Saponins are secondary metabolites found in many plant species. The name derives from the Latin word “sapo,” meaning soap, owing to their ability to produce foam in water (Sparg et al., 2004; Vincken et al., 2007). They can be found in the bark, leaves, stems, roots, and even flowers of plants (Moghimipour and Handali, 2015; Rai et al., 2021). Saponins are divided into two main classes, namely, triterpenoids and steroid glycosides, with specific structures characterized by the number and position of the attached sugar units (Sparg et al., 2004; Vincken et al., 2007; Jolly et al., 2024). These natural glycosides have both a hydrophobic region, consisting of a steroid or triterpenoid skeleton called an aglycone, and a hydrophilic region, consisting of a sugar chain containing glucose, glucuronic acid, xylose, rhamnose or methyl pentose attached to the aglycone through ether or ester linkages (Supplementary Figure S1) (Sparg et al., 2004; Vincken et al., 2007; Moghimipour and Handali, 2015). This combination of polar and nonpolar elements endows saponins with soap-like behaviour in aqueous solutions (Sparg et al., 2004; Vincken et al., 2007). Saponins from Quillaja saponaria (QSap) contain a triterpenic aglycone, most frequently quillaic acid, which is glycosylated at the C-3 and C-28 positions of the aglycone (Fleck et al., 2019). The specific limitation of the use of saponins is their cytotoxicity, which correlates with their haemolytic activity, which is influenced by the affinity of the aglycone to cholesterol in cell membranes (Sparg et al., 2004; Lorent et al., 2014). However, saponins are particularly useful as vaccine adjuvants (Kensil, 1996; Sun et al., 2009; Shen et al., 2023).

Adjuvants, substances that aid in the effectiveness of vaccines, have greatly increased the protection provided by vaccine immunizations. Adjuvants play important roles in the type, duration and effectiveness of immune responses to vaccines (Awate et al., 2013; Shi et al., 2019; Verma et al., 2023; Zhao et al., 2023). In 1925, Ramon was the first to describe immunological adjuvants as substances that, when combined with a specific antigen, produce a stronger immune response than produced by the antigen alone. He reported increased yields of tetanus and diphtheria antitoxins produced in horses when the animals developed an abscess at the injection site (Awate et al., 2013; Verma et al., 2023; Chippaux, 2024). By injecting starch, breadcrumbs or tapioca with inactivated toxin, sterile abscesses were induced at the site of injection, increasing antitoxin production. He confirmed that substances able to induce local inflammation at the injection site were also able to enhance the immune response (Di Pasquale et al., 2015; Verma et al., 2023).

Despite numerous advancements in the isolation of antigens and vaccine production, only a limited number of adjuvants have been approved for use in humans. Aluminium salts, which were developed nearly 100 years ago, are still the most widely used adjuvant in licenced human vaccines (Di Pasquale et al., 2015; Shi et al., 2019).

The adjuvant effect of saponins was reported in the initial work of Ramon in 1925; in fact, the adjuvant effects of breadcrumbs, tapioca and starch oil were probably due to the presence of saponins. Years later, in 1951, Espinet used a crude commercially available Quillaja saponaria saponin preparation to increase the potency of foot-and-mouth disease vaccines (Espinet, 1951; Barr et al., 1998).

After Espinet’s work in foot-and-mouth disease vaccines, in 1974, Dalsgaard successfully isolated the commercially available saponin Quil A^® from the cortex of the South American tree Quillaja saponaria Molina. He reported that the addition of Quil A^® to vaccine preparations stimulated both humoral and cellular immunity and induced differential antibody isotypes (Dalsgaard, 1974; Dalsgaard, 1977). Unfortunately, saponins also have strong haemolytic effects, increasing adverse reactions.

Therefore, efforts have focused on purifying a defined saponin molecule that maintains the adjuvant effect with less haemolytic effects. In 1991, Kensil and coworkers patented QS-21 (Kensil and Marciani, 1991). They further purified saponins by reverse chromatography, and 22 fractions were obtained. The most predominant fractions (QAs 7, 18, 19, and 21) had adjuvant activity (Kensil and Marciani, 1991; Kensil et al., 1991; Wang et al., 2019; Wang, 2021). QA21 (now QS-21) was the fraction that exhibited a better balance between adjuvant activity and low toxicity (Kensil and Marciani, 1991; Kensil, 2001).

Morein and coworkers reported that the formulation of saponins into lipidic nanoparticles, called immune-stimulating complexes (ISCOMs), decreased the haemolytic activity, whereas the adjuvant activity was unaltered (Morein et al., 1984; Barr and Mitchell, 1996). ISCOMs are spherical, open cage-like structures approximately 40 nm in size (Barr and Mitchell, 1996). The formulation of nanoparticles removes the need for complicated purification processes and allows for the safe use of defined mixes of saponins (Barr and Mitchell, 1996). If an antigen is included in a nanoparticle formulation with cholesterol, phospholipids and saponins, the resulting particle is called ISCOM. It is also feasible to create nanoparticles without antigens and combine the nanoparticles and antigens later before delivery. In this case, the nanoparticles are called the ISCOM matrix or simply the matrix (Lövgren Bengtsson et al., 2011). Many studies have shown that adjuvant activity is conserved independently of the use of ISCOM or ISCOM Matrices plus antigen (Fossum et al., 1990; Stertman et al., 2023).

In 1995, Cox, Morein and coworkers patented an ISCOM particle (ISCOM Matrix) that included fractions A and C of saponins from QSap; within the next years, this product was named Matrix-M (Cox et al., 1995). The Matrix-M adjuvant is the third generation of ISCOM technology and consists of two different types of physically stable nanoparticles mixed at a defined ratio (85% Matrix-A + 15% Matrix-C) (Cox et al., 1995; Lövgren Bengtsson et al., 2011; Bai et al., 2024). Matrix-A (nanoparticle of fraction A) and Matrix-C™ (nanoparticle of fraction C) contain different QSap molecules with complementary properties. According to structural analysis, fraction C is composed mainly of QS-21. Compared with those from fraction C, fraction A saponins have weaker adjuvant activity but are less reactive and more easily form nanoparticles. The combination of the two types of particles reduces the reactogenicity while preserving the adjuvant activity (Cox et al., 1995; Lövgren Bengtsson et al., 2011).

Currently, there are two main QSap adjuvant preparations licenced for human use. AS01 and AS02 from GlaxoSmithKline combine QS-21 with other immunostimulatory molecules (Vandepapeliere, 2013). AS01 contains 3-O-desacyl-4′-monophosphoryl lipid A (MPL), and QS-21 is formulated as a liposome, whereas AS02 contains MPL combined with QS-21 in an oil-in-water emulsion; both are designed to induce strong humoral and T-cell-mediated responses (Didierlaurent et al., 2017; Garçon and Di Pasquale, 2017).

AS01 was designed to strengthen the CD8⁺ response and is included in two licenced human vaccines (Shingrix™ for herpes virus and Mosquirix™ for malaria); it is also currently used in a candidate HIV and tuberculosis vaccine (Garçon and Di Pasquale, 2017; Lacaille-Dubois, 2019; Alving et al., 2020). AS02 has been evaluated in vaccines targeting complex pathogens that require a strong T-cell response and induce strong humoral and cellular immune responses (e.g., against hepatitis B, malaria, and HIV) (Garçon and Di Pasquale, 2017).

Matrix-M (Novavax) is also used in licenced human vaccines such as the Novavax COVID-19 vaccine (Nuvaxovid^™) and the antimalarian R21/Matrix-M in collaboration with Oxford University and the Serum Institute of India (Stertman et al., 2023). Nuvaxovid is a recombinant protein vaccine composed of SARS-CoV-2 spike trimer nanoparticles formulated with Matrix-M^™ adjuvant (Underwood et al., 2023; Lenart et al., 2024). Matrix-M is also being evaluated in clinical trials for new influenza vaccines (Pedersen et al., 2014; Shinde et al., 2022).

Although QS-21 exhibits limited haemolytic activity in vitro, higher doses may lead to side effects. Saponins can disrupt cell membranes, leading to haemolysis, as demonstrated by pore formation in erythrocyte membranes (Petrovsky, 2015). To reduce this toxicity, saponins can be encapsulated in lipid nanoparticles. This formulation strategy has been shown to reduce saponin-related adverse effects (Ragupathi et al., 2011; Petrovsky, 2015; Bigaeva et al., 2016).

Adverse reactions reported for licensed products, such as QS-21 or ISCOMs, indicate a low incidence of side effects comparable to those of other adjuvants. Saponin-based adjuvants have been associated with local adverse effects, including pain, redness, swelling, and erythema, as well as mild systemic effects such as fever and flu-like symptoms (Ragupathi et al., 2011; Petrovsky, 2015; Bigaeva et al., 2016).

However, owing to overexploitation of the QSap bark of Chilean forests, which has caused important ecological damage and resulted in the scarcity of available supplies, considerable efforts have been undertaken to discover sources of new saponins with improved adjuvant activity and reduced toxicity (Schlotterbeck et al., 2015; Fleck et al., 2019). Another species that belongs to the family Quillajaceae, Quillaja brasiliensis (now Quillaja lancifolia D. Don), a native tree distributed in southern Brazil, northern Uruguay, northeastern Argentina and eastern Paraguay, has been shown to be a better source of saponins because the saponins can be purified from leaves (Luebert, 2013; Schlotterbeck et al., 2015; Fleck et al., 2019). At the laboratory scale, saponins from Quillaja brasiliensis (QBr) presented characteristics and adjuvant activity similar to those of saponins extracted from QSap, opening a new avenue for the development of saponin-based adjuvants (Cibulski et al., 2016b; Cibulski et al., 2022; Fleck et al., 2019; Magedans et al., 2019; Rivera-Patron et al., 2021).

The precise mechanism of action of QSap or QBr saponins in their role as an adjuvant remains unclear. However, recent investigations have begun to delineate potential modes of action and signalling pathways. Saponins possess the capacity to induce both pro-inflammatory Th1/Th2 and anti-inflammatory Th2 immune responses (Marciani, 2018; Wang, 2021). Structure-activity relationship studies have elucidated that the presence of imine-forming carbonyl groups within the saponin structure is indispensable for T cell activation and the subsequent induction of Th1/Th2 responses. While saponins with diverse triterpenoid aglycons and oligosaccharide chains can activate dendritic cells (DCs) to induce both Th1 and Th2 responses, the presence of fucopyranosyl residues within their oligosaccharide chains can bias DC activation towards a Th2-dominant response through engagement of the Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) receptor (Guo et al., 2004; Aguilar and Rodríguez, 2007; Chea et al., 2012; Marciani, 2018; Marciani, 2022; Marciani, 2024). Glycosides like QS-21 can interact separately with T cells and dendritic cells. T cells are co-stimulated by the glycoside’s aldehyde, while dendritic cells are activated by interactions with the triterpene group and fucosyl residue, respectively (Soltysik et al., 1995; Liu et al., 2002; Ragupathi et al., 2011; Marciani, 2018; Marciani, 2024).

Lacalle-Dubois propose that QS-21 mechanism of action could be synthetized as follow: stimulation of Th2 humoral and Th1 cell-mediated immune responses through action on antigen presenting cells (APCs) and T cells; release of Th1 cytokines participating in the elimination of intracellular pathogens; activation of the NLRP3 inflammasome and the release of caspase-1 dependent cytokines IL 1β and IL-18 (Marty-Roix et al., 2016; Coccia et al., 2017; Lacaille-Dubois and Wagner, 2017; Marciani, 2018; Lacaille-Dubois, 2019). A growing body of evidence is continually revealing the mechanisms of action of QSap saponins, indicating their complex interactions with multiple signalling pathways.

In this review, we focus on the most common purification methods and formulations of saponins for vaccine adjuvants.

There are two classic methods for the preparation of these nanoparticles: dialysis and centrifugation. The protocols are identical for the formulations of the ISCOMs and matrix except that the latter does not introduce the antigen into the nanoparticle; the antigen is added later. Among the two methods, dialysis has gained favour over centrifugation because of its simplicity and ease of scaling up, and currently, it is the widely accepted method for ISCOM/matrix formulation (Barr and Mitchell, 1996). The main important factor in the formation of optimal ISCOMs is the correct ratio of the various components that are combined at the start. According to Barr and Michell, the optimal weight ratios of cholesterol, phospholipid and QSap saponin should be 1:1:5 for the matrix and ratios of 1:1:5:0.1 to 1:1:5:1 when the antigen is included. The dialysis protocol is very simple: solubilized proteins (in the case of ISCOMs) are added to cholesterol and phospholipid dissolved in a nonionic detergent (MEGA-10 is the most commonly used), and saponins are added and mixed. The detergent is subsequently removed from the mixture by extensive dialysis or diafiltration (Barr and Mitchell, 1996; Barr et al., 1998; Rivera-Patron et al., 2022). Compared to other process, dialysis produces more homogeneous formulations with a narrow particle size distribution (Lendemans et al., 2005; Myschik et al., 2006).

Conversely, the centrifugation method is more complex and includes a sucrose gradient and ultracentrifugation, and it is rarely used (Barr and Mitchell, 1996; Hu et al., 1998). Other methods, such as ether or ethanol injection and lipid-film hydration, are described but are not as popular as the dialysis method (Bangham et al., 1965; Batzri and Korn, 1973; Pons et al., 1993; Copland et al., 2005; Myschik et al., 2006; Pham et al., 2006; Demana et al., 2010; Lendemans et al., 2010).

Saponins in combination with other immunostimulants or formulated as lipid nanoparticles have become new protagonists in human vaccine adjuvants (Kensil, 2001; Garçon and Di Pasquale, 2017; Stertman et al., 2023; Marciani, 2024). Table 1 presents the most commonly used saponin preparations as vaccine adjuvants. Adjuvants formulated with saponins have the advantage of generating a strong and balanced immune response, including strong cellular responses that make them suitable for use in viral vaccines (Sjölander et al., 1997; Cibulski et al., 2016a; Verma et al., 2023; Zhao et al., 2023).

With respect to saponin purification for human vaccines, improvements have been made in two different ways. First, the purification or purification/modification of defined saponin molecules can achieve a strong immune response with few side effects (Kensil, 1996; Garçon and Di Pasquale, 2017; Marciani, 2024). Second, the purification of a defined mixture of saponins that can be formulated into nanoparticles maintains immune activity and decreases side effects (Cox et al., 1995; Stertman et al., 2023).

The primary challenges in saponin production for adjuvant use are the sustainability of Q. saponaria tree cultivation and the need for better characterized and purified compounds that retain adjuvant potency while minimizing adverse effects (Ragupathi et al., 2011; Fleck et al., 2019).

The extraction of saponins from Q. brasiliensis leaves has proven to be the most efficient and environmentally friendly method. Nevertheless, alternative sustainable methods are currently under investigation. Lv and coworkers, reports the successful production of QS-21, through plant cell culture. The study demonstrates that plant cell culture can provide a sustainable and scalable alternative for producing QS-21 with comparable chemical and biological properties to the bark-derived product (Lv et al., 2024).

In this way, Martin et al. propose the complete biosynthesis of QS-21 saponin. The study successfully reconstituted the entire 20-step pathway in tobacco, demonstrating the production of QS-21 in a heterologous expression system (Martin et al., 2024).

In relation with the molecular structure, Marciani emphasizes the need to view adjuvants, particularly saponins like QS-21, as a distinct class of drugs with specific immunopharmacological properties shaped by their functional groups (Marciani, 2024). Specifically, QS-21 has been shown to induce a pro-inflammatory Th1 response through its aldehyde group, challenging the misconception that its adjuvanticity is solely due to its particulate nature (Soltysik et al., 1995; Aguilar and Rodríguez, 2007). The fucose residue is also critical, influencing the type of immune response elicited, although it is often mischaracterized as merely structural. Research into synthetic analogues could help clarify the roles of these components, as changes in sugar moieties may shift immune response (Ragupathi et al., 2011; Marciani, 2024). To fully understand the mechanisms of action of complex adjuvants, it is essential to identify the specific cell receptors they target. Understanding these interactions and their immunological effects can lead to improved adjuvant design (Borriello et al., 2022; Marciani, 2024).