Development of Improved Vaccine Adjuvants Based on the Saponin Natural Product QS-21 through Chemical Synthesis

Abstract

Vaccines based on molecular subunit antigens are increasingly being investigated due to their improved safety and more precise targeting compared to classical whole-pathogen vaccines. However, subunit vaccines are inherently less immunogenic; thus, coadministration of an adjuvant to increase the immunogenicity of the antigen is often necessary to elicit a potent immune response. QS-21, an immunostimulatory saponin natural product, has been used as an adjuvant in conjunction with various vaccines in numerous clinical trials, but suffers from several inherent liabilities, including scarcity, chemical instability, and dose-limiting toxicity. Moreover, little is known about its mechanism of action. Over a decade-long effort, beginning at the University of Illinois at Urbana-Champaign and continuing at the Memorial Sloan Kettering Cancer Center (MSKCC), the group of Prof. David Y. Gin accomplished the total synthesis of QS-21 and developed a practical semisynthetic approach to novel variants that overcome the liabilities of the natural product. First, semisynthetic QS-21 variants were designed with stable amide linkages in the acyl chain domain that exhibited comparable in vivo adjuvant activity and lower toxicity than the natural product. Further modifications in the acyl chain domain and truncation of the linear tetrasaccharide domain led to identification of a trisaccharide variant with a simple carboxylic acid side chain that retained potent adjuvant activity, albeit with reemergence of toxicity. Conversely, an acyl chain analogue terminating in a free amine was inactive but enabled chemoselective functionalization with radiolabeled and fluorescent tags, yielding adjuvant-active saponin probes that, unlike inactive congeners, accumulated in the lymph nodes in vaccinated mice and internalized into dendritic cells. Subtle variations in length, stereochemistry, and conformational flexibility around the central glycosidic linkage provided QS-21 variants with adjuvant activities that correlated with specific conformations found in molecular dynamics simulations. Notably, deletion of the entire branched trisaccharide domain afforded potent, truncated saponin variants with negligible toxicity and improved synthetic access, facilitating subsequent investigation of the triterpene core. The triterpene C4-aldehyde substituent, previously proposed to be important for QS-21 adjuvant activity, proved to be dispensable in these truncated saponin variants, while the presence of the C16 hydroxyl group enhanced activity. Novel adjuvant conjugates incorporating the small-molecule immunopotentiator tucaresol at the acyl chain terminus afforded adjuvant-active variants but without significant synergistic enhancement of activity. Finally, a new divergent synthetic approach was developed to provide versatile and streamlined access to additional linear oligosaccharide domain variants with modified sugars and regiochemistries, opening the door to the rapid generation of diverse, synthetically accessible analogues. In this Account, we summarize these multidisciplinary studies at the interface of chemistry, immunology, and medicine, which have provided critical information on the structure-activity relationships (SAR) of this Quillaja saponin class; access to novel, potent, nontoxic adjuvants for use in subunit vaccines; and a powerful platform for investigations into the mechanisms of saponin immunopotentiation.

Figure 1

Structure of QS-21 and four structural domains.

Figure 2

Structures and semisynthetic modifications of main active components of GPI-0100.

Scheme 1. Semisynthesis of QS-7 from Partially Purified Quillaja saponaria Extract Quil A

Scheme 2. Synthesis of Modified Linear Tetrasaccharide Domain via (a) Preparation of Protected 4-Azido-4-deoxygalactose 10 and (b) Carbohydrate Assembly

Scheme 3. Synthesis of Acyl Chain Domain Variants with Amide Linkages by (a) Acylation of Amine 22 with (b–d) the Corresponding Acyl Chain Analogues

Figure 3

Immunological evaluation of acyl chain domain variants with amide linkages. (a–c) Antibody titers after three vaccinations and booster and (d) median weight loss after first vaccination. Mice vaccinated with GD3-KLH (10 μg) and saponin (10 μg); horizontal bars indicate median titers; statistical significance compared to no-adjuvant control assessed using two-tailed unpaired Student’s t test with 95% confidence interval, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. NQS-21 = natural QS-21.

Figure 4

Structures of additional acyl chain domain variants. Four-number SQS (synthetic Quillaja saponin) codes designate structural variants in each of the four corresponding structural domains of QS-21, left to right, with 0 assigned to the natural product structure.

Figure 5

Immunological evaluation of additional acyl chain domain variants (5 μg GD3-KLH, 5 μg MUC1-KLH, 10 μg saponin). SQS-21 = synthetic QS-21 (2:1 mixture of 1 and 2).

Figure 6

(a) Structures of progressively truncated linear tetrasaccharide domain variants. (b) Representative synthesis of linear trisaccharide variant 32.

Figure 7

Immunological evaluation of truncated linear tetrasaccharide domain variants (5 μg GD3-KLH, 2.5 μg MUC1-KLH, 20 μg OVA, 20 μg saponin).

Scheme 4. Synthesis of Functionalized Acyl Chain Domain Variants

Figure 8

Immunological evaluation of functionalized acyl chain domain variants (2.5 μg MUC1-KLH, 10 μg saponin).

Figure 9

Structures of central glycosidic linkage variants.

Scheme 5. Synthesis of Central Glycosidic Linkage Variants via (a) Preparation of Linear Trisaccharide Glycosyl Donors and (b) Installation on [PQPS]-CO₂H Core (5) Using Glycosyl Acceptor as an Electrophile or (c) Nucleophile

Figure 10

Immunological evaluation of central linkage variants (5 μg GD3-KLH, 2.5 μg MUC1-KLH, 20 μg OVA, 5 or 20 μg saponin). Anti-GD3 responses and data for inactive α/β-carbamates (45, 47) not shown.

Figure 11

Conformational ensembles and central glycosidic linkage dihedral angle distributions (arbitrary axis zero-points) from unrestrained molecular dynamics simulations of (a) QS-21-Api (1) and (b) representative saponin variants, distinguishing active and inactive saponins.

Scheme 6. Synthesis of Aryl Iodide Acyl Chain Domain Variants

Figure 12

Immunological evaluation of aryl iodide acyl chain domain variants (2.5 μg MUC1-KLH, 20 μg OVA, 20 or 50 μg saponin).

Figure 13

Structures of triterpene domain variants with independent modifications of C4-aldehyde and C16-hydroxyl group.

Figure 14

Immunological evaluation of triterpene domain variants (5 μg GD3-KLH, 2.5 μg MUC1-KLH, 20 μg OVA, 20 or 50 μg saponin). Data for attenuated oleanolic acid variant 77 (SQS-1-7-5-18) not shown.

Figure 15

Mechanistic studies of saponin variants. (a) Biodistribution of active and inactive radioiodinated saponins in mice (24 h post injection; 20 μg OVA, 20 μg unlabeled saponin, ≈25 μCi radiolabeled saponin); statistical significance shown only for injection site and lymph nodes. (b) In vivo fluorescence imaging of active fluorescein-labeled saponin 43 and inactive nonfluorescent precursor 42, each coadministered with Alexa-467-OVA (A647-OVA), in mice (24 h post injection; 20 μg A647-OVA, 10 μg saponin) and (c) fluorescence imaging of dissected lymph nodes (mice injected in left flank; right lymph nodes are negative controls). (d) Confocal microscopy imaging of subcellular localization of active and inactive fluorescent saponins and fluorescent glycine methyl ester (GlyOMe) negative controls in immature dendritic cells.

Scheme 7. Synthesis of Saponin–Tucaresol Conjugates

Figure 16

Streamlined synthetic access using (a) linear oligosaccharide domain variants derived from readily available carbohydrate precursors and (b) a divergent synthetic strategy.

Figure 17

Summary of saponin structure–adjuvant activity relationships.