Elucidating the Mechanisms of Action of Saponin-Derived Adjuvants

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Review

Elucidating the Mechanisms of Action of Saponin-Derived Adjuvants Dante J. Marciani1,* Numerous triterpenoid saponins are adjuvants that modify the activities of T cells and antigen-presenting cells, like dendritic cells (DCs). Saponins can induce either proinflammatory Th1/Th2 or sole anti-inflammatory Th2 immunities. Structure–activity relationships (SARs) have shown that imine-forming carbonyl groups are needed for T cell activation leading to induction of Th1/ Th2 immunities. While saponins having different triterpenoid aglycons and oligosaccharide chains can activate DCs to induce Th1/Th2 immunoresponses, fucopyranosyl residues from their oligosaccharides by binding to the DC-SIGN receptor can bias DCs toward a sole Th2 immunity. Here we discuss the mechanisms of action of these saponins in view of new information, which may serve as a basis to design improved adjuvants and related drugs.

Highlights Stimulation of proinflammatory Th1 and anti-inflammatory Th2 immunities should be considered different pharmacological effects. Triterpene saponin adjuvants like QS21 may act independently or in concert on T cells and dendritic cells, via receptor-mediated and non-receptormediated mechanisms. The aldehyde pharmacophore can induce beneficial or damaging effects, depending on the compound and type of immune cell.

Immunity and Vaccine Adjuvants During evolution, metazoan organisms have developed ways to protect themselves against toxins, infections by pathogens, and the presence of aberrant cells, such as cancer. This collection of protective approaches is known as ‘immunity’. Because of the myriad of agents that the body needs to be protected against, the immune system and its protective response have become intricate and complex [1] (Box 1). Nonetheless, use of immunity to avert infectious diseases has been one of the most successful strategies in the history of medicine.

Saponin adjuvants, depending on their oligosaccharides, may induce Th1 or Th2 immunities, thus mechanisms of action (MOAs) must be established only after the type of immune response has been certainly identified. It is very unlikely that a global MOA would be applicable to the large variety of saponin adjuvants and newly created analogs.

An effective way to prevent disease is to stimulate a protective immunity by pre-exposing the immune system to a pathogen or some of its fractions (i.e., vaccination). Vaccines can be live or inactivated; live vaccines are usually attenuated pathogens unable to cause disease, while inactivated vaccines contain either killed pathogens or some of their components or antigens (see Glossary) responsible for infection and/or their pathological effects, the latter type being called subunit vaccines. Inactivated vaccines while safe are poorly immunogenic, as besides an antigen(s) they need an adjuvant to boost the immunoresponse, a critical requisite in the very young and elderly [2]. In inactivated vaccines, while the antigen serves to target the immunoresponse, it is the adjuvant which defines its nature, that is, proinflammatory Th1 or anti-inflammatory Th2, and the potency of such response, regardless of the antigen’s nature [2]. Because inflammatory immunity appeared millions of years before anti-inflammatory immunity, a wide variety of adjuvants inducing Th1 immunity are derived from pathogens such as bacteria and viruses. Indeed, these adjuvants are exogeneous innate immunity ligands (e.g., lipid A and flagellin), which are recognized by TLRs [3]. Moreover, these ligands after activating innate immunity play a role in inducing adaptive immunity [1]. Although Th1 immunity effectively destroys cancer or virally infected cells, it may also cause tissue damage, thus it is always followed by the repairing Th2 immunity; but, because this immunity was developed much later during evolution, adjuvants that induce sole Th2 immunity

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The contributions of each saponin adjuvant’s component should be considered when establishing MOAs and/ or designing new immune modulatory saponin analogs.

1

Qantu Therapeutics, Inc., 612 East Main Street, Lewisville, TX 75057, USA

*Correspondence: [email protected] (D.J. Marciani).

https://doi.org/10.1016/j.tips.2018.03.005 © 2018 Elsevier Ltd. All rights reserved.

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Box 1. Innate and Acquired Immunities There are two kinds of immunoresponses: innate and adaptive or acquired immunity. Innate immunity is a nonspecific rapid protective inflammatory response induced by well-conserved motifs found only in pathogens, which are recognized by cellular receptors like TLRs [49]. Upon recognition of these motifs, several genes encoding cytokines, adhesion molecules, and other factors that promote the activation of leukocytes needed for removal of pathogens are induced. Adaptive immunity occurs only after exposure to a foreign antigen (e.g., pathogen); while it takes longer than innate immunity for a response to be established, it is more specific. However, adaptive immunity requires information from innate immunity to be activated. There are two main types of adaptive immunoresponses: Th1 or cell-mediated immunity (controlled by activated T cells) and Th2 or humoral immunity (controlled by activated B cells and antibodies) [50]. Whereas Th1 is always followed by Th2 immunity, the latter may exist as a sole immunity type. Both T and B cells carry on their surfaces binding sites specific for an infective pathogen, which can be either killed directly by a Th1 proinflammatory response that involves cytotoxic T lymphocytes (CTLs), activated phagocytes, and certain cytokines, or alternatively, they are neutralized by antibodies against the pathogens that are produced by the Th2 humoral immunoresponse, eventually stopping the spread of infection. CTLs result from the activation of naïve CD8+ T cells by DCs. Adaptive immunity has an immunological memory, which gives the host a long-term protection against reinfection by the same pathogen by allowing a rapid response against it. A new type of immunity is that mediated by Th17 cells, which produce the strongly proinflammatory cytokine IL-17. Th17 cells have a role in mucosal immunity against extracellular pathogens like fungi, but they also seem to have some detrimental effects. Indeed, abnormal upregulation of Th17 cells plays a major aggravating role in inflammation and autoimmune diseases, like rheumatoid arthritis and multiple sclerosis. The role of Th17 immunity is still being elucidated and there is limited information about its mechanism of action or linking the available adjuvants to it [51].

are rare. The best known, alum, while safe is frequently ineffective in the elderly [2]. Actually, the most effective Th2 adjuvants are saccharides from parasitic helminths, characterized by the presence of fucopyranosyl residues that upon binding to DC-SIGN on DCs bias the response to Th2 immunity [4,5]. These fucosylated saccharides mimic those made during pregnancy, such as LewisX antigen, to avert fetus rejection, a strategy also used by certain cancer cells to prevent inflammatory responses that would kill them [6]. Some phosphorylcholine (PC) compounds favor Th2 and Th17 over Th1 immunity; however, certain PC derivatives can induce a strong Th17 inflammatory immunity, which may cause or worsen autoimmunity (Box 1) [6]. While there are several immunostimulatory saponins, the proinflammatory Th1 adjuvant QS-21 (Box 2), a purified fraction of two isomeric triterpene glycosides from the tree Quillaja saponaria – having arabinose (A) and xylose (X), that is, QS-21A and QS-21X, in a ratio of 2:1, is perhaps the best characterized adjuvant that is not an innate immunity ligand [7]. Since its discovery, numerous studies have tried to elucidate its mechanism of action (MOA) and prepare synthetic derivatives. However, possible deficient information about these adjuvants’ immunological activities has yielded conflicting conclusions [8]. Paradoxically, a new sole antiinflammatory Th2 adjuvant, QT-0101 (Box 2) [9], is composed of two derivatives prepared by chemically modifying some native triterpene glycosides from Quillaja saponins, like QS-18 and QS-21. These saponin-derived adjuvants interact in different ways with innate immunity receptors. Indeed, while the proinflammatory QS-18 and QS-21 do not interact directly with innate immunity receptors like TLRs, anti-inflammatory QT-0101 apparently interacts via its fucopyranosyl residue with DC-SIGN, an innate immunity pattern recognition receptor present on DCs [4,5], to induce Th2 immunity and secretion of Th2 cytokines by these cells. In this article, we review the available information concerning these compounds and consider some rational MOAs, which may serve as a basis to design improved adjuvants and related drugs – compounds that would have applications in the prevention and/or treatment of conditions like cancer and infectious and autoimmune diseases.

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Glossary Acyl group: derived by removing a hydroxyl group from a carboxylic acid. It has the formula R–C(O)–, where R is an alkyl group. Adjuvant: a compound(s) that when coadministered with an antigen elicits a higher proinflammatory (Th1), antiinflammatory (Th2), or Th1/Th2 immune response. Amphiphilic: an organic molecule that has covalently linked hydrophilic and lipophilic hydrophobic polar sectors. Antigen: any compound capable of evoking an immune response; can be self-antigens, that is, components of the body, or nonself-antigens, that is, foreign agents like bacteria. CD2: a transmembrane glycoprotein receptor found in various cells (e.g., T cells) that plays a role in T cell activation, by facilitating costimulation. Costimulation: delivery of a signal to naïve T cells, which together with a signal provided by interactions between the T cell receptor and peptide-MHC molecules found on APCs, induces the activation of T cells. Critical micelle concentration (CMC): the concentration of an amphiphilic surfactant above which micelles start to form. CTLA-4 (cytotoxicT lymphocyte associated protein 4): a protein receptor functioning as an immune checkpoint, which by binding CD80 or CD86 ligands downregulates immunoresponses. Cytokines: small proteins that regulate the immunological activity of cells carrying cell-surface receptors for specific cytokine(s). They induce proinflammatory or anti-inflammatory immunoresponses. Cytotoxic T lymphocytes (CTLs): these are activated CD8+ T cells that recognize antigens presented as complexes with MHCI molecules, affix to those cells, and destroy them. DC-SIGN (dendritic cell-specific intercellular adhesion molecule3-grabbing non-integrin): a C-type lectin receptor present on the surface of macrophages and DCs, which binds either mannose or fucose, to induce Th1 or Th2 immunity, respectively.

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Saponin Adjuvants and Their Structure Triterpene saponin adjuvants, such as QS-21, are amphiphilic glycosides with a lipophilic triterpene nucleus that usually carries one or two hydrophilic saccharide chains linked by ester bonds and/or O-heterosidic bonds, that is, monodesmosidic or bidesmosidic glycosides, respectively [10]. In QS-21, these oligosaccharide chains are linked at positions C-3 and C-28 of the triterpene aglycone, with the oligosaccharide at C-28 having an acylated fucopyranosyl residue and the acyl group’s structure depending on the selected glycoside. While QS21’s fucopyranosyl residue is covalently bound by an ester between its 1-hydroxyl and the carboxyl group at C-28 of the triterpene aglycone, and to a rhamnosyl residue at its 2-hydroxyl group, its 4-hydroxyl group is acylated by two 3,5-dihydroxy-6-methyl octanoic acids linked in tandem [10] (Box 2). Saponin adjuvants that induce Th1 immunity, such as QS-21, usually have an imine-forming carbonyl group, that is, an aldehyde linked to the triterpene C-4 [7,10]. However, in other saponins, such an imine-forming carbonyl group may be present at different sites, such as the acyl chains, and may be substituted by ketone or other groups [11]. In addition, depending on the saponin, the triterpene may have hydroxyl groups. Several analogs of QS-21 have been made. One is GPI-0100, a semisynthetic QS-21 analog where the original acyl group at the fucopyranosyl residue has been replaced by a dodecylamide linked to the glucuronic acid (at C-6) of the saccharide at the triterpene C-3 [12]. The complete chemical synthesis of GPI-0100 has been recently reported, confirming that it induces, albeit at doses five to ten times higher than QS-21, Th1 immunity [12,13]; however, substitution of the new alkyl chain by groups like cyclohexyl abrogates GPI-0100’s Th1 adjuvanticity. The synthesis of QS-21 and new saponin derivatives (e.g., monodesmosidic, differing lipophilic chains, with and without aldehyde groups; Box 2) that have been tested for adjuvanticity [14–16] ought in principle helps to elucidate the MOA for these saponins. In addition to the Quillaja saponin adjuvants, there are triterpene saponins derived from other plants [10] as well as new synthetic saponins that have immunostimulatory activity [14–16].

Box 2. Triterpene Saponins: Structures and Adjuvanticity The Quillaja saponin QS-21A, which has a triterpene aglycone with a C-4-aldehyde group (shown in black) and a C-28bound oligosaccharide with a fucosyl pyranose acylated at its 4-hydroxyl group, with the acyl chain having a terminal arabinose (Figure IA) [7,10], elicits Th1/Th2 immunity with CTL production. However, deacylation of its fucosyl pyranose produces QT-0101 (Figure IB) [9], which induces sole Th2 immunity. Various QS-21 analogs have been synthesized, where the fucosyl pyranose is substituted by a galactosyl pyranose residue, shown in violet [8,14,15]. In one analog (Figure IC) [14], substitution of the acyl by an alkyl chain with a terminal amino group abrogates adjuvanticity. Evidently, formation of intramolecular and/or intermolecular imine groups prevents the triterpene aldehyde from reacting with cell surface amino groups. Removal of the C3-oligosaccharide from IC, plus addition of an aryl group to the alkyl chain terminal amino group (shown in brown), yields the compound in Figure ID [8] that induces antibody production. Although removal of the aldehyde group does not eliminate adjuvanticity, it remains uncertain if the induced immunity is Th1, Th2, or if there is CTL production; yet, one might conclude that the C-4-aldehyde is irrelevant, while the C-16-hydroxyl group is important for adjuvanticity [8,15]. A further derivative having both the C-4-aldehyde and an additional aldehyde tucaresol (shown in brown; Figure IE) [28] did not show increased adjuvanticity, indicating no additive effects. It would have been useful to assess the tucaresol’s effects on the induction of Th1 immunity by the compound in Figure ID, but, without the C-4-aldehyde; of [354_TD$IF]significance, in view of the opinion that aldehydes are irrelevant for adjuvanticity [8,15], [35_TD$IF]despite of evidence to the contrary [20–23,39,40,52]. In addition, saponins’ adjuvanticity is altered by their lipophilic properties, as shown by the correlation of their adjuvanticity with the hydrophile/lipophile balance (HLB) [19,53]. Thus, new saponin analogs lacking the C3-oligosaccharide but having new alkyl and aryl groups [8,15], which alter their HLB and capacity to disrupt cellular membranes and antigen cross-presentation, should elicit different immunoresponses. That saponins missing both the C-4-aldehyde and C-16-hydroxyl, like the Chiococca alba saponin that has a 3b-hydroxyolean-12,15-diene-28-oic acid aglycone (Figure IF) having adjuvanticity [19], supports an MOA for QS-21 where it acts separately on T cells and DCs. Depending on the saponin’s structure, adjuvanticity may vary in both type and magnitude, thus, emphasizing the requirement for well-defined immunity types to establish accurate MOAs.

Dendritic cells (DCs): antigenpresenting cells that process proteins for presentation to T cells. Endocytosis: the process of actively transporting a substance into a cell by engulfing it with a membrane. Endosome: membrane-bound vesicles formed by endocytosis that serve as intracellular transport carriers. Imine: or ‘Schiff base’ is a product of a reaction between a primary amine and an aldehyde or ketone; it has a carbon–nitrogen double bond (R–HC¼N–R). Immune stimulating complexes (ISCOMs): open cagelike structures composed of cholesterol, phospholipids, and Quillaja saponins, which are immune stimulatory. Lysosome: membrane-bound organelles that contain acidic hydrolytic enzymes that digest macromolecules. Th1: immunoresponse promoted by Th1 helper cells that secrete proinflammatory cytokines, like IFN-g and IL-2, and are characterized by the activation of macrophages and CTL production (i.e., cell-mediated immunity). Th2: a humoral antibody immunoresponse promoted by Th2 helper cells that secrete antiinflammatory cytokines like IL-4 and IL-10. It follows Th1 immunity, but it may also exist alone. Toll-like receptors (TLRs): membrane spanning receptors present on macrophages and DCs that recognize and bind conserved molecules from pathogens, initiating the activation of the innate immune system. Triterpene: a pentacyclic lipophilic structure built by fusion of six isoprene units. In saponins, it may have an aldehyde, ketones, carboxylic, and other groups.

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(A) QS-21A

(B) QT-0101

(C)

(D)

(E)

(F) Chiococca alba saponin

Figure I. Structures of QS-21A and Other Triterpene Saponins. Panels A and B correspond to QS-21A and its deacylated derivative QT-0101, respectively. Panels C–E show the structures of different synthetic triterpene glycosides described in the text. Panel F shows the structure of a saponin adjuvant from C. alba. The triterpene aglycone is shown in red, C-3 oligosaccharide in purple, C-28-bound fucosyl pyranose in blue with the rest of that oligosaccharide in green, and alkyl chains and aromatic rings in brown. The galactosyl pyranose is shown in violet.

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Some, similar to QS-21, are bidesmosidic, have an aldehyde at their triterpene core and an acylated fucopyranosyl residue, whereas others are monodesmosidic, nonacylated, lack aldehyde groups, and may have various hydroxyl groups at their triterpene (Box 2). However, because their structures contain both hydrophilic and hydrophobic groups, these saponins are amphiphilic, which explains their capacity to disrupt the lipid bilayers of cell membranes causing cell lysis and in some cases their adjuvanticity, as will be discussed in this review.

Proposed Mechanisms of Action for QS-21 Since QS-21 has been tested in over 120 clinical trials, approved by the European Medicines Agency (EMA) for use as an AS01 combination in a vaccine against malaria [17], and is the best characterized saponin both structurally and functionally, it is an obvious choice as a prototype. That some of the proposed MOA for QS-21 disagree [7,8] points to basic issues like incomplete structural and/or functional information. For instance, despite major differences between the proinflammatory Th1, anti-inflammatory Th2, and inflammatory Th17 immunities, often the type of immunity elicited by QS-21 analogs is undefined [8,13]. Therefore, it is likely that ambiguities concerning the induced immunities (i.e., the pharmacological effect) have hindered the elucidation of the MOA for these adjuvants. This situation is complicated by the fact that QS-21 and closely related saponins may exert their immunomodulatory effects by acting on T cells, both CD4+ and CD8+ lymphocytes, as well as on antigen-presenting cells (APCs) like DCs (reviewed in [10]), via receptor and non-receptor-mediated mechanisms, respectively [18]. Hence, proposing an MOA would require unequivocal identification of the elicited immunity, Th1 or Th2, by determining the cytokine profile, cytotoxic T lymphocyte (CTL) production, and other immunity parameters (Box 3), a requirement seldom fulfilled due to the complexity of those studies. Indeed, the type of immunity is often decided solely by antibody (IgG) production, which while easier does not consider that expression of the various IgG isotypes is affected differently by the increase in adjuvant dose [12]. Indeed, while the presence of antigen-specific antibodies indicates stimulation of an immunoresponse, it does not distinguish between Th1 and Th2 immunities, or determine CTL production, which is important in devising MOAs for adjuvants. Since Th1 is characterized by responses mediated by immune cells, and Th2 by antigen-specific antibodies, a thorough determination of those responses and the type of the cytokines secreted [7,12,19] would reveal the nature of the induced immunity. Therefore, information obtained using a single or narrow dose range of adjuvant, without analyzing the dose effects on the ratio of the relevant IgG isotypes and confirming it by cytokine profiles, raises doubts about the reported immunities. However, reliable structure–activity relationships (SARs) for some active moieties of QS-21 have been established and will be compared with other proposed MOAs [7,18,20]. Role of the Triterpene Aldehyde Group The QS-21 aldehyde’s role in inducing Th1 immunity was demonstrated by the loss of adjuvanticity after its reduction to an alcohol, results confirmed by the abrogation of adjuvanticity after converting that aldehyde to a secondary amine [7]. In addition, of the several synthetic analogs of QS-21, the one lacking adjuvanticity had a free amino-terminal group at its acyl chain [14]. Indeed, the formation of imines between the triterpene’s aldehyde and the new amino group would prevent the aldehyde from interacting with the cellular e-amino groups needed to show adjuvanticity. Actually, masking that new amino group by acylation with fluorescein restored adjuvanticity [14]. Since the loss of adjuvanticity also occurred after modifying the aldehyde with glycine, which yielded an anionic carboxyl group, such loss is unrelated to charge. The aldehydes’ role in immune stimulation has been described previously [21–23]. It has been shown that formation of imine groups or Schiff bases by reacting aldehydes or ketones with amino groups, likely from the CD2 receptor on T cells, delivers a costimulatory Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

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Box 3. Differentiating between Th1 and Th2 Immunities Th1 or Th2 immunity? In mice, the expression of different IgG isotypes is affected by the cytokines secreted by Th1 or Th2 immunities. Indeed, production of IgG2a is boosted by the Th1 cytokine IFN-g, while the production of IgG1 is enhanced by Th2 cytokines. Methods to Determine Th1 Immunity Antigen-specific lymphoproliferative response: This measures the capacity of T cells to undergo clonal proliferation when exposed in vitro to an antigen that the immune system has mounted a previous immune response against. Proliferation is determined by the incorporation of H3[35_TD$IF]-thymidine into DNA [10,12]. Cytokine profile: Identification of the cytokines secreted by lymphocytes exposed in vitro to a specific antigen can be made using ELISA or by enzyme-linked immunospot (ELISPOT) assay. Production of cytokines like IL-2 and IFN-g indicates Th1 immunity, while other cytokines would indicate Th2 immunity as described below. CTL production: Antigen cross-presentation to CD8+ T cells, which results in their activation to become CTLs, is another Th1 immunity indicator. Production of CTLs can be determined using target cells carrying a specific surface antigen and that are loaded with chromium-51 radioactive isotope, which upon being recognized and lysed by CTLs would release the radioactive isotope. Alternatively, the presence of activated CD8+[356_TD$IF] cells may be determined by the peptide/MHCI-tetramer staining technique that uses fluorescently labeled tetrameric MHC with the peptide of interest, which binds to T cells specific for both the MHC and peptide and is detected by flow cytometry. Methods to Determine Th2 Immunity Cytokine profile: An effective method to establish the presence of Th2 immunity is by identifying Th2 cytokines, such as IL-4 and IL-10 using ELISA or ELISPOT. However, the increase in Th2 cytokines must be concomitant with a decrease in Th1 cytokines. Antibody ratios: A tentative determination of the induced type of immunity, Th1 or Th2, may be made using the changes in the ratio of IgG1/IgG2a isotypes, which is affected by [357_TD$IF]the type of cytokines, that is, Th1 or Th2. Since IgG2a production is boosted by increasing doses of Th1 adjuvants, like QS-21, while that of IgG1 falls or remains constant, a decrease in the IgG1/IgG2a ratio as a function of adjuvant dose indicates Th1 immunity. Alternatively, as increasing doses of Th2 adjuvant would augment IgG1 production, while decreasing that of IgG2a, an increasing IgG1/IgG2a ratio would imply Th2 immunity. However, these conclusions need to be confirmed by further tests.

signal that replaces the one derived from interactions between CD80/86 ligands and CD28 receptor on DC and T cells, respectively, proteins that are lost with aging [21] (Figure 1, Key Figure). Thus, delivery of an alternative costimulatory signal is critical for T cell activation and to prevent its anergy. While triterpene saponins having aldehydes, like QS-21, that induce Th1 immunity with secretion of Th1 cytokines and have a strong primary immune response [20,24], some Th1 triterpene saponin adjuvants lacking aldehydes have other types of imine-forming carbonyl groups, like oxo groups, in their structure [11]. In fact, some compounds having different imine-forming carbonyl groups are Th1 immune stimulators, which are effective [25]. Hence, a conclusion would be that these carbonyls’ roles in immunity are complicated, depending on the compound, type of immune cell, and other factors (Box 4). Consequently, MOAs should be considered only after a distinct characterization of the induced immunity by the different adjuvants. Like some immunological processes, costimulation during T cell activation is apparently a single-hit mechanism, where one or a very low number of signals elicit costimulation [26,27]. Hence, increasing the number of aldehyde groups acting on a single T cell should not boost activation, which would explain why synthetic QS-21 analogs having more than one aldehyde group (Box 2) induce an immunoresponse similar to that of QS-21 variants having a single triterpene aldehyde [28]. That imines are rather stable in an aqueous environment is shown by

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Key Figure

Proposed Mechanism of Action for QS-21 and Related SaponinDerived Adjuvants

DendriƟc cell

T cell CH = N

QS-21

Ag

CD2

K+ Na+

Early endosome

TCR

CHO

C D2

MHCI

H2N

MAPK ERK2

CD80

Proteosome

CD28

Late endosome

ER

QS-21

Th1 cytokines

Figure 1. Quillaja saponin adjuvants, like QS-21, act on both T cells and dendritic cells (DCs). T cell: The aldehyde group on QS-21[351_TD$IF]forms an imine with an e-amino group from a T cell surface receptor, most likely CD2, delivering a costimulatory signal to the T cell. This signal substitutes the one derived from interactions between the T cell’s CD28 receptor and the DC’s CD80 (B7-1 ligand). This signal converges with T cell receptor (TCR)-mediated signaling at the level of tyrosyl phosphorylation of the mitogen-activated protein (MAP) kinase (ERK2), which together with changes in the cells K+ and Na+ transport stimulates T cell activation biased to Th1 immunity with the resultant secretion of Th1 cytokines. DC: Quillaja saponins act on DCs, but, in a non-receptor-mediated manner. Exogenous protein antigens (Ag) and QS-21 enter DCs by endocytosis, where QS-21 disrupts the endosomal membrane, allowing early escape of the antigens for further processing inside the cell into peptides. Properly degraded antigens are loaded into MHCI by the vacuolar pathway, while antigens that need more processing are transported to the cytosol to be cleaved by the proteasome (cytosolic pathway). These cleaved proteins (i.e., peptides) are transported to the endoplasmic reticulum (ER), which after additional processing are loaded into MHCI. Peptides derived from either the vacuolar or cytosolic pathways after binding to MHCI are presented on the DC surface to naïve CD8+ T cells in a process called cross-presentation to yield cytotoxic T lymphocytes (CTLs). Adapted from Marciani [18] and Rhodes [23].

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Box 4. Role of Imine-Forming Carbonyl Groups in Immunity Reactive carbonyl groups, largely aldehydes and ketones, have multiple effects on immunity, which depend on both the compound and the type of affected immune cell. Compounds like tucaresol, dioxoheptanoic acid, and others enhance Th1 inflammatory immunity by activating T cells and inducing their production of Th1 cytokines [21,25], an effect apparently shared by saponins with aldehyde or ketone groups [10,11,24]. These compounds by forming imines or Schiff bases with e-amino groups from receptors such as CD2 on T cells deliver a costimulatory signal, prompting T cell activation [18,21,22]. That the proliferation of T cells induced by neuraminidase-galactose oxidase-treated autologous erythrocytes was abolished by antibodies against the CD2’s ligand LFA-3 suggests that such a response was initiated by interactions between CD2 and maybe other T cell surface receptors that bind to antigens carrying aldehydes or ketones forming imines [52]. However, aldehydes can also affect DCs if present in oxidized mannan, by binding to their mannose receptor. Indeed, conjugates of oxidized mannan–MUC1 antigen are internalized in DC without imine formation, initiating a rapid and efficient entry into the class I pathway [54,55]. Paradoxically, the administration of MUC1 with tucaresol, which also acts at the T cell level, caused downmodulation of MUC1-stimulated human mononuclear cells, abolishing their protective effects [56]. This situation shows the influence of various factors on the aldehyde effects on immunity, which suggests different MOAs for the same pharmacophore. Moreover, new findings have shown that oxidative stress leads to the formation of toxic lipid peroxidation-derived aldehydes, like 4hydroxy-trans-2-nonenal, malondialdehyde (MDA), and others, which interfere with the regulation of redox-sensitive transcription factors like NF-kB, leading to inflammation [57]. In fact, it has been shown that MDA induces significant changes in inflammatory cytokines and other molecules, directing to lymphocyte activation via the inflammatory pathway [58]. Hence, it can be concluded that aldehydes and ketones can have beneficial and damaging effects on immunity, which depend on both the compound and immune cells, thus emphasizing the need for accurate determination of the induced immunity type, to establish working MOAs.

the numerous biochemical reactions involving this group (e.g., pyridoxal, retinal, and the various drugs having this pharmacophore) [29]. Hence, it is evident that in QS-21 and other Th1 saponin-derived adjuvants, imine-forming carbonyl groups are essential for adjuvanticity; a viewpoint that diverges from other proposed MOAs [8,15] that will be addressed in this review. Role of the Triterpene Nucleus and Acyl Chain Some bidesmosidic triterpene saponin adjuvants, like QS-21, with one of their saccharide chains acylated upon deacylation, such as in QT-0101 (Box 2), lose their capacity to stimulate Th1 immunity [30]. Hence, it has been postulated that the lipophilic acyl chain facilitates the delivery of exogenous antigens into the APC for processing by the endogenous pathway and presentation to T cells, inducing immunity in a non-receptor-mediated manner (Figure 1) [10]. That QS-21 is endocytosed in a cholesterol-dependent mode, collecting in the endosomes– lysosomes and destabilizing them, leading to DC activation and antigen cross-presentation, is due to the triterpene’s high affinity for cholesterol and its ability to intercalate into the cholesterol-rich regions of cell membranes [31]. Indeed, it has been shown that QS-21 facilitates the endosomal escape of antigens, essential for cross-presentation [32]. In fact, saponins like QS-21 have also been shown to increase the translocation of toxins from late endosomes and lysosomes into the cytosol, augmenting their cytotoxicity [33]. Additional support for the role of saponins’ non-receptor-mediated activation is derived from the fact that the small aldehyde tucaresol, while inducing in vivo Th1 immunity and increasing pre-existing CTL production, cannot induce de novo production of CTL by itself [21]. Moreover, that some nonacylated triterpene saponins lacking imine-forming carbonyl groups have immune stimulatory properties, although lower than QS-21 (reviewed in [10]), is evidently a result of the endocytosis of saponin–antigen complexes and subsequent DC cross-presentation. Puzzlingly, while some nonacylated triterpene saponins induce Th1 immunity, upon deacylation, QS-21 and similar saponins lose their capacity to induce Th1, eliciting instead a sole Th2 immunity [9]. QS-21 Saccharide Moiety and Immune Modulation The paradox of deacylated QS-21 or QT-0101 (Box 2) may be explained by the fucopyranosyl residue present in these saponins’ C-28 oligosaccharide, which by binding to the DC-SIGN

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receptor on DC biases its response toward Th2 immunity, inducing secretion of Th2 cytokines [34], a receptor-mediated immune modulation that can be sterically hindered by acylating the fucose with large fatty acids or similar groups, as in QS-21. Although binding to DC-SIGN usually occurs with terminal fucopyranosyl residues, it is possible to conclude from X-ray and other studies that the crucial 3- and 4-hydroxyl groups of the fucopyranosyl residue are available in QT-0101 to interact with the Ca2+[358_TD$IF] ion within the DC-SIGN binding pocket, which is required for binding to this lectin [35]. Since the absence of fucopyranosyl residues apparently correlates with a saponin’s capacity to induce Th1 immunity, it is likely that removal or substitution of QT-0101’s fucopyranosyl residue by other sugars could abrogate its capacity to induce Th2 immunity and as a consequence may lead to the development of new effective Th1 adjuvants. Effects of Saponins’ Associative Properties and Conformation on Adjuvanticity Evidently, the amphiphilicity of QS-21 and related saponins plays a role in their non-receptormediated Th1 immunomodulating activity at the DC level, by allowing interactions with the DC membrane and subsequent cell internalization [31,32]. While these glycosides are capable of forming micelles, the effective adjuvant dose is below their critical micelle concentration (CMC), thus, QS-21 must be active as a monomer. Furthermore, the addition of nonionic surfactants to QS-21 drastically augments its adjuvanticity [36] and implies aggregation, where breaking down the putative aggregates into mixed micelles of QS-21/surfactant would raise the number of costimulatory centers that can activate T cells and hence adjuvanticity. Conceivably, triterpene saponins are sequestered at the administration site by lipids and/or lipid carriers, yielding complexes with high saponin density, a situation averted by the prior formation of mixed micelles. Indeed, a similar situation occurs with GPI-0100, where the effective adjuvant dose is above the CMC; yet, formation of mixed micelles with surfactants results in a sizeable adjuvanticity increase (D.J. Marciani, unpublished observations). These results would explain why adding more aldehyde groups to saponin molecules does not increase adjuvanticity, presumably the result of a single-hit costimulation as indicated earlier. However, mixed micelles in addition to T cells may also increase the number of APCs like DCs, activated by these saponins. Of interest are the studies about the role of these glycosides’ conformation in adjuvanticity, using variants with glycosidic linkage modifications. A major difference between these synthetic QS-21 variants and natural saponin(s) is that the C-28-b-fucopyranosyl residue linked to position C-28 of the aglycone in natural saponins (Box 2) has been replaced in the variants by a b-d-galactopyranosyl residue (Box 2) [15,37]. Whereas deacylated fucopyranosyl seems to bind DC-SIGN, biasing DCs to Th2 immunity like in QT-0101, such a change should not occur with galactopyranosyl residues. Still, an issue would be how the endocytosis by DCs of these saponin variants will be affected by the absence of the lipophilic side chain. Hence, these new saponin analogs offer the opportunity to test the roles of fucose and the acyl chain in the induction of Th2 and Th1 immunities, respectively.

Synthetic Q Saponin Analogs While synthetic analogs based on QS-21 present an opportunity to explain this and related saponin adjuvants’ MOA, this task is hindered by the undefined information about the nature of the induced immunity, that is, Th1, Th2, or Th17 [14,15,37]. Clearly, total IgG while indicating adjuvanticity cannot distinguish Th1 from Th2 immunity; also, use of IgG2a/IgG1 ratios determined at a single adjuvant dose or over a narrow dose range to decide the immunity type often delivers inconclusive results, a task aggravated by the fact that some of the used antigens have maleimide cross-linkers, which often interfere with immunity [38]; therefore, the immunity type Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

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should be always established from the cytokine profile. Moreover, since induction of Th1 immunity in rare cases does not warrant CTL production (D.J. Marciani, unpublished observations), the presence of CTLs should be determined using established procedures (Box 3) in order to generate meaningful SARs between CD8+ activation and the saponins’ structures. A conclusion derived from the work with synthetic analogs is that while the C-16-hydroxyl group plays an important role in triterpene saponins’ adjuvanticity, the C-4-aldehyde does not, such as in echinocytic acid derivatives [8,15]. Examination of other saponin adjuvants shows that while they do not have the C-16-hydroxyl, all have the C-4-aldehyde group [10,24]. That substitution of the QS-21 aldehyde by another group abrogates its adjuvanticity, despite the presence of the C-16hydroxyl group [7,8], casts doubt about this hydroxyl’s relevance in adjuvanticity. Yet, that triterpene saponins like those from Chiococca alba (Box 2), which lack both imine-forming carbonyls and C-16-hydroxyl groups, show adjuvanticity [19] raises the possibility of different MOA for these glycosides. Indeed, because of QS-21 and other saponins’ amphiphilic properties and their triterpene’s high cholesterol affinity, they may exercise their adjuvanticity by nonreceptor-mediated processes, that is, disrupting the membranes from different DC compartments (e.g., cellular and endosomal) [31–33]. Hence, QS-21’s adjuvanticity is evidently a result of receptor and non-receptor-mediated activities, working in concert on both T cells and DCs, which may explain QS-21’s superior adjuvanticity when compared with other saponins that act only at the DC level. In fact, QS-21 induces a strong Th1/Th2 immune response with CTL production in a relatively short time, as compared with other adjuvants. Another important moiety in saponin adjuvants is their carbohydrate one, as shown by the fact that glycosylated triterpenes enhance the escape of toxins from the endosome, increasing their effective cytotoxic concentration in the cytosol of cancer cells [33], an event comparable to the release of exogenous antigens from the endosome into the DC cytosol for cross-presentation. Apparently, the most effective saponins in facilitating endosomal translocation are bidesmosidic having at C3 a branched oligosaccharide with a glucuronic acid [33]. Of relevance is that as indicated before, saponins with an acylated fucopyranosyl residue, like QS-21, upon deacylation change their adjuvanticity from Th1/Th2 to a sole Th2 [9,35]. Thus, it would be worthwhile to compare the adjuvanticity of analogs carrying b-galactopyranose with those carrying fucose on the immune response.

Proposed MOA An effective MOA for QS-21 and closely related saponins, like QS-18, should take into account that this adjuvant(s) apparently acts on both T cells and DCs, but in different ways (Figure 1). Indeed, by forming imines with e-amino groups from certain T cell surface receptors, like CD2, their aldehyde delivers a T cell costimulatory signal by a mechanism that results in changes in Na+[359_TD$IF]/K+ transport, which together with T cell receptor (TCR)-mediated signaling at the level of phosphorylation of the mitogen-activated protein kinase ERK2 plus increased Ca2+ mobilization, promote T cell activation and boost the production of Th1 cytokines by T cells [21–23,39,40] (Box 4). Support for CD2’s role as an alternative costimulatory receptor upon imines formation comes from the fact that binding to its natural DCs’ CD58 ligand enhances phosphorylation of the kinase ERK2 and induces the production of Th1 cytokines, but like with aldehyde `costimulation, T cell activation occurs only in the presence of TCR signals [41]. Indeed, CD2 signaling can effectively substitute the CD28–CD80/86 costimulatory signal. Hence, QS-21’s potent Th1 adjuvanticity may be explained by DC activation plus increased Th1 cytokine secretion, and that different from the CD28–CD80/86 T cell costimulatory signal, its imine-derived signal is not downregulated by CTLA-4, a checkpoint for T cell activation [42]. Indeed, Quillaja saponins induce a faster and stronger primary immunoresponse than other saponins [24], important for 10

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vaccines, but seldom considered in adjuvant development [8,15]. While QS-21’s costimulatory effects are well defined, those on the DCs are complex, because the triterpene and acyl chain interact in a non-receptor-mediated manner with the plasma membrane’s lipids to allow entry into the cell of exogenous antigens for cross-presentation by either the cytosolic or vacuolar pathways [32]. That the entry and intracellular processing of protein antigens may depend on the DC subtype and its activation status [43] would explain why some saponins induce CTL production under some conditions, but not others. While findings based on immune stimulating complexes (ISCOMs) [44,45] and liposomes [32,46] having QS-21 assumed that endocytosis of particulate antigens and their cross-presentation by the cytosolic pathway are requirements to activate CD8+[360_TD$IF] T cells, actually, free exogenous antigens with monomeric QS-21 also induce CTL production [18,47]. Indeed, triterpene glycosides lacking adjuvanticity, although capable of forming ISCOMs, cannot stimulate immunity [24,48]. Therefore, a coherent QS-21’s MOA for DCs would comprise cholesterol-dependent endocytosis of antigen/QS-21 with traffic to the lysosomes, followed by QS-21 lysosomal destabilization; which allows a rapid antigen translocation to the cytosol for proteasome-independent cross-presentation of antigen–MHCI complexes to naïve CD8+ T cells (Figure 1). The antigen would be processed by lysosomal proteases, which vary depending on the DC subtype [43], and the saponin responsible for the membrane disruption. Hence, it is unlikely that QS-21 or closely related acylated saponins, like QS-18, would activate DCs by interacting directly with specific cell receptors, a likely event with T cells [18]; however, by altering the structure of cellular membranes and their permeability properties, these glycosides induce events leading to DC activation. That would explain why not all saponins are adjuvants or have similar adjuvanticities, a reason for questioning MOAs based on antibody production only. Actually, addition of aryl groups and removal of oligosaccharide chains as described for some QS-21 synthetic analogs (Box 2) [8,13] would alter their amphiphilicity [31] and cross-presenting properties, reducing CTL production – immune cells that are important in cancer and antiviral vaccines, since they search and destroy cells that are malignant or virally infected carrying foreign antigens. Indeed, the only reliable way to establish CTL production will be by determining the lysis of target cells by either effector cells or peptide/MHCI-tetramer staining (Box 3).

Concluding Remarks The potent proinflammatory Th1 adjuvant QS-21 has stimulated interest in the development of improved analogs, an objective hindered by the lack of suitable MOAs. Because of the numerous saponin adjuvants, it is unlikely that a single MOA could explain their adjuvanticities. Using QS-21 as a model it would be feasible to propose an MOA with DCs either in conjunction with T cells or alone, the latter situation rationalizing the adjuvanticity of some glycosides lacking imine-forming carbonyl groups. In addition, important are both the acyl chain and fucopyranosyl residue of QS-21 to explain its capacity upon deacylation to elicit a sole Th2 immunity [9]. The modification of these groups results in new adjuvant activities and offers an opportunity to develop improved immunomodulators (see Outstanding Questions). Hence, in proposing MOAs for QS-21 and adjuvant saponin analogs, in addition to the development of new structures, all of these glycosides’ moieties should be considered. Thus, to establish correct SARs and allow the design of better immunomodulators, it is of the upmost importance to accurately characterize the type of immunity induced, since new preventive/therapeutic vaccines must induce specific immune responses, but without side effects. Disclaimer Statement D.J.M. is a Founder, President, and Chief Scientific Officer, of Qantu Therapeutics, Inc. and has filed patent applications covering the use of deacylated saponins as adjuvants.

Outstanding Questions How relevant would it be to clearly identify the [362_TD$IF]cellular receptor(s) for active carbonyl groups? Its identification would allow the design of small molecules that could form imines or Schiff bases selectively with this receptor’s amino groups. These new compounds, which may be used as potentiators of Th1 immunity, could have uses in vaccines against infectious diseases and cancer. What is the oligosaccharides’ role in saponin adjuvants? New reports suggest that the saponins’ oligosaccharides by binding to cell surface lectin receptors bias immunity toward Th1 or Th2. The presence of fucosyl residues would allow binding to the DCSIGN’s fucose-binding site, biasing DCs to an anti-inflammatory Th2 immunity, as with QT-0101. However, mannose residues would allow binding to mannose lectin and the DC-SIGN’s mannose-binding site biasing DCs to a proinflammatory Th1 immunity. Hence, modification of the carbohydrate moiety could impact the saponins’ stimulated immunity and in some cases their toxicity. What is the importance of saponin adjuvants that induce sole Th2 immunity? Because of the increase in autoimmunity and neurodegenerative diseases, which are initiated or aggravated by agents inducing Th1 immunity, there is a need for immunomodulators that elicit Th2 while inhibiting Th1 immunity to avert or treat damaging inflammatory immune responses. Could a global MOA explain the triterpene saponins’ adjuvanticity? Unlikely, while triterpene’s affinity for cholesterol is crucial for cross-presentation, letting saponins to intercalate in the cell’s membrane, enter it by endocytosis, and disrupt endosomal membranes, not all saponins are adjuvants. Indeed, that groups such as aldehydes, hydroxyls, and others are either reactive, affecting cell receptors, or can influence the saponins’ hydrophile/lipophile balance, altering the disruption of cellular membranes and cross-presentation, implies that MOAs can be inclusive only for structurally closely related saponins, which is relevant to drug design.``

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References 1. Mills, C.D. et al. (2015) Sequential immune responses: the weapons of immunity. J. Innate Immun. 7, 443–449

23. Rhodes, J. (2002) Discovery of immunopotentiatory drugs: current and future strategies. Clin. Exp. Immunol. 130, 363–369

2. Bergmann-Leitner, E.S. and Leitner, W.W. (2014) Adjuvants in the driver’s seat: how magnitude, type, fine specificity and longevity of immune responses are driven by distinct classes of immune potentiators. Vaccines 2, 252–296

24. Bomford, R. et al. (1992) Adjuvanticity and ISCOM formation by structurally diverse saponins. Vaccine 10, 572–577

3. Toussi, D.N. and Massari, P. (2014) Immune adjuvant effect of molecularly-defined Toll-like receptor ligands. Vaccines 2, 323– 353 4. Okano, M. et al. (2001) Lacto-N-fucopentaose III found in Schistosoma mansori egg antigens functions as adjuvant for proteins by inducing Th2-type response. J. Immunol. 167, 442–450 5. Gringhuis, S.I. et al. (2014) Fucose-specific DC-SIGN signalling directs T helper cell type-2 responses via IKKe- and CYLDdependent BcI3 activation. Nat. Commun. 5, 3898 6. Marciani, D.J. (2015) Alzheimer’s disease vaccine development: a new strategy focusing on immune modulation. J. Neuroimmunol. 287, 54–63 7. Soltysik, S. et al. (1995) Structure/function studies of QS-21 adjuvant: assessment of triterpene aldehyde and glucuronic acid roles in adjuvant function. Vaccine 13, 1403–1410 8. Fernández-Tejada, A. et al. (2014) Development of a minimal saponin vaccine adjuvant based on QS-21. Nat. Chem. 6, 635–643 9. Marciani, D.J. (2014) New Th2 adjuvants for preventive and active immunotherapy of neurodegenerative proteinopathies. Drug Discov. Today 19, 912–920 10. Press, J.B. et al. (2000) Structure/function relationships of immunostimulating saponins. In Studies in Natural Products Chemistry (Vol. 24) (Rahman, A., ed.), In pp. 131–174, Elsevier 11. Castro-Díaz, N.L. et al. (2012) Saponins from the Spanish saffron Crocus sativus are efficient adjuvants for protein-based vaccines. Vaccine 30, 388–397 12. Marciani, D.J. et al. (2003) Fractionation, structural studies, and immunological characterization of the semi-synthetic Quillaja saponins derivative GPI-0100. Vaccine 21, 3961–3971 13. Wang, P. et al. (2016) Synthesis and evaluation of QS-21-based immunoadjuvants with a terminal-functionalized side chain incorporated in the west wing trisaccharide. J. Org. Chem. 81, 9560– 9566 14. Chea, E.K. et al. (2012) Synthesis and preclinical evaluation of QS-21 variants leading to simplified vaccine adjuvants and mechanistic probes. J. Am. Chem. Soc. 134, 13448–13457 15. Fernández-Tejada, A. et al. (2016) Development of improved vaccine adjuvants based on the saponin natural product QS21 through chemical synthesis. Acc. Chem. Res. 49, 1741–1756 16. Greatrex, B.W. et al. (2015) Synthesis, formulation, and adjuvanticity of monodesmosidic saponins with olenanolic acid, hederagenin and gypsogenin aglycones, and some C-28 ester derivatives. ChemistryOpen 4, 740–755 17. Lacaille-Dubois, M.A. and Wagner, H. (2017) New perspectives for natural triterpenoid glycosides as potential adjuvants. Phytomedicine 37, 49–57 18. Marciani, D.J. (2003) Vaccine adjuvants: role and mechanisms of action in vaccine immunogenicity. Drug Discov. Today 8, 934–943 19. Nico, D. et al. (2012) The adjuvanticity of Chiococca alba saponins increases with the length and hydrophilicity of their sugar chains. Vaccine 30, 3169–3179 20. Kensil, C.R. et al. (1998) QS-21 and QS-7: purified saponin adjuvants. Dev. Biol. Stand. 92, 41–47 21. Rhodes, J. et al. (1995) Therapeutic potentiation of the immune system by co-stimulatory Schiff-base forming drugs. Nature 377, 71–75 22. Simon, R.H. and Rhodes, J. (2001) Schiff-base mediated costimulation primes the T-cell-receptor-dependent calcium signaling pathway in CD4 T cells. Immunology 104, 50–57

12

25. Rhodes, J. Glaxo Wellcome Inc. Immunopotentiary agent and physiologically salts thereof, US5958980A 26. Sykulev, Y. et al. (1996) Evidence that a single peptide-MHC complex on a target cell can elicit a cytolytic T cell response. Immunity 4, 565–571 27. Henrickson, S.E. et al. (2008) T cell sensing of antigen dose governs interactive behavior with dendritic cells and sets a threshold for T cell activation. Nat. Immunol. 9, 282–291 28. Fernández-Tejada, A. et al. (2014) Design, synthesis, and immunologic evaluation of vaccine adjuvant conjugates based on QS21 and tucaresol. Bioorg. Med. Chem. 22, 5917–5923 29. Kajal, A. et al. (2013) Schiff bases: a versatile pharmacophore. J. Catal. 2013, 893512 30. Marciani, D.J. et al. (2001) Altered immunomodulating and toxicological properties of degraded Quillaja saponaria Molina saponins. Int. Immunopharmacol. 1, 813–818 31. Lorent, J.H. et al. (2014) The amphiphilic nature of saponins and their effects on artificial and biological membranes and potential consequences for red blood and cancer cells. Org. Biomol. Chem. 12, 8803–8822 32. Den Brok, M.H. et al. (2016) Saponin-based adjuvants induce cross-presentation in dendritic cells by intracellular lipid body formation. Nat. Commun. 7, 13324 33. Fuchs, H. et al. (2017) Glycosylated triterpenoids as endosomal escape enhancers in targeted tumor therapies. Biomedicines 5, E14 ^ 34. Svajger, U. et al. (2010) C-type lectin DC-SIGN: an adhesion, signalling and antigen-uptake molecule that guides dendritic cells in immunity. Cell Signal. 22, 1397–1405 35. Marciani, D.J. (2015) Is fucose the answer to the immunomodulatory paradox of Quillaja saponins? Int. Immunopharmacol. 29, 908–913 36. Kensil, C.R. and Beltz, G.A. Antigenics Inc. Compositions of saponin adjuvants and excipients, US6645495 37. Walkowicz, W.E. et al. (2016) Quillaja saponin variants with central glycosidic linkage modifications exhibit distinct conformations and adjuvant activities. Chem. Sci. 7, 2371–2380 38. Boeckler, C. et al. (1996) Immunogenicity of new heterobifunctional cross-linking reagents used in the conjugation of synthetic peptides to liposomes. J. Immunol. Methods 191, 1–10 39. Chen, H. et al. (1997) Convergence of Schiff base costimulatory signaling and TCR signaling at the level of mitogen-activated protein kinase ERK2. J. Immunol. 159, 2274–2281 40. Hall, S.R. and Rhodes, J. (2001) Schiff base-mediated costimulation primes the T-cell-receptor-dependent calcium signalling pathway in CD4 T cells. Immunology 104, 50–57 41. Leitner, J. et al. (2015) CD58/CD2 is the primary costimulatory pathway in human CD28CD8+ T cells. J. Immunol. 195, 477– 487 42. Buchbinder, E. and Hodi, F.S. (2015) Cytotoxic T lymphocyte antigen-4 and immune checkpoint blockade. J. Clin. Invest. 125, 3377–3383 43. Nierkens, S. et al. (2013) Antigen cross-presentation by dendritic cell subsets: one general or all sergeants? Trends Immunol. 34, 361–370 44. Schnurr, M. et al. (2009) ISCOMATRIX adjuvant induces efficient cross-presentation of tumor antigen by dendritic cells via rapid cytosolic antigen delivery and processing via tripeptidyl peptidase II. J. Immunol. 182, 1253–1259 45. Duewell, P. et al. (2011) ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells. J. Immunol. 187, 55–63

Trends in Pharmacological Sciences, Month Year, Vol. xx, No. yy

TIPS 1517 No. of Pages 13

46. Welsby, I. et al. (2017) Lysosome-dependent activation of human dendritic cells by the vaccine adjuvant QS-21. Front. Immunol. 7, 663 47. Marty-Roix, R. et al. (2016) Identification of QS-21 as an inflammasome-activating molecular component of saponin adjuvants. J. Biol. Chem. 291, 1123–1136 48. Daines, A.M. et al. (2009) Mannosylated saponins based on oleanolic and glycyrrhizic acids: Towards synthetic colloidal antigen delivery systems. Bioorg. Med. Chem. 17, 5207–5218 49. Akira, S. (2011) Innate immunity and adjuvants. Philos. Trans. R. Soc. Lond. B Biol. Sci. 366, 2748–2755 50. Clark, R. and Kupper, T. (2005) Old meets new: the interaction between innate and adaptive immunity. J. Invest. Dermatol. 125, 629–637 51. Noack, M. and Miossec, P. (2014) Th17 and regulatory T cell balance in autoimmune and inflammatory diseases. Autoimmun. Rev. 13, 668–677

and its complementary structure. Scand. J. Immunol. 27, 697–704 53. Oda, K. et al. (2003) Relationship between adjuvant activity and amphipathic structure of soyasaponins. Vaccine 21, 2145–2151 54. Apostolopoulos, V. et al. (2000) Aldehyde-mannan antigen complexes target the MHC class I antigen-presentation pathway. Eur. J. Immunol. 30, 1714–1723 55. Apostolopoulos, V. et al. (2014) Dendritic cell immunotherapy: clinical outcomes. Clin. Transl. Immunol. 3, e21 56. Wright, S.E. et al. (2014) Tucaresol down-modulation of MUC1stimulated human mononuclear cells. Immunol. Invest. 43, 160– 169 57. Yadav, U.C.S. and Ramana, K.V. (2013) Regulation of NF-kBinduced inflammatory signaling by lipid peroxidation-derived aldehydes. Oxid. Med. Cell. Longev. 2013, 690545 58. Raghavan, S. et al. (2012) Proinflammatory effects of malondialdehyde in lymphocytes. J. Leukoc. Biol. 92, 1055–1067

52. Ocklind, G. et al. (1988) Activation of human T cells by neuraminidase-galactose oxidase-treated erythrocytes involving CD2(T11)

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