Alberto Fernández-Tejada

The saponin natural product QS‐21 is a potent immunological adjuvant used in human vaccines to enhance the immune response but suffers from scarcity, instability, and dose‐limiting toxicity, precluding its advancement as a stand‐alone adjuvant. Further, putative cellular receptor(s) and its molecular mechanisms of action are unknown, which has prevented the rational design of saponin adjuvants, emphasizing the need for discovering these receptors and elucidating mechanisms. Herein, we have developed novel saponin photoaffinity probes for molecular target identification, merging synthetic chemistry, photoaffinity labeling, and chemical proteomics. One of these potent adjuvants generated superior antibody responses to QS‐21 with less toxicity and enabled identification of Cathepsin D (CatD) as the selective receptor driving saponin adjuvant effects. Binding to surface‐exposed CatD, lipid raft interactions, and lysosomal acidification were shown to be key for CatD‐mediated saponin‐induced immune activation, whereas neither photocrosslinking nor apparent binding was observed in comparative experiments with other lysosomal cathepsins, confirming the specificity of the interaction. These studies highlight the promise of this saponin probe as a potent vaccine adjuvant, unveiling CatD as the molecular target. This breakthrough sets the stage for elucidating the precise mechanisms of saponin immunopotentiation, facilitating rational adjuvant design and development of optimized adjuvant–antigen combinations for future vaccines.

The saponin natural product QS‐21 is a potent immunological adjuvant used in human vaccines to enhance the immune response but suffers from scarcity, instability, and dose‐limiting toxicity, precluding its advancement as a stand‐alone adjuvant. Further, putative cellular receptor(s) and its molecular mechanisms of action are unknown, which has prevented the rational design of saponin adjuvants, emphasizing the need for discovering these receptors and elucidating mechanisms. Herein, we have developed novel saponin photoaffinity probes for molecular target identification, merging synthetic chemistry, photoaffinity labeling, and chemical proteomics. One of these potent adjuvants generated superior antibody responses to QS‐21 with less toxicity and enabled identification of Cathepsin D (CatD) as the selective receptor driving saponin adjuvant effects. Binding to surface‐exposed CatD, lipid raft interactions, and lysosomal acidification were shown to be key for CatD‐mediated saponin‐induced immune activation, whereas neither photocrosslinking nor apparent binding was observed in comparative experiments with other lysosomal cathepsins, confirming the specificity of the interaction. These studies highlight the promise of this saponin probe as a potent vaccine adjuvant, unveiling CatD as the molecular target. This breakthrough sets the stage for elucidating the precise mechanisms of saponin immunopotentiation, facilitating rational adjuvant design and development of optimized adjuvant–antigen combinations for future vaccines.

Subunit cancer vaccines based on aberrant glyglycoproteins or glycolipids overexpressed on the tumor cell surface are limited by the inherent low immunogenicity of the corresponding glycan/peptide antigens. As such, they require the implementation of novel strategies that can boost the induced immune responses. One of these approaches exploits the conjugation of such antigenic fragments to immunoactive molecules, such as adjuvants and immunostimulatory epitopes, leading to unimolecular constructs with self‐adjuvanting properties. Herein, we report the development of novel di‐ and tricomponent conjugates built upon a streamlined, underexploited saponin scaffold derived from the QS‐21 adjuvant, which was covalently linked to carbohydrate and peptide antigens found on tumor‐associated glycolipids (e.g., Gb3 trisaccharide) and glycoproteins (e.g., Tn antigen and MUC1 tandem repeat sequence). Facile, late‐stage conjugation to a T‐helper peptide epitope resulted in self‐adjuvanting vaccine constructs that induced increased levels of antigen‐specific antibodies in mice recognizing the native antigen on cancer cells. The chemical versatility of our synthetically accessible saponin platform, together with the nontoxicity and immunogenicity of these new tricomponent candidates, emphasizes the potential of our novel saponin‐based, self‐adjuvanting approach for the development of molecular vaccine prototypes with prospective applications in cancer immunotherapy.

A generalizable semisynthetic vaccine approach to increase antigen presentation and immune recognition of poorly immunogenic tumor-associated MUC1 glycopeptides.

In this study, we formulated an alternative to AS01b by combining FP20, a synthetic TLR4 agonist, and QS21v, a minimal saponin adjuvant, aiming to improve the vaccine efficacy and stability. The phase transition temperature of FP20 was determined by using differential scanning calorimetry to be 43.9 °C, providing a foundation for the formulation process. The coformulation was prepared using a dry film method for even adjuvant distribution. Characterization by dynamic light scattering and nanoparticle tracking analysis revealed a uniform particle size distribution of ∼120 nm. Cryogenic electron microscopy (CryoEM) revealed nanosized interactions between FP20 and QS21v, forming stable structures that likely enhanced the antigen presentation and immune activation. These physicochemical properties contributed to a robust in vivo synergy, where the coformulation elicited significantly higher antigen-specific antibody titers compared to individual adjuvants. These findings suggest that the FP20+QS21v coformulation provides a potent, stable, and safer alternative to traditional adjuvants, enhancing both vaccine efficacy and immunogenicity.

The overexpression of aberrantly glycosylated tumor-associated mucin-1 (TA-MUC1) in human cancers makes it a major target for the development of anticancer vaccines derived from synthetic MUC1-(glyco)peptide antigens. However, glycopeptide-based subunit vaccines are weakly immunogenic, requiring adjuvants and/or additional immunopotentiating approaches to generate optimal immune responses. Among these strategies, unimolecular self-adjuvanting vaccine constructs that do not need coadministration of adjuvants or conjugation to carrier proteins emerge as a promising but still underexploited approach. Herein, we report the design, synthesis, immune-evaluation in mice, and NMR studies of new, self-adjuvanting and self-assembling vaccines based on our QS-21-derived minimal adjuvant platform covalently linked to TA-MUC1-(glyco)peptide antigens and a peptide helper T-cell epitope. We have developed a modular, chemoselective strategy that harnesses two distal attachment points on the saponin adjuvant to conjugate the respective components in unprotected form and high yields via orthogonal ligations. In mice, only tri-component candidates but not unconjugated or di-component combinations induced significant TA-MUC1-specific IgG antibodies able to recognize the TA-MUC1 on cancer cells. NMR studies revealed the formation of self-assembled aggregates, in which the more hydrophilic TA-MUC1 moiety gets exposed to the solvent, favoring B-cell recognition. While dilution of the di-component saponin–(Tn)MUC1 constructs resulted in partial aggregate disruption, this was not observed for the more stably-organized tri-component candidates. This higher structural stability in solution correlates with their increased immunogenicity and suggests a longer half-life of the construct in physiological media, which together with the enhanced antigen multivalent presentation enabled by the particulate self-assembly, points to this self-adjuvanting tri-component vaccine as a promising synthetic candidate for further development.

The O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation of proteins is an essential and dynamic post-translational modification in mammalian cells that is regulated by the action of two enzymes. O-GlcNAc transferase (OGT) incorporates this monosaccharide on serine/threonine residues, whereas O-GlcNAcase (OGA) removes it. This modification is found on thousands of intracellular proteins involved in vital cellular processes, both under physiological and pathological conditions. Aberrant expression of O-GlcNAc has been implicated in diseases such as Alzheimer, diabetes, and cancer, and growing evidence over the last decade has also revealed key implications of O-GlcNAcylation in immunity. While some key signaling pathways involving O-GlcNAcylation in immune cells have been discovered, a complete mechanistic understanding of how O-GlcNAcylated proteins function in the immune system remains elusive, partly because of the difficulties in mapping and quantifying O-GlcNAc sites. In this minireview, we discuss recent progress on chemical biology tools and approaches to investigate the role of O-GlcNAcylation in immune cells, with the intention of encouraging further research and developments in chemical glycoimmunology that can advance our understanding of O-GlcNAc in immunity.

Vaccine adjuvants are key for optimal vaccine efficacy, increasing the immunogenicity of the antigen and potentiating the immune response. Saponin adjuvants such as the carbohydrate-based QS-21 natural product are among the most promising candidates in vaccine formulations, but suffer from inherent drawbacks that have hampered their use and approval as stand-alone adjuvants. Despite the recent development of synthetic derivatives with improved properties, their full potential has not yet been reached, allowing the prospect of discovering further optimized saponin variants with higher potency. Herein, we have designed, chemically synthesized, and immunologically evaluated novel oxime-derivatized saponin adjuvants with targeted structural modifications at key triterpene functionalities. The resulting analogues have revealed important findings into saponin structure-activity relationships, including adjuvant mechanistic insights, and have shown superior adjuvant activity in terms of significantly increased antibody response augmentation compared to our previous saponin leads. These newly identified saponin oximes emerge as highly promising synthetic adjuvants for further preclinical development towards potential next generation immunotherapeutics for future vaccine applications.

Versatile chemical strategy for the development of novel conjugates based on streamlined saponin adjuvants covalently linked to a synthetic antigen (Tn) that induced moderate antibody responses in mice.

This review describes the recent developments in chemical probing of O-GlcNAcylation with a special focus on its molecular, structural and mechanistic implications.