients.

Impact of the saponin adjuvant QS-21 and aluminium hydroxide on the immunogenicity of recombinant OspA and OspB of Borrelia burgdorferi.

The impact of the adjuvants QS-21 and aluminium hydroxide (alum) on the immunogenicity of recombinant outer surface proteins A (OspA) and B (OspB) of Borrelia burgdorferi was investigated. Both non-acylated OspA and OspB derived from strain B31 were expressed in Escherichia coli and purified by reversible citraconylation and anion-exchange chromatography. Antisera to OspA or OspB were prepared in mice with antigens formulated with QS-21 or alum, and evaluated for specific immunoglobulin G isotypes, agglutination and borreliacidal activity. QS-21 significantly enhanced IgG2a and IgG2b antibody responses to OspA and OspB, and IgG1 response to OspA when compared with the formulation containing antigen alone. In contrast, alum significantly inhibited the induction of IgG2a and IgG2b responses to OspA. Alum had no significant effect on IgG1 response to OspA, or IgG2a and IgG2b responses to OspB, but significantly enhanced IgG1 antibody response to OspB. Antisera to OspA or OspB formulated by QS-21 possessed higher titres of agglutinating antibody than antisera to OspA or OspB alone. Borreliacidal activity was eight- to 64-fold higher in antisera to OspA formulated with QS-21 than in antisera to OspA formulated with or without alum. These antisera were highly borreliacidal to New York strain B31, a California isolate CA-2-87, German isolate Fr, and Swedish isolate G25. Antisera to OspB formulated with QS-21 were highly borreliacidal to strains B31 and Fr, but not to CA-2-87 and G25. Antisera to OspB formulated with alum were borreliacidal only to B31. Thus, OspA was superior to OspB and QS-21 superior to alum at eliciting functional antibody responses.(ABSTRACT TRUNCATED AT 250 WORDS)