Synthesis and Preclinical Evaluation of QS-21 Variants Leading to Simplified Vaccine Adjuvants and Mechanistic Probes

Chea, Eric K.; Fernández‐Tejada, Alberto; Damani, Payal; Adams, Michelle M.; Gardner, Jeffrey R.; Livingston, Philip O.; Ragupathi, Govind; Gin, David Y.

doi:10.1021/ja305121q

Cited by 57 publications

(79 citation statements)

References 40 publications

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“…QS-21-triggered NLRP3 responses are likely not due to particulate formation because the amount of QS-21 used here is ϳ20-fold lower than the critical micelle concentration (30). QS-21 should be present in all current saponin containing vaccines, hence the inflammasome-inducing action of the heterogeneous saponins may be due to QS-21 and similar compounds (26,69).…”

Section: Discussionmentioning

confidence: 99%

Identification of QS-21 as an Inflammasome-activating Molecular Component of Saponin Adjuvants

Marty-Roix

Vladimer

Pouliot

et al. 2016

Journal of Biological Chemistry

161

154

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Many immunostimulants act as vaccine adjuvants via activation of the innate immune system, although in many cases it is unclear which specific molecules contribute to the stimulatory activity. QS-21 is a defined, highly purified, and soluble saponin adjuvant currently used in licensed and exploratory vaccines, including vaccines against malaria, cancer, and HIV-1. However, little is known about the mechanisms of cellular activation induced by QS-21. We observed QS-21 to elicit caspase-1-dependent IL-1␤ and IL-18 release in antigen-presenting cells such as macrophages and dendritic cells when co-stimulated with the TLR4-agonist adjuvant monophosphoryl lipid A. Furthermore, our data suggest that the ASC-NLRP3 inflammasome is responsible for QS-21-induced IL-1␤/IL-18 release. At higher concentrations, QS-21 induced macrophage and dendritic cell death in a caspase-1-, ASC-, and NLRP3-independent manner, whereas the presence of cholesterol rescued cell viability. A nanoparticulate adjuvant that contains QS-21 as part of a heterogeneous mixture of saponins also induced IL-1␤ in an NLRP3-dependent manner. Interestingly, despite the role NLRP3 plays for cellular activation in vitro, NLRP3-deficient mice immunized with HIV-1 gp120 and QS-21 showed significantly higher levels of Th1 and Th2 antigen-specific T cell responses and increased IgG1 and IgG2c compared with wild type controls. Thus, we have identified QS-21 as a nonparticulate single molecular saponin that activates the NLRP3 inflammasome, but this signaling pathway may contribute to decreased antigenspecific responses in vivo.Because many protein antigens do not elicit strong immune responses on their own, vaccines often contain stimulatory adjuvants that enhance cell-mediated and humoral immune responses to help confer stronger protection. However, despite widespread use, there is little known regarding the pathways affected by many adjuvants. A better understanding of the mechanisms involved in adjuvant-generated protection can assist in the design of better vaccines against infections that currently lack effective immunization.Adjuvants activate an innate immune response, which in turn determines the strength and quality of the adaptive immune response. This response is first mediated by activation of antigen-presenting cells (APCs) such as dendritic cells and macrophages. Engagement of pattern recognition receptors, such as extracellular, membrane-bound Toll-like receptors (TLRs) 2 and cytosolic inflammasome-stimulating Nod-like receptors (NLRs) by their ligands elicits an inflammatory milieu and can eventually lead to a honed adaptive immune response.The NLR inflammasomes are multiprotein complexes that upon activation license the proteolytic processing of the zymogen pro-caspase-1 into mature caspase-1 (1). caspase-1 can then activate pro-forms of the inflammatory cytokines IL-1␤ and IL-18 into mature proteins, which are then secreted through unknown pathways. IL-1␤ and IL-18 are potent proinflammatory cytokines that can, for instance, promote T helper 17 ...

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Section: Discussionmentioning

confidence: 99%

Identification of QS-21 as an Inflammasome-activating Molecular Component of Saponin Adjuvants

Marty-Roix

Vladimer

Pouliot

et al. 2016

Journal of Biological Chemistry

161

154

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“…In an earlier confocal microscopy study, Chea et al showed internalization of 43 to a distinct cellular compartment of immature dendritic cells, in contrast to related attenuated variants bearing BODIPY (SQS-0-0-5-17) and Cascade Blue (SQS-0-0-5-15) fluorophores, and other fluorescent negative controls (Figure 15d). 31 Taken together, these results suggest a role for active adjuvants in the trafficking of OVA by antigen-presenting cells to the draining lymph nodes, a known site of immune cell maturation, and provide early insights into the mechanisms of saponin immunopotentation.…”

Section: Development Of Improved Synthetic Saponin Adjuvantsmentioning

confidence: 63%

“…Thus, Chea et al investigated additional acyl chain domain variants (Figure 4). 31 Dodecanedioic acyl variant 30 (SQS-0-0-4-5) was designed to improve water solubility while cholestanyl variant 31 (SQS-0-0-4-6) was devised to probe a hypothesis that interaction of QS-21 with cell membrane-bound cholesterol may be important in initiating adjuvant activity. Mouse vaccinations with GD3-KLH and the immunogenic peptide MUC1 (prostate and breast cancer antigen, nonglycosylated tandem repeat) conjugated to KLH (MUC1-KLH) revealed that 30 elicited antibody responses comparable to natural and synthetic QS-21 across all antigens, while 31 was less active (Figure 5a–c).…”

Section: Development Of Improved Synthetic Saponin Adjuvantsmentioning

confidence: 99%

“…inactive acyl chain domain amine variant 42 [SQS-0-0-5-11]). 31 Thus, Fernández-Tejada et al pursued more conservative structural modifications, replacing the C4-aldehyde substituent in quillaic acid variant 72 (SQS-1-0-5-18) with a hydroxymethyl group in caullophylogenin variant 73 (SQS-1-11-5-18) or a methyl group in echinocystic acid variant 74 (SQS-1-8-5-18). The triterpene C16-hydroxyl group was also independently deleted in the corresponding gypsogenin 75 (SQS-1-9-5-18), hederagenin 76 (SQS-1-10-5-18), and oleanolic acid 77 (SQS-1-7-5-18) variants (Figure 13).…”

Section: Development Of Improved Synthetic Saponin Adjuvantsmentioning

confidence: 99%

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

2016

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ConspectusVaccines 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 Accoun...

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“…Our studies have revealed that significant structural modifications of the bridging fucose residue within the linear tetrasaccharide and of the acyl chain of QS-21 cause little or no impairment of adjuvant activity [9, 10, 12]. Additionally, systematic structure–function studies of the carbohydrate regions have indicated that the terminal sugar within the linear tetrasaccharide as well as the entire branched trisaccharide domain are not required for adjuvant activity [10, 11]. This led us to identify highly simplified acyl chain variants and a truncated linear trisaccharide congener that were equipotent to QS-21 in a preclinical vaccination model.…”

Section: Introductionmentioning

confidence: 99%

Semisynthesis of Analogues of the Saponin Immunoadjuvant QS-21

Fernández‐Tejada

Walkowicz

Tan

et al. 2016

Methods in Molecular Biology

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Saponins are triterpene glycoside natural products that exhibit many different biological properties, including activation and modulation of the immune system, and have therefore attracted significant interest as immunological adjuvants for use in vaccines. QS-21 is the most widely used and promising saponin adjuvant but suffers from several liabilities, such as scarcity, dose-limiting toxicity, and hydrolytic instability. Chemical synthesis has emerged as a powerful approach to obtain homogeneous, pure samples of QS-21 and to improve its properties and therapeutic profile by providing access to optimized, synthetic saponin variants. Herein, we describe a general method for the semisynthesis of these molecules from QS-21, with detailed synthetic protocols for two saponin variants developed in our recent work.

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Synthesis and Preclinical Evaluation of QS-21 Variants Leading to Simplified Vaccine Adjuvants and Mechanistic Probes

Cited by 57 publications

References 40 publications

Identification of QS-21 as an Inflammasome-activating Molecular Component of Saponin Adjuvants

Identification of QS-21 as an Inflammasome-activating Molecular Component of Saponin Adjuvants

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

Semisynthesis of Analogues of the Saponin Immunoadjuvant QS-21

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