molecules Review Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities Juliane Deise Fleck 1 , Andresa Heemann Betti 2 , Francini Pereira da Silva 1 , Eduardo Artur Troian 1 , Cristina Olivaro 3 , Fernando Ferreira 4 and Simone Gasparin Verza 1, * 1 2 3 4 * Molecular Microbiology Laboratory, Institute of Health Sciences, Feevale University, Novo Hamburgo 93525-075, RS, Brazil; [email protected] (J.D.F.); [email protected] (F.P.d.S.); [email protected] (E.A.T.) Bioanalysis Laboratory, Institute of Health Sciences, Feevale University, Novo Hamburgo 93525-075, RS, Brazil; [email protected] Science and Chemical Technology Department, University Center of Tacuarembó, Udelar, Tacuarembó 45000, Uruguay; [email protected] Organic Chemistry Department, Carbohydrates and Glycoconjugates Laboratory, Udelar, Mondevideo 11600, Uruguay; [email protected] Correspondence: [email protected]; Tel.: +55-51-3586-8800 Academic Editors: Vassilios Roussis and Efstathia Ioannou Received: 25 October 2018; Accepted: 28 December 2018; Published: 4 January 2019 Abstract: Quillaja saponaria Molina represents the main source of saponins for industrial applications. Q. saponaria triterpenoids have been studied for more than four decades and their relevance is due to their biological activities, especially as a vaccine adjuvant and immunostimulant, which have led to important research in the field of vaccine development. These saponins, alone or incorporated into immunostimulating complexes (ISCOMs), are able to modulate immunity by increasing antigen uptake, stimulating cytotoxic T lymphocyte production (Th1) and cytokines (Th2) in response to different antigens. Furthermore, antiviral, antifungal, antibacterial, antiparasitic, and antitumor activities are also reported as important biological properties of Quillaja triterpenoids. Recently, other saponins from Q. brasiliensis (A. St.-Hill. & Tul.) Mart. were successfully tested and showed similar chemical and biological properties to those of Q. saponaria barks. The aim of this manuscript is to summarize the current advances in phytochemical and pharmacological knowledge of saponins from Quillaja plants, including the particular chemical characteristics of these triterpenoids. The potential applications of Quillaja saponins to stimulate further drug discovery research will be provided. Keywords: triterpenoids; Quillaja saponaria; Quillaja brasiliensis; antimicrobial; antiviral vaccine; immunoadjuvant; 1. Introduction Quillaja saponaria Molina is commonly found in Peru, Chile, and Bolivia. It was first described in 1782 due to the saponin content in the bark. The expression ‘saponin’ is Latin in origin: the word sapo that means ‘soap’, since saponins form foams when mixed with water [1,2]. The amphiphilic structure of saponins, due to their lipophilic aglycones and hydrophilic saccharide side chains, is responsible for the foam formation and chemical properties [1,2]. The most commonly reported aglycone from Q. saponaria, i.e., quilaic acid [3,4], is characterized by an aldehyde group at the C-23 position. The acyl side chain, another characteristic of Quillaja saponins, seems to also play an important role in some biological activities [5]. Molecules 2019, 24, 171; doi:10.3390/molecules24010171 www.mdpi.com/journal/molecules Molecules 2019, 24, 171 2 of 29 Saponins from Q. saponaria have a wide range of applications. Historically, Quillaja bark saponins have been used as a detergent. Nowadays they are approved for use as food additives in 187 signatory countries of the Codex Alimentarius, including the European Union, the United Kingdom, the United States, China, and Japan [6], the latter of which also allows its use in cosmetics [7]. The European Commission of Cosmetic Ingredients (CosIng) database has listed some Quillaja products as cosmetic ingredients, such as the bark, bark extract, root extract, and wood extract. Depending on the material used, different functions are attributed, such as antidandruff, cleansing, emulsifying, foaming, masking, moisturizing, skin conditioning, and surfactant [8]. Nowadays, there is increasing interest in using saponins as natural emulsifiers, alone or blended with others. Numerous studies have reported that Quillaja saponins are good emulsifiers for O/W emulsions [7,9,10] and nanoemulsions [10]. Recently, a food ingredient containing Quillaja bark saponins (Q-Naturale® ) was approved by the FDA as an effective emulsifier for beverages [6,10]. In addition to their detergent and emulsifying properties, other pharmacological activities, including antiviral [11–13], antifungal [14], antibacterial [15–17], and antiparasitic ones, have been reported for Quillaja saponins. Regarding its biological properties, Q. saponaria has been included in the European and Brazilian Pharmacopoeias. The raw material is recommended as a cough reliever and expectorant, anti-inflammatory, hypocholesterolemic, and hemolytic agent. Topically, it has been indicated to treat scalp diseases (dandruff and hair loss) and other dermatological disorders. In the African Pharmacopoeia, Quillaja bark has been indicated as an emulsifying agent only. Meanwhile, the strong immunoadjuvant activity reported for Quil-A® , a crude extract of Q. saponaria bark, and its derivatives, makes them ideal substances for vaccine development [5,18–21]. Since Quil-A® is a heterogeneous mixture of saponins, its various components may exhibit different biological properties, including toxicological effects [5]. Thus, more purified saponin fractions have been obtained, especially QS-21, which has been applied in preclinical studies and clinical trials [5,21]. An improvement to reduce saponin toxicity and enhance immunoadjuvant activity has been reported with the development of immunostimulating complexes (ISCOMs). ISCOMS are cage-like particles (40 nm), composed of saponins derived from Q. saponaria, phospholipids, cholesterol, and the targeted antigen [22–24]. Recently, a purified fraction of Quillaja brasiliensis saponins was also tested in ISCOMs, and the typical cage-like structures were viewed [25]. It is important to note that these structures are described only for Quillaja species. Q. brasiliensis is native to Northern Uruguay, Southern Brazil, Northeastern Argentina, and Eastern Paraguay. It has also been studied as an immunoadjuvant, with crude extracts and purified saponin fractions being evaluated and shown to stimulate a great immune response to viral antigens, as observed with saponins from Q. saponaria [25–28]. Saponins are also abundant in the leaves of Q. brasiliensis, which represents a renewable source of these substances, since in Q. saponaria the main source of saponins is the barks of the tree. Nowadays, the overexploitation of native forests has been replaced and other alternative sources are available such as synthetic analogues of QS-21 saponin. Meanwhile, natural alternative sources of these products are also important [29]. Considering the relevance and biological properties already described for Quillaja species, a systematic review of published trials was accomplished with respect to their chemical characteristics and biological activities. To perform the review, the PubMed database was searched using the keywords Quillaja saponins, chemical characteristics, biological activity, adjuvant, antiviral, antifungal, antibacterial, and toxicity, either alone or in combination. 2. Chemical Structure/Characterization Despite their widespread use, the structure of Q. saponaria saponins was not completely elucidated until 1986, when Higuchi et al. [3] published the structure of two deacetylated saponins from Q. saponaria bark (Figure 1, Figure 2 and Table 1). Further isolation and structural studies allowed for the identification of nearly 60 saponins from Q. saponaria, sharing several structural motifs. These saponins contain a triterpenic aglycone belonging to the β-amyrin series, most frequently quillaic acid (Figure 1). Molecules 2019, 24, 171 Molecules Molecules 2019, 2019, 24, 24, xx 3 of 29 33 of of 30 30 Figure 1. Reported triterpene aglycones in Quillaja saponaria and/or Figure1. 1.Reported Reportedtriterpene triterpeneaglycones aglyconesin inQuillaja Quillajasaponaria saponariaand/or and/or Q. Q. brasiliensis brasiliensis saponins. saponins. Figure Figure brasiliensis saponins. Figure 2. 2. General General structure structure of of Q. Q. saponaria saponaria and and Q. Q. brasiliensis brasiliensissaponins. saponins. These These saponins are bidesmosides, glycosylated at the C-3 and C-28 positions of the aglycone These saponins saponins are are bidesmosides, bidesmosides, glycosylated glycosylated at at the the C-3 C-3 and and C-28 C-28 positions positions of of the the aglycone aglycone (Figure 2). The C-3 position of the aglycone is most frequently glycosylated by the disaccharide residue (Figure (Figure 2). 2). The The C-3 C-3 position position of of the the aglycone aglycone is is most most frequently frequently glycosylated glycosylated by by the the disaccharide disaccharide βD -Galp-(1 2)-β-D-GlcAp, most frequently branchedbranched at the O-3 the glucuronic acid unit residue β-Galp-(1→2)-βD most at the position of residue β-D D→ -Galp-(1→2)-βD-GlcAp, -GlcAp, most frequently frequently branched atposition the O-3 O-3 of position of the the glucuronic glucuronic with a αL -Rhap or a βD -Xylp residue. In a few cases, the saponin lacks the C-28 linked oligosaccharide acid acid unit unit with with aa αα-LL-Rhap -Rhap or or aa ββ-D D-Xylp -Xylp residue. residue. In In aa few few cases, cases, the the saponin saponin lacks lacks the the C-28 C-28 linked linked (saponins 3–5, Table 1), but3–5, normally position is glycosylated through an acetal-ester linkage to a oligosaccharide (saponins Table 1), normally this is through an oligosaccharide (saponins 3–5, Table this 1), but but normally this position position is glycosylated glycosylated through an acetalacetalcomplex oligosaccharide. oligosaccharide hasoligosaccharide a conserved moiety the disaccharide ester to oligosaccharide. This has conserved composed ester linkage linkage to aa complex complexThis oligosaccharide. This oligosaccharide has aacomposed conservedofmoiety moiety composed αL -Rhap-(1 → 2)-βD -Fucp with different decorations [30]. More often this disaccharide is extended by of of the the disaccharide disaccharide αα-LL-Rhap-(1→2)-β-Rhap-(1→2)-β-D D-Fucp -Fucp with with different different decorations decorations [30]. [30]. More More often often this this adisaccharide β-D-Xyl-(1→is residue, which bear a second β-D-Xylp →3)bear residue or a β→3) residue. by D residue, which aa second β-Xylp (1→3) residue disaccharide is4)extended extended by aa ββ-can D-Xyl-(1→4) -Xyl-(1→4) residue, which(1can can bear second β-DD D-Apif -Xylp(1 (1→3) residue The βD -Fucp residue of the constant disaccharide can present either a αL -Rhap or a βD -Glcp at O-3, or aa or aa ββ-D D-Apif -Apif (1→3) (1→3) residue. residue. The The ββ-D D-Fucp -Fucp residue residue of of the the constant constant disaccharide disaccharide can can present present either either and can be acetylated or not. The βD -Fucp residue can be acylated at O-4, simply with an acetate αα-LL-Rhap -Rhap or or aa ββ-D D-Glcp -Glcp at at O-3, O-3, and and can can be be acetylated acetylated or or not. not. The The ββ-D D-Fucp -Fucp residue residue can can be be acylated acylated at at residue or a branched ester dimer composed of two units 3,5-dihidroxy-octanoic acid, called Fa, O-4, with residue or ester composed units of O-4, simply simply with an an acetate acetate residue or aa branched branched esterofdimer dimer composed of of two two units of 3,53,5- dihidroxy-octanoic dihidroxy-octanoic acid, acid, called called Fa, Fa, which which can can be be glycosylated glycosylated in in the the terminal terminal 5-hidroxy 5-hidroxy group group by by aa ββD D-Araf -Araf residue residue (Figure (Figure 2) 2) [30]. [30]. This This terminal terminal ββ-D D-Araf -Araf residue residue can can also also bear bear aa (1→2)-β(1→2)-β-D D-Glcp -Glcp residue. residue. Molecules 2019, 24, 171 4 of 29 which can be glycosylated in the terminal 5-hidroxy group by a β-D-Araf residue (Figure 2) [30]. This terminal β-D-Araf residue can also bear a (1→2)-β-D-Glcp residue. The β-D-Fucp residue linked to C-28 can be acylated at O-4 (R4 in Figure 1, see Table 1). However, the corresponding regioisomers acylated at the O-3 position can be found in solution due to a transesterification reaction between these two cis-vicinal hydroxyl groups, as has been observed in the adjuvant saponin QS-21. The rate of this isomerization is higher at pH > 5.5, leading to an equilibrium mixture of both isomers [31]. Phytochemical studies of the less known Quillaja brasiliensis tree, even though it is a well-known rich source of saponins, started relatively later. In a pioneering study, a prosapogenin composed of quillaic acid glycosylated at C-3 with a β-D-GlcAp residue was isolated from the products of partial hydrolysis, suggesting that these saponins might also present structures like those from Q. saponaria [32]. Recently, Wallace et al. [29] presented the first structural studies of complete Q. brasiliensis saponins using electrospray ionization ion trap-multiple stage mass spectrometry (ESI-IT-MSn). These authors identified 27 bidesmosidic triterpenic saponins, with either quillaic acid, gypsogenin, phytolaccinic acid, or 23-O-acetyl-phytolaccinic acid as aglycones (Figure 1). The Q. brasiliensis saponins described present glycosylation patterns at positions C-3 and C-28 of the aglycone analogous to those of Q. saponaria saponins with the same conserved motifs (Figure 2). Some saponins presented different degrees of acylation, and regioisomers with the Fa acyl substituent at the O-4 or the O-3 positions of the β-D-Fucp residue were found, as well as the presence of the QS-21 Xyl and QS-21 Api saponins (S4/S6, Table 1). Table 1. Structures of Quillaja saponaria saponins. Nº Saponin Structure Mi Aglycone 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 DS-1 DS-2 * * * 4 5 6 7 8 9 10 11a 11b 12a 12b 13a 13b 14a 14b 15a 15b 16a 16b 17a 17b 1512.0 1674.0 824.0 970.0 956.0 1436.6 1582.7 1568.7 1714.7 1700.7 1714.7 1700.7 1598.7 1584.7 1628.7 1614.7 1730.7 1716.7 1760.8 1746.7 1640.7 1626.7 1802.8 1788.7 1744.8 1730.7 Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Ref. R0 R1 R2 R3 Xyl Xyl H Rha Xyl H Rha Xyl Rha Xyl Rha Xyl Rha Xyl Rha Xyl Rha Xyl Rha Xyl Rha Xyl Rha Xyl Rha Xyl Api-Xyl Api-Xyl Xyl Xyl Xyl Xyl-Api Xyl-Api Xyl-Xyl Xyl-Xyl Xyl Xyl H H Xyl-Api Xyl-Api Xyl Xyl Xyl Xyl Xyl Xyl Xyl Xyl H H H H H H H H H H H Glc Glc Glc Glc Glc Glc H H Glc Glc Glc Glc H Glc Rha Rha Rha Rha Rha Rha Rha Glc Glc Glc Glc H H Glc Glc Glc-Ac Glc-Ac Glc-Ac Glc-Ac Rha Rha R4 Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac [3] [3] [33] [33] [34] [34] [34] [34] [34] [34] [34] [34] [35] [35] [35] [35] [35] [35] [35] [35] [35] [35] [35] [35] [35] [35] Molecules 2019, 24, 171 5 of 29 Table 1. Cont. Nº Saponin Structure Mi Aglycone 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 18a 18b S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 B1 B2 B3 B4 B5 B6 B7 B8 QS-III 20a 20b 21a 21b 22a 22b 19 23 S13 1876.8 1862.8 1870.9 1856.9 2002.9 1988.9 2002.9 1988.9 1912.9 1898.9 2045.0 2030.9 2045.0 2030.9 2033.0 2018.9 2165.0 2151.0 2165.0 2151.0 1886.9 2018.9 2297.0 1656.7 1642.7 1788.8 1774.8 1978.9 1964.9 1392.7 1732.8 1560.7 Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q-OH Q-OH Q-OH Q-OH Q-OH Q-OH P E P-Ac R0 R1 R2 Rha Xyl Rha Xyl Rha Xyl Rha Xyl Rha Xyl Rha Xyl Rha Xyl Rha Xyl Rha Xyl Rha Xyl H H Xyl Rha Xyl Rha Xyl Rha Xyl H Xyl H Xyl-Api Xyl-Api Xyl Xyl Xyl-Xyl Xyl-Xyl Xyl-Api Xyl-Api Xyl Xyl Xyl-Xyl Xyl-Xyl Xyl-Api Xyl-Api Xyl Xyl Xyl-Api Xyl-Api Xyl-Xyl Xyl-Xyl Xyl Xyl-Api Xyl-Api Xyl Xyl Xyl-Api Xyl-Api Xyl-Api Xyl-Api H Xyl H Glc Glc H H H H H H H H H H H H Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc Glc H Glc MeBu Ref. R3 R4 Rha Ac Rha Ac H Fa-Ara H Fa-Ara H Fa-Ara H Fa-Ara H Fa-Ara H Fa-Ara Ac Fa-Ara Ac Fa-Ara Ac Fa-Ara Ac Fa-Ara Ac Fa-Ara Ac Fa-Ara H Fa-Ara H Fa-Ara H Fa-Ara H Fa-Ara H Fa-Ara H Fa-Ara H Fa-Ara H Fa-Ara Fa-Ara-Rha H H MeBu H MeBu H MeBu H MeBu Rha OHMeHex Rha OHMeHex Glc MeBu Glc Ac Glc MeBu [35] [35] [36] [36] [36] [36] [36] [36] [4] [4] [4] [4] [4] [4] [36] [36] [36] [36] [37] [37] [37] [37] [3] [34] [34] [34] [34] [34] [34] [34] [34] [4] *: Monodesmosidic C-3 saponins. DS: deacylateded saponins, Q: quillaic acid, Q-OH: quillaic acid, 22β-OH, P: phytolaccinic acid, P-Ac: phytolaccinic acid, 23-O-Ac, E: echynocystic acid. Fa-Ara: see Figure 2, QS_III: quillaja saponin with highest molecular mass, MeBu: 2-methylbutanoyl, OHMeHex: 3-hydroxy-4-methylhexanoyl. Adapted from Kite et al. [30]. 3. Biological Activities Regarding the biological activities, Quillaja saponins have been widely studied. The main activities described are reviewed below, especially the antibacterial, antiviral, antifungal, antiparasitic, antitumor, hepatoprotective, and immunoadjuvant ones. 3.1. Antimicrobial Activities of Quillaja Saponins The antimicrobial activities of saponins from Quillaja are described in several studies, especially their antibacterial, antifungal, and antiviral ones [11,12,14,15,38–40]. The main reason for these activities is the affinity of the aglycone with cell membrane cholesterol, leading to changes in the membrane and, consequently, preventing microorganism infection of the cell [41,42]. 3.1.1. Antibacterial Activity Saponins present detergent properties by destroying membrane lipids and, for this reason, they can make the bacterial cell membranes permeable facilitating the influx of molecules through the bacterial wall, such as antibiotics [43–45]. On the other hand, a study conducted with clinical Molecules 2019, 24, 171 6 of 29 strains of multiresistant Escherichia coli isolated from human urine demonstrated that commercial saponin from Q. saponaria barks (12 µg/mL) significantly increased the growth of the six strains tested. In susceptibility tests of E. coli strains against ampicillin, streptomycin, and ciprofloxacin, saponins increased the colony forming units per mL (CFU/mL), even in the presence of antibiotics [46]. Saponin extracts from Q. saponaria are sold in the market and produced by different brands, and their composition can vary according to the process used to obtain them. Therefore, the antimicrobial activity may be altered since the constitution and nature of the saponins differs. Sen et al. [16] evaluated the anti E. coli action of Q. saponaria saponins from three different commercial companies (Carl Roth GmbH® , Karlsruhe, Germany; Sigma® , Saint Louis, MO, USA; Quillaja 300 Powder® , Nor-feed, Hvidovre, Denmark). The study showed different results between the three brands used, in which the CFU increased with the different concentrations of saponins used. The percentage of bacterial growth was 151%, 31%, and 97% for the Roth, Sigma, and Nor-feed brands, respectively. The activity of Q. saponaria saponins, in the induction of bacterial growth, indicates that they may act by facilitating the transport of nutrients from the bacterial growth media into the microorganism by increasing bacterial permeability though the cell membranes, thus stimulating the colonies’ growth [16,46]. In contrast, Hassan et al. [38] showed the antibacterial activity of a commercial extract rich in Quillaja saponins (Sigma® ) against Salmonella typhimurium, Staphylococcus aureus, and E. coli. Similarly, Sewlikar and D’Souza [47] observed the strong antibacterial activity of the aqueous extract of Q. saponaria against four strains of E. coli producers of Shiga toxin (STEC) O157:H7 and six non-O157 STEC serotypes. At the time of treatment with the extracts, the 4 O157:H7 strains had an initial CFU of approximately 7.5 log and after 16 h of treatment, the remaining counts were reduced to 6.79 and 3.5 log CFU at room temperature. The non-O157 STECs treated with Q. saponaria showed reductions of 6.81 to 4.55 log CFU after 16h at room temperature. Additional tests showed that incubation at 37 ◦ C reduced the CFU to undetectable levels (control without any significant reduction) after 1h of treatment for all O157:H7 strains and 30 min post-treatment for non-O157. Scanning electron microscopy analysis revealed the damage to bacteria cell membranes subjected to the treatment. The commercial extract of Q. saponaria saponins (Sigma® ) was shown to be a relevant agent for increasing the efficiency of the disinfection process of the bacteria genus Asaia. Pretreatment with 1% saponin solution resulted in increased sensitivity of the bacterial cultures tested. Cultures pre-exposed to saponin solution and submitted to N-ethyl-N,N-dimethylhexadecylammonium bromide (QAC) showed half of the minimum inhibitory concentration (MIC) compared to non-saponin-treated bacteria. In tests performed with peracetic acid, the difference in MICs was 4 to 8-times lower than the bacterial strains without saponin pre-treatment. With these findings, the authors suggest that treatment with saponin solution prior to disinfection may reduce the need for high concentrations of the antimicrobials tested. Concerning the inhibition of bacterial biofilms, experiments conducted in this same study revealed that the extract reduced the live cells number in biofilms by approximately 4 to 5 log [48]. Different studies on the antibacterial activity of Q. saponaria saponins suggest that saponins’ biological activity is related to their chemical constitution, thus different microorganisms have different susceptibilities to saponin action [49,50]. 3.1.2. Antiviral Activity Two extracts of Q. saponaria, Ultra Dry 100 Q (a spray-dried purified aqueous extract, consisting of 65% saponins) and Vax sap (purified medical grade material, obtained by an additional purification of Ultra Dry 100 Q, with saponin content greater than 90%), showed antiviral activity in vitro and in vivo. Ultra Dry 100 Q showed strong antiviral action against herpes simplex virus type 1, vaccinia virus, human immunodeficiency viruses 1 and 2, varicella zoster virus, rhesus rotavirus (RRV), and reovirus. Very low concentrations of the extract were able to prevent the virus from infecting their host cells and were still able to maintain their blocking activity and stop the virus binding to the cells for up to 16 h after their removal from the culture medium. Both extracts studied prevented the binding of RRV and reovirus to host cells pretreated with them, also inhibiting the propagation of infectious Molecules 2019, 24, 171 7 of 29 particles to the uninfected neighboring cells. This cell protecting effect persisted for up to 16 h after the extracts were removed from the cell monolayers [11,13]. The action of saponins is possibly due to the cell membrane and the affinity of the aglycone portion with the membrane cholesterol. Consequently, without the cholesterol, the fluidity of the cell membrane increases, causing it to be beyond the control of the enzymatic activities. Thus, saponins may act in cellular proteins by preventing specific binding of viral receptors [41,51]. Confirming the evidence shown in the in vitro tests, in which the saponin-rich extracts of Q. saponaria Molina are able to inhibit RRV infection in host cells by some modification in the cell membrane proteins preventing binding of viral receptors, in vivo experiments with Vax sap extract presented strong anti-RRV action. Newborn mice were exposed to the aqueous extract for two days and, afterwards, RRV was administered for another five consecutive days. Doses of 0.015 mg/mouse reduced the diarrhea caused by the virus by 68% in mice exposed to 500 plaque forming units (PFU). The experiments revealed that lower concentrations of the extract do not have a strong antiviral effect when administered in higher viral titers (5000 PFU). In addition, in the case of a higher inoculant (50,000 PFU), an extract concentration of 0.03 mg/mouse blocked virus infection, also reducing diarrhea in the animals [12]. 3.1.3. Antifungal Activity Membranes of regenerated cellulose nanofibers supplemented with Q. saponaria saponins are described as having antifungal activity. Dixit et al. [14] verified that, after 24 h, the nanofiber membranes containing dead spores of Penicillium roguefortii Thom caused an 80.4% grown reduction and Aspergillus ochraceus anamorph resulted in a 53.6% reduction. In addition, Chapagain et al. [52] described moderate activity of Q. saponaria bark saponins against different fungal species: Alternaria solani (47.5%), Pythium ultimum (59.12%), Fusarium oxysporum (56.11%), and Verticillium dahliae (35.92%), but not for Colletotrichum coccodes species. 3.2. Antiparasitic Activity The saponins from Q. saponaria barks showed antiparasitic activity, in vitro, in a ruminal fermentation system [15,39,40]. A commercial extract of Q. saponaria revealed an antiparasitic effect inversely proportional to the extract concentrations tested [17]. Quillaja saponins were also evaluated against cloned xenic cultures of different isolates of Blastocystis sp., Tetratrichomonas gallinarum, and Histomonas meleagridis. The in vitro assays for antiprotozoal activity showed that the effect of this plant’s saponins are directed towards the protozoa, although more studies are necessary to elucidate the mode of action of saponins though the parasites [53]. A study performed to evaluate the effect of commercial saponins obtained from Q. saponaria reduced the viability of the protozoan Trichomonas vaginalis by 100% at a 0.025% concentration of the extract in culture medium. The aqueous extract of Q. brasiliensis leaves also showed anti-T. vaginalis activity (0.1% inhibited 98% of the microorganism’s growth). Further experiments demonstrated that saponins induced damage to the parasite membrane, suggesting that the extracts acted by inhibiting the viability of T. vaginalis [54]. This antiparasitic effect may be due to the detergent action of the saponins from the genus Quillaja in the cholesterol of the protozoa membranes, resulting in cell lysis. However, the activity may not occur in all protozoa species, since some species may not be susceptible to the toxic effects of saponins or the parasite may adapt to saponin effects [42,50,55]. 3.3. Antitumor Activity of Quillaja Saponins Fractions rich in Quillaja saponins are also described as therapeutic agents for treating and preventing cancerous diseases [56]. One of the first studies to demonstrate the saponins’ antitumor potential was conducted with Quil-A® , where the fraction prolonged the survival of mice with leukemia [57]. Since saponins interact with the cholesterol of cell membranes and, consequently, Molecules 2019, 24, 171 8 of 29 cause membrane damage, cell lysis, and necrosis, they present high toxicity for clinical practice. These toxic effects have been reversed by converting Q. saponaria fractions into stable nanoparticles, binding them to cholesterol. The QS fraction 21 (QS-21) (a more hydrophobic fraction with an acyl chain-ASAP) formulated in killing and growth inhibiting (KGI) particles selectively killed nine of the ten tumor cell lines at a concentration 30 times lower than the concentration required to kill normal monocytes. In addition, the ASAP’s lytic effect on red blood cells was inhibited by the formulation with KGI particles; cell death occurred through the apoptosis mechanism [58]. 3.4. Hepatoprotective Activity of Quillaja Saponins Thought-provokingly, Abdel-Reheim et al. [59] demonstrated that bark saponins from Q. saponaria are interesting hepatoprotective agents. In this study, the authors verified that saponin significantly decreased the elevation of ALT (achieving 57% hepatotoxicity control), AST (66%), ALP (76%), GGT (60%), NOx (77%), TC (70%), MDA (65%), LDH (54%), TG (54%), and total (54%), direct (54%), and indirect (54%) bilirubin, coupled with increased GSH (219%) and albumin (159%) levels. Furthermore, saponin from Quillaja bark significantly decreased liver MDA and NOx production, reinforcing that saponin has antioxidant properties, since it is able to scavenge excessive radicals and thus suppress oxidative stress [60,61]. The hepatoprotective effect of Quillaja bark saponin is probably due to the amelioration of oxidative stress and suppression of NOS expression, maintaining hepatocyte integrity and functioning. The decrease in TG and TC serum levels can be explained by the decrease in intestinal cholesterol absorption and by the reduction in cholesterol levels, accompanied by an increase in the HMG-CoA reductase activity and LDL receptor levels in the liver [62]. In addition, the immunohistochemical results help to explain Quillaja bark saponins’ protective activity, since saponins suppressed NOS expression, attenuating oxidative and nitrosative stress [59]. 3.5. Immunoadjuvant Activity of Quillaja Saponins One remarkable biological activity is their ability to modulate immunity towards a more cell mediated response or to enhance antibody production [18,21,63]. This property is mainly correlated with vaccinology, since saponins can act as immunoadjuvants. Despite both steroid and triterpenoid saponins from different plant species demonstrating immunogenic activity, Quillaja triterpenoid saponins are the most extensively studied [18]. Because of their amphiphilic structure, both aglycone and branched sugar radicals play important roles in stimulating the immune system. In contrast to other saponin structures, Quillaja saponins feature a fatty acid chained to β-D-Fucp residue (C-28) and, at position 4, an aldehyde group, which increases their immunogenic activity leading to a more intense stimulation of the cytotoxic T lymphocytes (CTL) [20,64]. Although the immunogenic mechanism is not yet clearly understood, it is known that they can activate different pathways, including Nod-like Receptors (NLR) and Toll-like Receptors (TLR) [18,20]. Saponins can stimulate antigen-presenting cells (APC) by inducing inflammasome formation [21,65]. The inflammasome is a set of receptors and sensors that induce inflammation by activating caspase-1. Saponins can stimulate the secretion of IL-6 and IL-1β in vitro despite requiring the activation of TLR4, leading to active forms of caspase-1. Also, saponins can stimulate the expression of TLR2 interferon-α leading to active forms of IL-18 and IL-1β [66,67]. Taken together, Marty-Roix et al. (2016) found that saponins can cause an enlargement of the lymph node, indicating cell recruitment or cell proliferation through cytokine signaling [68]. The fatty acid in their structure boosts the cytotoxicity of the CD8 lymphocyte by increasing the interaction of the saponins with the cell membrane through cholesterol, inducing the formation of cell pores and, possibly, signaling to CTLs [18–20,69]. As for the humoral response, saponins can suppress secretion of IL-4, thus lowering IgE levels. On the other hand, they can shift a Th2 response to a Th1 response, inducing the secretion of IgG2a rather than IgG1, indicating a more pronounced Th1 response than Th2 response through changes in cytokine secretion (in this context IL-2 and IFNγ) [20,70–72]. This mechanism may occur in Molecules 2019, 24, 171 9 of 29 two different ways: first, through the deacylated aldehyde that binds to T cell surface receptors (mostly CD2), leading the cell to Th1 immunity; and second, through endocytosis of the saponin with an antigen, disrupting the endosomal membrane of the processed antigen and facilitating the antigen Molecules 2019, 24, x presentation process (Figure 3) [20,69,73]. 9 of 30 Figure 3. The endocytosis of the saponin with an antigen can cause disruption to the endosomal membrane of the processed antigen, facilitating the antigen presentation process by extravasation of the antigen and its transportation to the endoplasmic reticulum with posterior complexation with MHCI. Figure 3. Thedeacylated endocytosis of the saponin with anCD4 antigen can cause to the endosomal Also, saponins can interact with naïve T-cells through CD2disruption receptors, stimulating T membrane the processed antigen, theconsequent antigen presentation by extravasation of cell of activation biased towards a Th1facilitating response with secretion of IL-2process and IFNγ. the antigen and its transportation to the endoplasmic reticulum with posterior complexation with Despite the immunogenic activity, the considerable toxicity restricts its use in humans. MHCI. Also, deacylated saponins can interact with naïve CD4 T-cells through CD2 receptors, An alternative for reducing saponin toxicity and increasing its immunoadjuvancy is to formulate stimulating T cell activation biased towards a Th1structures responsecalled with Immune consequent secretion of IL-2 and it with cholesterol and phospholipids into cage-like Stimulating Complexes IFNγ. (ISCOMs) [74]. These structures were first described in 1971 and shown to enhance antibody production when compared to saponins, mostly because they can be easily transported to lymph nodes due to their nanoscale size (up to 80 nanometers) [75–77]. ISCOMs can modulate immune response in Despite the immunogenic activity, the considerable toxicity restricts its use in humans. An different ways according to the antigen. It is known that some antigens can increase or decrease the alternative for reducing saponin toxicity and increasing its immunoadjuvancy is to formulate it with production of IL-10, pushing the immune response towards a Th2 or Th1 response, respectively [78,79]. cholesterol and phospholipids into cage-like structures called Immune Stimulating Complexes ISCOMs can also stimulate CTLs through cytokine modulation [77]. The immunogenic mechanism (ISCOMs) [74]. These structures first related described in 1971 to enhance is not clearly understood, but it were is probably to saponins, sinceand they shown cause similar changes inantibody production when compared to to saponins, because they[77,78]. can be easily transported to lymph cytokine expression, leading different Tmostly lymphocyte activation In their vaccine systems, saponins be nanometers) used alone or [75–77]. complexed with different molecules immune nodes due to nanoscale size (up can to 80 ISCOMs can modulate oil/water, in different structures (liposomes) in ISCOMs [81,82]). semisynthetic response(i.e., in in different ways according to the antigen.[80], It isorknown that someAlso, antigens can increase or saponins can be used as adjuvants in order to reduce toxicity and maintain their immunoadjuvant decrease the production of IL-10, pushing the immune response towards a Th2 or Th1 response, properties [83]. Considering their effects when used as adjuvants in vaccines, they are promising respectively [78,79]. ISCOMs can also stimulate CTLs through cytokine modulation [77]. The candidates and have featured in plenty of pre-clinical studies for vaccines, as mentioned in Table 2. immunogenic mechanism not clearly understood, but it is probably related to saponins, since they The studies reported inisTable 2 are arranged by year of publication. cause similar changes in cytokine expression, leading to different T lymphocyte activation [77,78]. In vaccine systems, saponins can be used alone or complexed with different molecules (i.e., in oil/water, in different structures (liposomes) [80], or in ISCOMs [81,82]). Also, semisynthetic saponins can be used as adjuvants in order to reduce toxicity and maintain their immunoadjuvant properties [83]. Considering their effects when used as adjuvants in vaccines, they are promising candidates and have featured in plenty of pre-clinical studies for vaccines, as mentioned in Table 2. The studies reported in Table 2 are arranged by year of publication. Molecules 2019, 24, 171 10 of 29 Table 2. Pre-clinical studies using Quillaja saponins as an adjuvant in vaccine formulations targeting different antigens. Antigen Adjuvant PorA P1.6 (membrane porin of Neisseria meningitidis) Quil-A® S. bovis and Lactobacillus spp. Quil-A® Model Findings Ref. Mice PorA supplemented with Quil-A® resulted in four to seven times higher antibody titers (IgG1, IgG2a, and IgG2b) when compared with lipopolysaccharide as an adjuvant. [84] Cattle Immunization of feedlot steers against S bovis and Lactobacillus spp with vaccines incorporating Freund adjuvant, Quil-A® , dextran, or alum as an adjuvant effectively induced high IgG concentrations when dextran was used as an adjuvant. [85] [86] 38-kDa mycobacterial protein ISCOMs Mice An increase in antibody titers was observed after a booster injection, with significant levels of IgG2a. The ISCOM formulations induced this in the same way. Regarding the induction of CTL responses, the differences shown comparing various ISCOMS formulations were minimal. Tetanus toxoid QS-21 Mice Administration of QS-21 p.o. as an adjuvant elicits strong serum IgM and IgG Ab responses. [87] Babesian equi immunogen Quil-A® Donkeys A mixture of Quil A® and Babesia equi immunogen was optimal in generating a significant immune response and reducing the lethality. [88] OVA QS-21 and deacylated QS-21 (DS-1) Mice Despite DS-1 requiring a higher dose to induce IgG1 responses, it did not induce IgG2a or CTL responses. Lower doses of QS-21 induced higher IgG titers, including IgG2a and CTL responses. [89] FML * Quil-A® Dogs The formulation protected dogs against canine kala-azar in the field with robust immunogenicity. [90] Measles virus ISCOMs QA-22 Rats and Macaques Vaccines induced high levels of antibodies, which showed no decrease during a 2-year follow up. [91] Herpes virus Quillaja saponins Mice Saponins’ deacylation significantly reduced antibody production and increased mortality rates during viral challenge test. [92] Opa J from Neisseria meningitidis Quil-A® Mice Quil-A® formulated with OpaJ was highly immunogenic, inducing the production of Opa-specific antibodies. [93] Non-fimbrial adhesin hemagglutinin B (HagB) Semi-synthetic saponin of Quillaja GPI-0100 Mice GPI-0100 showed better immunoadjuvancy than monophosphoryl lipid A and alum, as a mucosal and systemic adjuvant, in inducing serum anti-HagB. [94] FML * Quil-A® Dogs The formulation was shown be effective in immunotherapy against visceral leishmaniasis of asymptomatic infected dogs. All animals showed significantly increased CD8 lymphocyte percentages. [95] Molecules 2019, 24, 171 11 of 29 Table 2. Cont. Antigen Adjuvant Model Findings Ref. Human respiratory syncytial virus (HRSV) HRSV-ISCOMs prepared with Quillaja saponin fractions: QH-A; QH-C; QH-A + QH-C (QH-AC) Mice In general, the ISCOMs tested were well tolerated. However, the combination of QH-A + QH-C ISCOMs was lethal in neonates despite the QH-A or QH-C fractions alone being well tolerated. [96] FML * Quillaja saponaria sapogenins containing aldehyde Mice Vaccines elicited high levels of antibodies and cellular specific response to FML and IFNγ sera levels and protection against L. donovani murine infection was shown. [97] [98] Ovalbumin (OVA) Quil-A® Mice Increasing amounts of Quil-A® (20% to 70%) were tested in liposomes. Higher doses of Quil A reduced the particle sizes formed, thus decreasing antigen incorporation and uptake by DC. Liposomes containing 20% Quil-A® were more effective as immunostimulants, and more toxic in cell cultures too, when compared with those containing 70% Quil-A® . Mtb72F (Mycobacterium tuberculosis) AS02A Mice Increase in both Th1 and Th2 response, with Th1 response being more pronounced. [99] LSP PbCS 242–310 ** QS-21 Mice The use of LSP PbCS 242–310 combined with QS-21 induced a satisfactory immune response similar to the one generated with the injection of radiation-attenuated sporozoites. [100] HRSV ISCOMs formulated with Quillaja saponin fractions Mice All three formulations favor Th1 responses to different degrees with IFN-c being produced up to 50 times more than Il-4 and IL-5. The HRSV 703 ISCOMs induced the most pronounced innate and acquired response with the most prominent Th1 profile. [101] HIV-1 DNA prime/protein–Env and Gag *** QS-21 Rhesus macaques and mice Cellular and humoral responses were observed when Polyvalent DNA prime/protein boost vaccine was administered. CD8+ CTL, CD4+ T-helper cells and Th1 cytokines were involved in cellular immune response. [102] FML * Fractions of the Riedel de Haen saponin mixture (QS-21 saponin fraction; two deacylsaponins mixture; a mixture of glucose, rutin, quercetin, and sucrose). Mice QS-21 and the deacylsaponins induced the most significant reduction of parasite burden in the liver, demonstrating a promising adjuvant potential in the Riedel de Haen saponin mixture, which contains deacylated saponins that are not toxic and induce robust immunity. [103] Plant-made measles virus hemagglutinin (MV-H) protein Cholera toxin (CTB/CT); LT(R192G) ****; Saponin extracted from Quillaja bark (Sigma; S-4521) Mice Despite both LT(R192G) and the crude saponins showing strong adjuvant activity, the crude extract had superior immunostimulatory properties. [104] Molecules 2019, 24, 171 12 of 29 Table 2. Cont. Antigen Molecules 2019, 24, x Adjuvant Model Findings Ref. 12 of 30 Mice immunized with ADAD system with Qs had a survival rate of Fh15 (recombinant 15 kDa 50–62.5% while animals without Qs had a survival rate of 40–50%. ADAD System ***** Mice and sheep [105] Fasciola hepatica protein) Sheep immunized with ADAD system with Qs showed less hepatic Table 2. Cont. damage compared to control group. Fh15 (recombinant 15 Mice immunized with ADAD system with Qs had a survival rate of 50–62.5% while and Molecules Molecules2019, 2019, Molecules Molecules Molecules 24, 24,xx 2019, 2019, 2019, 24, 24, 24, xxMice x 12 12ofof30 30 12 1 Despite nonspecific reactions being observed in the immunized kDa Fasciola hepatica ADAD System ***** animals without Qs had a survival rate of 40–50%. Sheep immunized with ADAD [105] FML * QS-21 Dogs animals (similar to other veterinary vaccines), a significant decrease [106] sheep protein) system with Qs showed less hepatic damage to control group. was showncompared with subsequent doses. Table Table2.2. Cont. Table Table Table 2.2.2. Cont. Cont. Cont. animals (similar to other Despite nonspecific reactions being observed inCont. the immunized FML * QS-21 Dogs Mice vaccinated with a combination of plant-made LTB-ESAT-6[106] veterinary vaccines), a significant decrease was shown with subsequent doses. Fh15 Fh15(recombinant (recombinant Fh15 Fh15 Fh15 (recombinant (recombinant (recombinant 15 15 15 15 15 Mice Mice immunized immunized Mice Mice Mice immunized immunized with immunized with ADAD ADAD with with with system system ADAD ADAD ADAD with with system system system Qs Qs had had with with with aasurvival survival Qs Qs Qs had had had rate arate asurvival asurvival survival ofof50–62.5% 50–62.5% rate rate rate ofofof while 50–62.5% while 50–62.5% 50–62.5% while while while fusion BCG oral adjuvant had significantly more IL-10 production Miceaand and Mice Mice Mice and and and Mice vaccinated Mice with combination of plant-made LTB-ESAT-6 fusion BCG oral kDa kDaFasciola Fasciola kDa kDa kDa hepatica hepatica Fasciola Fasciola Fasciola hepatica hepatica hepatica ADAD ADADSystem System ADAD ADAD ADAD ***** ***** System System System ***** ***** ***** when compared animals animals without without animals animals animals Qs Qs without without had without hadaasurvival survival Qs Qs Qs had had had arate arate survival asurvival survival ofof40–50%. 40–50%. rate rate rate of Sheep of Sheep of 40–50%. 40–50%. 40–50%. immunized immunized Sheep Sheep Sheep immunized immunized with immunized withADAD ADAD with with with ADAD ADAD ADAD [105] [105] LTB-ESAT-6 ;; and with mice vaccinated without adjuvant. However, LTB-ESAT-6 and Bacillus Quillaja extract [107] sheep sheep sheep sheep sheep adjuvantMice had significantly more production when during compared with mice test vaccinated Calmette-Guérin noIL-10 protection was shown the challenge with M. protein) protein) protein) Mice system system with with system system system Qs Qsshowed showed with with with Qs Qs less Qs less showed showed showed hepatic hepatic less less damage less damage hepatic hepatic hepatic compared compared damage damage damage to compared to compared control controlgroup. to group. toto control control control group. group. group. Bacillus Calmette- (BCG) Quillajaprotein) extract protein) [107] compared without adjuvant. However,tuberculosis, no protectioninwas shown during the challenge test with M. groups treated withbeing oral BCG (with or without Despite Despitenonspecific nonspecific Despite Despite Despite nonspecific nonspecific reactions nonspecific reactions reactions being reactions reactions observed observed being being being in observed in observed the observed the immunized immunized ininin the the the immunized immunized animals immunized animals(similar (similar animals animals animals toto(similar other (similar other (similar tototo other other other Guérin (BCG) FML FML** FML FML FML *** QS-21 QS-21 tuberculosis, QS-21 QS-21 QS-21 Dogs Dogs Dogs Dogswith oral BCG (with [106] [106] inDogs groups treated or without saponin). saponin). veterinary veterinaryveterinary vaccines), veterinary vaccines), veterinary avaccines), avaccines), significant vaccines), significant aasignificant adecrease significant decrease significant was was decrease decrease decrease shown shown was was with was with shown shown shown subsequent subsequent with with with subsequent subsequent doses. subsequent doses. doses. doses. doses. GD3-KLH GPI-0100 ; sQS-21 Mice Both synthetic Quillaja molecules produced antibodies in mice.antibodies [108] Molecules Molecules Molecules Molecules 2019, 2019, 2019, 2019, 24, 24, 24, 24, xxxx 12 12 12 12 of ofof30 of30 30 30 LTB-ESAT-6 Bothsaponin synthetic Quillaja saponin molecules produced in Mice Mice vaccinated vaccinated Mice Mice Mice vaccinated with vaccinated with vaccinated aacombination combination with with with aacombination acombination combination ofofplant-made plant-made ofofof plant-made plant-made LTB-ESAT-6 plant-made LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 fusion fusionBCG BCG fusion fusion oral fusion oral BCG BCG BCG oral oral oral Molecules Molecules 2019, 2019, 24, 24,xx ;;; 12 12ofof3 GD3-KLH GPI-0100 sQS-21 [108] LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 ;;;and and and and and FALVAC-1A (different QS-21 Mice was the second adjuvant that induced the most significant levels of antibodies, mice. adjuvant adjuvant had had adjuvant adjuvant adjuvant significantly significantly had had had significantly significantly significantly more more IL-10 IL-10 more more production more production IL-10 IL-10 IL-10 production production when production when compared compared when when when compared with compared with compared mice micewith vaccinated with vaccinated with mice mice mice vaccinated vaccinated vaccinated AIPO4, QS-21, Montanide Bacillus BacillusCalmetteCalmetteBacillus Bacillus Bacillus CalmetteCalmetteCalmetteQuillaja extract extract Quillaja Quillaja Quillaja extract extract extract Mice Mice Mice Mice Micealso induced the highest levels of IL-4 compared [107] [107] epitopes from MiceQuillaja inducing predominantly IgG2c. QS-21 [109] without adjuvant. adjuvant. without without without However, adjuvant. However, adjuvant. adjuvant. However, no However, However, protection protection no no no protection was protection was protection shown shown was was during was during shown shown shown the theduring challenge during challenge during the the the test challenge test challenge challenge with withM. M. test test test with with with M. M. M. ISA-720, or CRL-1005 QS-21 without was the second adjuvant thatno induced the most significant Table Table Table Table 2.2.2. Cont. 2. Cont. Cont. Cont. Guérin Guérin(BCG) (BCG) Guérin Guérin Guérin (BCG) (BCG) (BCG) FALVAC-1A (different Plasmodium falciparum) to the other adjuvants, indicating and Th2 responses. Table TableTh1 2.2.tuberculosis, Cont. Cont. tuberculosis, tuberculosis, tuberculosis, tuberculosis, iningroups groupstreated in treated inin groups groups groups with with treated treated treated oral oral BCG with BCG with with (with oral (with oral oral BCG or BCG or BCG without without (with (with (with or saponin). or saponin). or without without without saponin). saponin). saponin). AIPO4, QS-21, Montanide ISA-720, or levels of antibodies, inducing predominantly IgG2c. QS-21 also epitopes from Plasmodium Mice [109] The vaccine induced robust immunogenicity with high levels ofrate FML-seroconversion, as Fh15 Fh15 Fh15 Fh15 (recombinant (recombinant (recombinant (recombinant 15 15 15 15 Mice Mice Mice Mice immunized immunized immunized immunized with with with with ADAD ADAD ADAD ADAD system system system system with with with Qs Qs Qs Qs had had had had aaBoth survival aBoth survival aBoth survival survival rate rate rate ofof of 50–62.5% of 50–62.5% 50–62.5% 50–62.5% while while while while CRL-1005 induced the with highest levels of IL-4 compared to the other adjuvants, GD3-KLH GD3-KLH GD3-KLH GD3-KLH GD3-KLH GPI-0100 GPI-0100 GPI-0100 ; GPI-0100 GPI-0100 sQS-21 sQS-21 ; ; ; sQS-21 sQS-21 sQS-21 Mice Mice Mice Mice Mice Both Both synthetic synthetic Quillaja synthetic Quillaja synthetic synthetic saponin saponin Quillaja Quillaja Quillaja molecules molecules saponin saponin saponin molecules produced molecules produced molecules antibodies produced antibodies produced produced antibodies in antibodies in antibodies mice. mice. ininin mice. mice. mice. [108] [108] FML * QS-21 Dogs [110] Fh15 Fh15 (recombinant (recombinant 15 15 Mice Mice immunized immunized with withADAD ADAD system system with with Qs Qs had had aasurvival survival rate rate of of 50–62.5% 50–62.5% while while Mice Mice Mice and and and and ; Molecules Molecules Molecules Molecules Molecules 2019, 2019, 2019, 2019, 2019, 24, 24, 24, 24, x24, xxxxMice 12 12 12 12 of12 ofo3o falciparum) demonstrated by CD8+ and CD4+ T cell populations. Mice and and kDa kDa kDa kDa Fasciola Fasciola Fasciola Fasciola hepatica hepatica hepatica hepatica ADAD ADAD ADAD ADAD System System System System ***** ***** ***** ***** animals animals animals animals without without without without Qs Qs Qs Qs had had had had aMice asurvival asurvival asurvival survival rate rate rate rate of of of 40–50%. of 40–50%. 40–50%. 40–50%. Sheep Sheep Sheep Sheep immunized immunized immunized immunized with with with with ADAD ADAD ADAD ADAD [105] [105] [105] [105] indicating Th1 and Th2 responses. FALVAC-1A FALVAC-1A FALVAC-1A FALVAC-1A FALVAC-1A (different (different (different (different (different QS-21 QS-21 was was QS-21 the QS-21 the QS-21 second second was was was the adjuvant the adjuvant the second second second that that adjuvant adjuvant adjuvant induced induced that that that the the induced induced induced most most significant significant the the the most most most significant levels significant levels significant ofofantibodies, antibodies, levels levels levels of of of antibodies, antibodies, antibodies, kDa kDa Fasciola Fasciola hepatica hepatica ADAD ADAD System System ***** ***** animals animals without without Qs Qs had had a a survival survival rate rate of of 40–50%. 40–50%. Sheep Sheep immunized immunized with with ADAD ADAD [105] [105 sheep sheep sheep sheep AIPO4, AIPO4,QS-21, QS-21, AIPO4, AIPO4, AIPO4, Montanide Montanide QS-21, QS-21, QS-21, Montanide Montanide Montanide AD-472 or HSV-2 Guinea Both formulations reduced clinical disease; however, GPI-0100 improved the sheep sheep protein) protein) protein) protein) system system system system with with with with Qs Qs Qs Qs showed showed showed showed less less less hepatic hepatic hepatic hepatic damage damage damage damage compared compared compared compared tototo control to control control control group. group. group. group. epitopes epitopes from from epitopes epitopes epitopes from from from Mice Mice Mice Mice inducing Mice inducing predominantly inducing predominantly inducing inducing predominantly predominantly predominantly IgG2c. IgG2c. QS-21 QS-21 IgG2c. IgG2c. IgG2c. also also QS-21 induced QS-21 induced QS-21 also also also the the induced induced induced highest highest the the levels the levels highest highest highest ofof IL-4 IL-4 levels levels levels compared compared ofofof IL-4 IL-4 IL-4 compared compared compared [109] [109] Theless vaccine induced robust immunogenicity with high levels of GPI-0100 protein) [111] protein) system system with with Qs Qsshowed showed less less hepatic hepatic damage damage compared compared toto control control group. group. ISA-720, ISA-720, or or ISA-720, ISA-720, CRL-1005 ISA-720, CRL-1005 or or or CRL-1005 CRL-1005 CRL-1005 Table Table Table Table Table 2. 2. 2. Cont. 2. Cont. 2. Cont. Cont. Cont. animals glycoprotein D pigs glycoprotein-D formulation only. Despite Despite Despite Despite nonspecific nonspecific nonspecific nonspecific reactions reactions reactions reactions being being being being observed observed observed observed in in in the in the the the immunized immunized immunized immunized animals animals animals (similar (similar (similar (similar to to to other to other other other FML * QS-21 Dogs FML-seroconversion, as demonstrated by CD8+ and CD4+ T cell [110] Plasmodium Plasmodium Plasmodium Plasmodium falciparum) Plasmodium falciparum) falciparum) falciparum) falciparum) to to the the other other to to adjuvants, to adjuvants, the the the other other other adjuvants, indicating adjuvants, indicating adjuvants, indicating Th1 indicating Th1 indicating and and Th2 Th2 Th1 Th1 Th1 responses. responses. and and and Th2 Th2 Th2 responses. responses. responses. Despite Despitenonspecific nonspecificreactions reactionsbeing beingobserved observedininthe the immunized immunized animals animals(similar (similartotoother other FML FML FML FML **** QS-21 QS-21 QS-21 QS-21 Dogs Dogs Dogs Dogs [106] [106] [106] [106] MATRIX-M shifted athe immune response to anshown IgG2a response (towards adoses. Th1 FML FML** QS-21 QS-21 Dogs DogsThe [106] [106 veterinary veterinary veterinary veterinary vaccines), vaccines), vaccines), vaccines), asignificant asignificant asignificant significant decrease decrease decrease was was was was shown shown shown with with with with subsequent subsequent subsequent subsequent doses. doses. doses. populations. Thedecrease vaccine vaccine The The The induced induced vaccine vaccine vaccine robust robust induced induced induced immunogenicity immunogenicity robust immunogenicity immunogenicity immunogenicity with high high levels with levels with with high high FML-seroconversion, FML-seroconversion, levels levels levels of of of FML-seroconversion, FML-seroconversion, FML-seroconversion, as as as as Fh15 Fh15 Fh15 Fh15 Fh15 (recombinant (recombinant (recombinant (recombinant (recombinant 15 15 15 15 15 Mice Mice Mice Mice Mice immunized immunized immunized immunized immunized with with with with with ADAD ADAD ADAD ADAD ADAD system system system system system with with with with with Qs Qs Qs Qs had Qs had had had had ashown asurvival ahigh survival aof survival aof survival survival rate rate rate rate rate of of of 50–62.5% of 50–62.5% of 50–62.5% 50–62.5% 50–62.5% while while while while while as Aluminium phosphate; veterinary veterinary vaccines), vaccines), arobust arobust significant significant decrease decrease was was shown with with subsequent subsequent doses. doses. + FML FML** FML FML FML *** QS-21 QS-21 QS-21 QS-21 QS-21 Dogs Dogs Dogs Dogs Dogs [110] [110] Mice Mice Mice and and and and and response). On the other hand, the CD8 T-cell response could be improved using Mice Mice Mice Mice vaccinated vaccinated vaccinated vaccinated with with with with aacombination aMice combination aMice combination combination ofofof plant-made of plant-made plant-made plant-made LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 fusion fusion fusion fusion BCG BCG BCG BCG oral oral oral oral demonstrated demonstrated demonstrated demonstrated demonstrated by by and and by by by CD8+ CD4+ CD8+ CD4+ CD8+ and Tof and Tand cell cell CD4+ CD4+ populations. CD4+ populations. T T T cell cell cell populations. populations. populations. kDa kDa kDa kDa kDa Fasciola Fasciola Fasciola Fasciola Fasciola hepatica hepatica hepatica hepatica hepatica ADAD ADAD ADAD ADAD System System System System System ***** ***** ***** ***** ***** animals animals animals animals animals without without without without without Qs Qs Qs Qs had Qs had had acombination aCD8+ survival aCD8+ survival asurvival ahowever, survival survival rate rate rate rate rate of of of 40–50%. of 40–50%. 40–50%. 40–50%. 40–50%. Sheep Sheep Sheep Sheep Sheep immunized immunized immunized immunized immunized with with with with with ADAD ADAD ADAD ADAD ADAD [105] [105 [10 [1[1 AD-472 or HSV-2 aluminium hydroxide; Both formulations reduced clinical disease; GPI-0100 H9N2 MF59 Mice ADAD [112] Mice Mice vaccinated vaccinated with with ahad ahad combination of of plant-made plant-made LTB-ESAT-6 LTB-ESAT-6 fusion fusion BCG BCG oral oral LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 ;;; and ; and and and GPI-0100# ;;and Guinea pigs [111] sheep sheep sheep sheep sheep MATRIX-M or MF59. Among all tested molecules, MATRIX-M was themice most effective in LTB-ESAT-6 LTB-ESAT-6 and adjuvant adjuvant adjuvant adjuvant had had had had significantly significantly significantly significantly more more more more IL-10 IL-10 IL-10 IL-10 production production production production when when when when compared compared compared compared with with with with mice mice mice vaccinated vaccinated vaccinated vaccinated glycoprotein improved the glycoprotein-D formulation only. AD-472 AD-472 AD-472 AD-472 AD-472 or or HSV-2 HSV-2 or or or HSV-2 HSV-2 HSV-2 Guinea Guinea Guinea Guinea Guinea Both Both formulations formulations Both Both Both formulations formulations formulations reduced reduced clinical clinical reduced reduced reduced disease; disease; clinical clinical clinical however, however, disease; disease; disease; GPI-0100 however, GPI-0100 however, however, improved GPI-0100 improved GPI-0100 GPI-0100 the improved the improved improved the the the protein) protein) protein) protein) protein) system system system system system with with with with with Qs Qs Qs Qs showed Qs showed showed showed showed less less less less less hepatic hepatic hepatic hepatic hepatic damage damage damage damage damage compared compared compared compared compared to to to control to control to control control control group. group. group. group. group. ; andextract MATRIX-M adjuvant adjuvant had had significantly significantly more more IL-10 IL-10 production production when when compared compared with with mice mice vaccinated vaccinated Bacillus Bacillus Bacillus Bacillus CalmetteCalmetteCalmetteCalmette-D Quillaja Quillaja Quillaja Quillaja extract extract extract Mice Mice Mice Mice GPI-0100 [107] [107] [107] [107] GPI-0100 GPI-0100 GPI-0100 GPI-0100 [111] [111][107] inducing the immune response, followed by MF59 and aluminium-based adjuvants. Bacillus Bacillus CalmetteCalmetteQuillaja Quillaja extract extract Mice Mice [107 without without without without adjuvant. adjuvant. adjuvant. adjuvant. However, However, However, However, no no no protection protection protection protection was was was was shown shown shown shown during during during during the the the the challenge challenge challenge challenge test test test test with with with with M. M. M. M. glycoprotein glycoprotein glycoprotein glycoprotein glycoprotein D D DDD pigs pigsno pigs pigs pigsDespite glycoprotein-D glycoprotein-D glycoprotein-D glycoprotein-D glycoprotein-D formulation formulation formulation formulation formulation only. only. only. only. only. Despite Despite Despite Despite nonspecific nonspecific nonspecific nonspecific nonspecific reactions reactions reactions reactions reactions being being being being being observed observed observed observed observed ininin the in the in the the immunized the immunized immunized immunized immunized animals animals animals animals animals (similar (similar (similar (similar (similar to toto other to other to other other other without without adjuvant. adjuvant. However, However, no no protection protection was was shown shown during during the the challenge challenge test testwith with M. M. Guérin Guérin Guérin Guérin (BCG) (BCG) (BCG) (BCG) MATRIX-M shifted the immune response to an IgG2a response TM FML FML FML FML FML **;*** QS-21 QS-21 QS-21 QS-21 QS-21 Dogs Dogs Dogs Dogs Dogs [106] [106 [10 [1[1 ISCOMATRIXTMGuérin ; Guérin ISCOM ISCOMATRIXTM and ISCOMTM vaccines, using two different antigens and different (BCG) (BCG) tuberculosis, tuberculosis, tuberculosis, tuberculosis, in inin groups in groups groups groups treated treated treated treated with with with with oral oral oral oral BCG BCG BCG BCG (with (with (with (with or or or without or without without without saponin). saponin). saponin). saponin). +an MATRIX-M MATRIX-M MATRIX-M shifted shifted the the shifted immune shifted immune shifted the response response immune immune immune to to response response response an IgG2a IgG2a to to response to an response an an IgG2a IgG2a IgG2a (towards (towards response response response a(towards aTh1 (towards Th1 (towards aaTh1 aTh1 Th1 veterinary veterinary veterinary veterinary veterinary vaccines), vaccines), vaccines), vaccines), vaccines), aasignificant asignificant athe significant athe significant significant decrease decrease decrease decrease decrease was was was was was shown shown shown shown shown with with with with with subsequent subsequent subsequent subsequent subsequent doses. doses. doses. doses. doses. tuberculosis, tuberculosis, in in groups groups treated treated with oral oral BCG BCG (with (with or or without without saponin). saponin). (towards MATRIX-M aMATRIX-M Th1 response). On the other hand, the with CD8 T-cell Aluminium Aluminium Aluminium Aluminium phosphate; Aluminium phosphate; phosphate; phosphate; phosphate; HpaA and catalase Cholera toxin; and aluminium Mice delivery systems (intranasal or subcutaneous), have the same efficacy in reducing H. + + + [108] [113] Aluminium phosphate; GD3-KLH GD3-KLH GD3-KLH GD3-KLH GPI-0100 GPI-0100 GPI-0100 GPI-0100 ; ; ; ; sQS-21 sQS-21 sQS-21 sQS-21 aluminium Mice Mice Mice Both Both Both Both synthetic synthetic synthetic synthetic Quillaja Quillaja Quillaja Quillaja saponin saponin saponin saponin molecules molecules molecules molecules produced produced produced produced antibodies antibodies antibodies antibodies in in mice. in mice. mice. mice. [108] [108] [108] + + response). response). response). On response). On response). the the other other On On On hand, hand, the the the the the CD8 hand, CD8 hand, hand, T-cell T-cell the the the CD8 CD8 response CD8 response T-cell T-cell T-cell could could response response response be be improved improved could could could be be be using improved using improved improved using using using [108] response could beMice improved using MATRIX-M or MF59. Among all Mice Mice Mice Mice vaccinated vaccinated vaccinated vaccinated vaccinated with with with with with aaother combination aother combination aother combination acombination combination of ofof plant-made of plant-made of plant-made plant-made plant-made LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 fusion fusion fusion fusion fusion BCG BCG BCG BCG BCG oral oral oral oral oral GD3-KLH GD3-KLH GPI-0100 GPI-0100 ; ;Mice sQS-21 sQS-21 MF59 Mice MiceMice Both Both synthetic synthetic Quillaja Quillaja saponin saponin molecules molecules produced produced antibodies antibodies in in mice. mice. [112] H9N2 hydroxide; MF59 ;and and H9N2 H9N2 H9N2 H9N2 H9N2 aluminium aluminium aluminium aluminium hydroxide; aluminium hydroxide; hydroxide; hydroxide; MF59 hydroxide; MF59 MF59 MF59 Mice Mice Mice Mice [112] [112] [108 hydroxide pylori colonization as that the gold standard cholera toxin (CT) adjuvant LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 ; ;; and ; and ; and FALVAC-1A FALVAC-1A FALVAC-1A FALVAC-1A (different (different (different (different QS-21 QS-21 QS-21 QS-21 was was was was the the the the second second second second adjuvant adjuvant adjuvant adjuvant that that that induced induced induced induced the the the the most most most most significant significant significant significant levels levels levels levels ofofof antibodies, of antibodies, antibodies, antibodies, tested molecules, MATRIX-M was the most effective in inducing the MATRIX-M MATRIX-M MATRIX-M MATRIX-M MATRIX-M or or MF59. MF59. Among Among or or or MF59. MF59. MF59. all all Among tested Among tested Among molecules, all molecules, all all tested tested tested molecules, MATRIX-M molecules, MATRIX-M molecules, MATRIX-M MATRIX-M MATRIX-M was was the themost most was was was effective effective the the the most most most in in effective effective effective ininin adjuvant adjuvant adjuvant adjuvant adjuvant had had had had had significantly significantly significantly significantly significantly more more more more more IL-10 IL-10 IL-10 IL-10 IL-10 production production production production production when when when when when compared compared compared compared compared with with with with with mice mice mice mice mice vaccinated vaccinated vaccinated vaccinated vaccinated # FALVAC-1A FALVAC-1A (different (different QS-21 QS-21 was was the the second second adjuvant adjuvant that that induced induced the the most most significant significant levels levels of of antibodies, antibodies, AIPO4, AIPO4, AIPO4, AIPO4, QS-21, QS-21, QS-21, QS-21, Montanide Montanide Montanide Montanide # # # # # MATRIX-M ; ; and and ; ; MATRIX-M ; MATRIX-M and and and MATRIX-M MATRIX-M MATRIX-M Bacillus Bacillus Bacillus Bacillus Bacillus CalmetteCalmetteCalmetteCalmetteCalmetteQuillaja Quillaja Quillaja Quillaja Quillaja extract extract extract extract extract Mice Mice Mice Mice Mice [107] [107 [10 [1[1 Prophylactic vaccination with adjuvanted TA-CIN protected the mice from viral AIPO4, QS-21, QS-21, Montanide Montanide epitopes epitopes epitopes epitopes from from from from Mice Mice Mice MiceAIPO4, inducing inducing inducing inducing predominantly predominantly predominantly predominantly IgG2c. IgG2c. IgG2c. IgG2c. QS-21 QS-21 QS-21 QS-21 also also also also induced induced induced induced the the the the highest highest highest highest levels levels levels levels of of of IL-4 of IL-4 IL-4 IL-4 compared compared compared compared [109] [109] [109] [109] immune response, followed by MF59 and aluminium-based inducing inducing the inducing the inducing inducing immune immune the the the response, response, immune immune immune followed response, followed response, response, by by followed followed followed MF59 MF59 and and by by by MF59 aluminium-based MF59 aluminium-based MF59 and and and aluminium-based aluminium-based aluminium-based adjuvants. adjuvants. adjuvants. adjuvants. adjuvants. without without without without without adjuvant. adjuvant. adjuvant. adjuvant. adjuvant. However, However, However, However, However, no no no no protection no protection protection protection protection was was was was was shown shown shown shown shown during during during during during the the the the challenge the challenge challenge challenge challenge test test test test test with with with with with M. M. M. M. M.[109] epitopes epitopesfrom from Mice Mice inducing inducing predominantly predominantly IgG2c. IgG2c. QS-21 QS-21 also also induced induced the the highest highest levels levels of ofIL-4 IL-4 compared compared [109 ISA-720, ISA-720, ISA-720, ISA-720, or or or CRL-1005 or CRL-1005 CRL-1005 CRL-1005 Guérin Guérin Guérin Guérin Guérin (BCG) (BCG) (BCG) (BCG) (BCG) Mice and challenge, whereas vaccination without adjuvant was almost ineffective. Moreover, GPIISA-720, ISA-720, or or CRL-1005 CRL-1005 Plasmodium Plasmodium Plasmodium Plasmodium falciparum) falciparum) falciparum) falciparum) to to to the to the the the other other other other adjuvants, adjuvants, adjuvants, adjuvants, indicating indicating indicating indicating Th1 Th1 Th1 Th1 and and and and Th2 Th2 Th2 Th2 responses. responses. responses. responses. adjuvants. TM TM TM TM TM TM TM TM TM TM ISCOMATRIX ISCOMATRIX ISCOMATRIX ISCOMATRIX ISCOMATRIX ; ;ISCOM ISCOM ;; ;ISCOM ; ;ISCOM ISCOM ;;; ISCOMATRIXTM ISCOMATRIXTM ISCOMATRIXTM ISCOMATRIXTM ISCOMATRIXTM and and ISCOMTM ISCOMTM and and and ISCOMTM ISCOMTM vaccines, ISCOMTM vaccines, using vaccines, using vaccines, vaccines, two two using different using different using two two two antigens antigens different different different and and antigens antigens antigens different different and and and different different different tuberculosis, tuberculosis, tuberculosis, tuberculosis, tuberculosis, in in in groups in groups in groups groups groups treated treated treated treated treated with with with with with oral oral oral oral oral BCG BCG BCG BCG BCG (with (with (with (with (with or or or without or without or without without without saponin). saponin). saponin). saponin). saponin). TA-CIN ## GPI-0100 [114] Plasmodium Plasmodium falciparum) falciparum) totothe the other other adjuvants, adjuvants, indicating indicating Th1 Th1 and and Th2 Th2 responses. responses. monkeys The 0100 boosted IFN-γrobust secreting CD8+ T cell response in mice compared to the formulation The The The vaccine vaccine vaccine vaccine induced induced induced induced robust robust robust immunogenicity immunogenicity immunogenicity immunogenicity with with with with high high high high levels levels levels levels ofofof FML-seroconversion, of FML-seroconversion, FML-seroconversion, FML-seroconversion, as as as as HpaA HpaA and and HpaA catalase HpaA catalase HpaA and and and catalase catalase catalase Cholera Cholera toxin; toxin; Cholera Cholera Cholera and and toxin; aluminium toxin; aluminium toxin; and and and aluminium aluminium aluminium Mice Mice Mice Mice delivery delivery systems systems delivery delivery delivery (intranasal (intranasal systems systems systems (intranasal or (intranasal or (intranasal subcutaneous), subcutaneous), or or or subcutaneous), subcutaneous), subcutaneous), have have the the same same have have have efficacy efficacy the the the same same same ininefficacy reducing efficacy reducing efficacy in in H. in H. reducing reducing reducing [113] [113] H. H. ISCOMATRIXTM and ISCOMTM vaccines, using two different GD3-KLH GD3-KLH GD3-KLH GD3-KLH GD3-KLH GPI-0100 GPI-0100 GPI-0100 GPI-0100 ; ; ; ; sQS-21 ; sQS-21 sQS-21 sQS-21 sQS-21 Mice Mice Mice Mice Mice Mice Both Both Both Both Both synthetic synthetic synthetic synthetic synthetic Quillaja Quillaja Quillaja Quillaja Quillaja saponin saponin saponin saponin saponin molecules molecules molecules molecules molecules produced produced produced produced produced antibodies antibodies antibodies antibodies antibodies ininin mice. in mice. in mice. mice. mice. [108] [108 [10 [1[1 The Thevaccine vaccine induced induced robust robust immunogenicity immunogenicity with with high high levels levels of of FML-seroconversion, FML-seroconversion, as asH. FML FML FML FML **** QS-21 QS-21 QS-21 QS-21 Dogs Dogs Dogs DogsGPI-0100 [110] [110] [110] [110] without adjuvant. Vaccination of different macaques induced specific T cell responses. TM FML FML ** TM ; Cholera hydroxide QS-21 QS-21 Dogs Dogs [110] [110 demonstrated demonstrated demonstrated demonstrated by by by by CD8+ CD8+ CD8+ CD8+ and and and and CD4+ CD4+ CD4+ CD4+ Tcolonization TT cell T cell cell cell populations. populations. populations. populations. antigens and delivery systems (intranasal or subcutaneous), ISCOMATRIX ; ISCOM hydroxide hydroxide hydroxide hydroxide pylori pylori colonization pylori pylori pylori colonization colonization colonization as as the the gold gold as as standard as standard the the the gold gold gold cholera cholera standard standard standard toxin toxin cholera cholera cholera (CT) (CT) toxin adjuvant toxin adjuvant toxin (CT) (CT) (CT) adjuvant adjuvant adjuvant FALVAC-1A FALVAC-1A FALVAC-1A FALVAC-1A FALVAC-1A (different (different (different (different (different QS-21 QS-21 QS-21 QS-21 QS-21 was was was was was the the the the second the second second second second adjuvant adjuvant adjuvant adjuvant adjuvant that that that that that induced induced induced induced induced the the the the most the most most most most significant significant significant significant significant levels levels levels levels levels of of of antibodies, of antibodies, of antibodies, antibodies, antibodies, demonstrated demonstrated by by CD8+ CD8+ and and CD4+ CD4+ T T cell cell populations. populations. HpaA and catalase Mice [113] AIPO4, AIPO4, AIPO4, AIPO4, AIPO4, QS-21, QS-21, QS-21, QS-21, QS-21, Montanide Montanide Montanide Montanide Montanide AD-472 AD-472 AD-472 AD-472 or or or HSV-2 or HSV-2 HSV-2 HSV-2 Guinea Guinea Guinea Guinea Both Both Both Both formulations formulations formulations formulations reduced reduced reduced reduced clinical clinical clinical clinical disease; disease; disease; disease; however, however, however, however, GPI-0100 GPI-0100 GPI-0100 GPI-0100 improved improved improved improved the the the the have the same efficacy in reducing H. pylori colonization as the gold toxin; andepitopes aluminium hydroxide Prophylactic Prophylactic Prophylactic Prophylactic Prophylactic vaccination vaccination vaccination vaccination with vaccination with adjuvanted adjuvanted with with with adjuvanted adjuvanted TA-CIN adjuvanted TA-CIN protected protected TA-CIN TA-CIN TA-CIN the protected the protected protected mice mice from from the the the mice viral mice viral mice from from from viral viral viral [109] epitopes epitopes epitopes epitopes from from from from Mice Mice Mice Mice Mice inducing inducing inducing inducing inducing predominantly predominantly predominantly predominantly predominantly IgG2c. IgG2c. IgG2c. IgG2c. IgG2c. QS-21 QS-21 QS-21 QS-21 QS-21 also also also also also induced induced induced induced induced the the the the highest the highest highest highest highest levels levels levels levels levels of of of IL-4 of IL-4 of IL-4 IL-4 IL-4 compared compared compared compared compared [109 [10 [1[1 AD-472 AD-472 from or or HSV-2 HSV-2 Guinea Guinea Both Both formulations formulations reduced reduced clinical clinical disease; disease; however, however, GPI-0100 GPI-0100 improved improved the the GPI-0100 GPI-0100 GPI-0100 GPI-0100 [111] [111] [111] [111] ISA-720, ISA-720, ISA-720, ISA-720, or or or CRL-1005 or CRL-1005 or CRL-1005 CRL-1005 CRL-1005 standard cholera toxin (CT) adjuvant GPI-0100 GPI-0100 [111] [111 glycoprotein glycoprotein glycoprotein glycoprotein DDDD pigs pigs pigs pigs ISA-720, glycoprotein-D glycoprotein-D glycoprotein-D glycoprotein-D formulation formulation formulation formulation only. only. only. only. Mice Mice and and Mice Mice Mice challenge, challenge, and and and challenge, whereas challenge, whereas challenge, vaccination vaccination whereas whereas whereas vaccination vaccination without vaccination without adjuvant adjuvant without without without was was adjuvant adjuvant adjuvant almost almost was was ineffective. was ineffective. almost almost almost ineffective. ineffective. Moreover, ineffective. Moreover, Moreover, GPIMoreover, GPIMoreover, GPIGPIGPIPlasmodium Plasmodium Plasmodium Plasmodium Plasmodium falciparum) falciparum) falciparum) falciparum) falciparum) to to to the to the to the the other the other other other other adjuvants, adjuvants, adjuvants, adjuvants, adjuvants, indicating indicating indicating indicating indicating Th1 Th1 Th1 Th1 Th1 and and and and and Th2 Th2 Th2 Th2 Th2 responses. responses. responses. responses. responses. glycoprotein glycoprotein D D pigs pigs glycoprotein-D glycoprotein-D formulation formulation only. only. ## ## ## ## ## TA-CIN TA-CIN TA-CIN TA-CIN TA-CIN GPI-0100 GPI-0100 GPI-0100 GPI-0100 GPI-0100 [114] [114] MATRIX-M MATRIX-M MATRIX-M MATRIX-M shifted shifted shifted shifted the the the the immune immune immune immune response response response response to to to an to an an an IgG2a IgG2a IgG2a IgG2a response response response response (towards (towards (towards aresponse aTh1 a+Th1 a+Th1 +(towards +T monkeys monkeys monkeys monkeys monkeys 0100 0100 boosted boosted 0100 0100 0100 IFN-γ IFN-γ boosted boosted boosted secreting secreting IFN-γ IFN-γ IFN-γ CD8 secreting CD8 secreting secreting Timmunogenicity cell cell CD8 CD8 response CD8 T+Th1 TT cell cell cell in in response response mice response mice compared compared in in in mice mice mice to compared to compared the the formulation formulation tototo the the formulation formulation formulation The The The The The vaccine vaccine vaccine vaccine vaccine induced induced induced induced induced robust robust robust robust robust immunogenicity immunogenicity immunogenicity immunogenicity with with with with with high high high high high levels levels levels levels levels ofofcompared of FML-seroconversion, of FML-seroconversion, of FML-seroconversion, FML-seroconversion, FML-seroconversion, as as as as as MATRIX-M MATRIX-M shifted shifted the the immune immune response response to to an an IgG2a IgG2a response response (towards (towards athe aTh1 Th1 Aluminium Aluminium Aluminium Aluminium phosphate; phosphate; phosphate; phosphate; + +T-cell +T-cell +T-cell FML FML FML FML FML ***** QS-21 QS-21 QS-21 QS-21 QS-21 Dogs Dogs Dogs Dogs Dogs [110] [110 [11 [1[1 Aluminium Aluminium phosphate; phosphate; response). response). response). response). On On On On the the the the other other other other hand, hand, hand, hand, the the the the CD8 CD8 CD8 CD8 T-cell response response response response could could could could be be be be improved improved improved improved using using using using without without adjuvant. adjuvant. without without without Vaccination adjuvant. Vaccination adjuvant. adjuvant. Vaccination Vaccination Vaccination ofof macaques macaques of of of macaques induced macaques induced macaques induced induced induced T T cell specific cell specific specific responses. responses. TTT cell cell cell responses. responses. responses. +and +and demonstrated demonstrated demonstrated demonstrated by by by by CD8+ by CD8+ CD8+ CD8+ CD8+ and and and CD4+ CD4+ CD4+ CD4+ CD4+ Tspecific Tspecific T cell T cell T cell cell cell populations. populations. populations. populations. populations. response). response). On Ondemonstrated the the other otherhand, hand, the the CD8 CD8 T-cell T-cell response response could could be be improved improved using using H9N2 H9N2 H9N2 H9N2 aluminium aluminium aluminium aluminium hydroxide; hydroxide; hydroxide; hydroxide; MF59 MF59 MF59 MF59 Mice Mice Mice Mice [112] [112] [112] [112] H9N2 H9N2 aluminium aluminium hydroxide; hydroxide; MF59 MF59 Mice Mice [112] [112 MATRIX-M MATRIX-M MATRIX-M MATRIX-M or or or MF59. or MF59. MF59. MF59. Among Among Among Among all all all all tested tested tested tested molecules, molecules, molecules, molecules, MATRIX-M MATRIX-M MATRIX-M MATRIX-M was was was was the the the the most most most most effective effective effective effective in in in in # # or # or # or AD-472 AD-472 AD-472 AD-472 AD-472 HSV-2 or HSV-2 or HSV-2 HSV-2 HSV-2 Guinea Guinea Guinea Guinea Guinea MATRIX-M Both Both Both Both Both formulations formulations formulations formulations formulations reduced reduced reduced reduced reduced clinical clinical clinical clinical clinical disease; disease; disease; disease; disease; however, however, however, however, however, GPI-0100 GPI-0100 GPI-0100 GPI-0100 GPI-0100 improved improved improved improved improved the the the the theinin MATRIX-M or or MF59. MF59.Among Among all alltested tested molecules, molecules, MATRIX-M MATRIX-M was wasthe the most most effective effective ; ; ; ; and and and and MATRIX-M MATRIX-M MATRIX-M MATRIX-M ## GPI-0100 GPI-0100 GPI-0100 GPI-0100 GPI-0100 [111] [111 [11 [1[1 ; ; and and MATRIX-M MATRIX-M inducing inducing inducing inducing the the the the immune immune immune immune response, response, response, response, followed followed followed followed by by by by MF59 MF59 MF59 MF59 and and and and aluminium-based aluminium-based aluminium-based aluminium-based adjuvants. adjuvants. adjuvants. adjuvants. glycoprotein glycoprotein glycoprotein glycoprotein glycoprotein DDDDD pigs pigs pigs pigs pigs glycoprotein-D glycoprotein-D glycoprotein-D glycoprotein-D glycoprotein-D formulation formulation formulation formulation only. only. only. only. only. inducing inducingthe theimmune immuneresponse, response, followed followedformulation by by MF59 MF59and and aluminium-based aluminium-based adjuvants. adjuvants. TM TM TM TM TM TM ISCOMATRIX ISCOMATRIX ISCOMATRIX ISCOMATRIX ; TM ;ISCOM ;ISCOM ;ISCOM ISCOM ; TM ;;; ISCOMATRIXTM ISCOMATRIXTM ISCOMATRIXTM ISCOMATRIXTM and and and and ISCOMTM ISCOMTM ISCOMTM ISCOMTM vaccines, vaccines, vaccines, vaccines, using using using using two two two two different different different different antigens antigens antigens antigens and and and and different different different different TM TM MATRIX-M MATRIX-M MATRIX-M MATRIX-M MATRIX-M shifted shifted shifted shifted shifted the the the the immune the immune immune immune immune response response response response response tototo an to an to an an IgG2a an IgG2a IgG2a IgG2a IgG2a response response response response response (towards (towards (towards (towards (towards adifferent Th1 aTh1 aTh1 aTh1 Th1 ISCOMATRIX ISCOMATRIXTM ; ;ISCOM ISCOMTM ;; ISCOMATRIXTM ISCOMATRIXTM and and ISCOMTM ISCOMTM vaccines, vaccines, using using two two different different antigens antigens and andadifferent Aluminium Aluminium Aluminium Aluminium phosphate; phosphate; phosphate; phosphate; phosphate; HpaA HpaA HpaA HpaA and and and and catalase catalase catalase catalase Cholera Cholera Cholera Cholera toxin; toxin; toxin; toxin; and and and and aluminium aluminium aluminium aluminium Mice Mice Mice Mice Aluminium delivery delivery delivery delivery systems systems systems systems (intranasal (intranasal (intranasal (intranasal or or or subcutaneous), or subcutaneous), subcutaneous), subcutaneous), have have have have the the the the same same same same efficacy efficacy efficacy efficacy ininin reducing in reducing reducing reducing H. H. H. [113] [113] [113] [113] +H. +T-cell +T-cell +T-cell +T-cell response). response). response). response). response). On On On On the On the the the other the other other other other hand, hand, hand, hand, hand, the the the the CD8 the CD8 CD8 CD8 CD8 T-cell response response response response response could could could could could be be be be improved be improved improved improved improved using using using using using HpaA HpaAand andcatalase catalase Cholera Choleratoxin; toxin;and andaluminium aluminium Mice Mice delivery delivery systems systems (intranasal (intranasal or or subcutaneous), subcutaneous), have have the thesame same efficacy efficacy in inreducing reducing H. H. [113] [113 H9N2 H9N2 H9N2 H9N2 aluminium aluminium aluminium aluminium aluminium hydroxide; hydroxide; hydroxide; hydroxide; hydroxide; MF59 MF59 MF59 MF59 MF59 as Mice Mice Mice Mice Mice [112] [112 [11 [1[1 hydroxide hydroxide hydroxide hydroxide H9N2 pylori pylori pylori pylori colonization colonization colonization colonization as as the as the the the gold gold gold gold standard standard standard standard cholera cholera cholera cholera toxin toxin toxin toxin (CT) (CT) (CT) (CT) adjuvant adjuvant adjuvant adjuvant MATRIX-M MATRIX-M MATRIX-M MATRIX-M MATRIX-M or or or MF59. or MF59. or MF59. MF59. MF59. Among Among Among Among Among all all all all tested all tested tested tested tested molecules, molecules, molecules, molecules, molecules, MATRIX-M MATRIX-M MATRIX-M MATRIX-M MATRIX-M was was was was was the the the the most the most most most most effective effective effective effective effective in in in in in hydroxide hydroxide pylori pylori colonization colonization as as the the gold gold standard standard cholera cholera toxin toxin (CT) (CT) adjuvant adjuvant ##### ; ; ; ; ; and and and and and MATRIX-M MATRIX-M MATRIX-M MATRIX-M MATRIX-M Prophylactic Prophylactic Prophylactic Prophylactic vaccination vaccination vaccination vaccination with with with with adjuvanted adjuvanted adjuvanted adjuvanted TA-CIN TA-CIN TA-CIN TA-CIN protected protected protected protected the the the the mice mice mice mice from from from from viral viral viral viral inducing inducing inducing inducing inducing the the the the immune the immune immune immune immune response, response, response, response, response, followed followed followed followed followed by by by by MF59 by MF59 MF59 MF59 MF59 and and and and and aluminium-based aluminium-based aluminium-based aluminium-based aluminium-based adjuvants. adjuvants. adjuvants. adjuvants. adjuvants. Prophylactic Prophylactic vaccination vaccination with withadjuvanted adjuvanted TA-CIN TA-CIN protected protected the themice micefrom from viral viral Mice Mice Mice Mice and and and and challenge, challenge, challenge, challenge, whereas whereas whereas whereas vaccination vaccination vaccination vaccination without without without without adjuvant adjuvant adjuvant adjuvant was was was was almost almost almost almost ineffective. ineffective. ineffective. ineffective. Moreover, Moreover, Moreover, Moreover, GPIGPIGPIGPITM TM TM TM TM TM TM TM TM TM ######## Molecules Molecules2019, 2019,24, 24,xx 12 12ofof3 Molecules 2019, 24, 171 13 of 29 Table Table2.2.Cont. Cont. Antigen TA-CIN ## Inactivated Chlamydophila abortus sLEa -KLH (glycolipid/glycoprotein expressed on cancer cell surface) Tetanus toxoid Fh15 Fh15(recombinant (recombinant15 15 kDa kDaFasciola Fasciolahepatica hepatica protein) protein) Adjuvant ADAD ADADSystem System***** ***** FML FML** QS-21 QS-21 GPI-0100 ;;and LTB-ESAT-6 LTB-ESAT-6 and Bacillus BacillusCalmetteCalmetteGuérin Guérin(BCG) (BCG) Quillaja Quillajaextract extract Mice Miceimmunized immunizedwith withADAD ADADsystem systemwith withQs Qshad hadaasurvival survivalrate rateofof50–62.5% 50–62.5%while while animals animalswithout withoutQs Qshad hadaasurvival survivalrate rateofof40–50%. 40–50%.Sheep Sheepimmunized immunizedwith withADAD ADAD sheep sheep system systemwith withQs Qsshowed showedless lesshepatic hepaticdamage damagecompared compared controlgroup. group. Findings Ref.totocontrol Despite Despitenonspecific nonspecificreactions reactionsbeing beingobserved observedininthe theimmunized immunizedanimals animals(similar (similartotoother other Dogs Dogs Prophylactic vaccination with adjuvanted TA-CIN protected the mice veterinary veterinaryvaccines), vaccines),aasignificant significantdecrease decreasewas wasshown shownwith withsubsequent subsequentdoses. doses. from viral challenge, whereas vaccination without adjuvant was Mice Micevaccinated vaccinatedwith withaacombination combinationofofplant-made plant-madeLTB-ESAT-6 LTB-ESAT-6fusion fusionBCG BCGoral oral almost ineffective. Moreover, GPI-0100 boosted IFN-γ secreting CD8+ [114] adjuvant adjuvant had had significantly significantly more more IL-10 IL-10 production production when when compared compared with with mice mice vaccinated vaccinated T cell response in mice compared to the formulation without Mice Mice without withoutof adjuvant. adjuvant. However, However, noprotection protection wasshown shownduring duringthe thechallenge challengetest testwith withM. M. adjuvant. Vaccination macaques inducedno specific T cellwas responses. tuberculosis, tuberculosis,iningroups groupstreated treatedwith withoral oralBCG BCG(with (withor orwithout withoutsaponin). saponin). The formulation induced the proliferation of B cells, which is Mice Mice Both Bothsynthetic synthetic Quillaja saponin saponin molecules molecules produced produced antibodies antibodiesininmice. mice. important to confer immunity to theQuillaja bacteria rather than cellular [115] QS-21 QS-21was wasthe the second second adjuvant adjuvant that that induced induced the the most most significant significant levels levels of of antibodies, antibodies, response. Mice Mice inducing inducingpredominantly predominantlyIgG2c. IgG2c.QS-21 QS-21also alsoinduced inducedthe thehighest highestlevels levelsofofIL-4 IL-4compared compared totothe theother otheradjuvants, adjuvants,indicating indicatingTh1 Th1and andTh2 Th2responses. responses. Immunized animals produced high titers of highly reactive IgM and [116] The Thevaccine vaccine inducedantibodies. robust robustimmunogenicity immunogenicitywith withhigh highlevels levels ofofFML-seroconversion, FML-seroconversion,as as IgG induced specific Dogs Dogs demonstrated demonstratedby byCD8+ CD8+and andCD4+ CD4+TTcell cellpopulations. populations. Guinea Guinea Both Bothformulations formulationsreduced reducedclinical clinicaldisease; disease;however, however,GPI-0100 GPI-0100improved improvedthe the Formulations with Quillaja saponins did not increase mucosal IgA. pigs pigs glycoprotein-D glycoprotein-D formulation formulation only. only. On the other hand, the saponins induced the highest systemic IgG [117] MATRIX-M MATRIX-Mshifted shiftedthe theimmune immuneresponse responsetotoan anIgG2a IgG2aresponse response(towards (towardsaaTh1 Th1 titers. response). response).On Onthe theother otherhand, hand,the theCD8 CD8+ +T-cell T-cellresponse responsecould couldbe beimproved improvedusing using The Mice Miceformulation with ISCOMs induced high specificity CTLs [118]was MATRIX-M MATRIX-Mor orMF59. MF59.Among Amongall alltested testedmolecules, molecules,MATRIX-M MATRIX-M wasthe themost mosteffective effectiveinin targeting different tumoral cells. inducing inducingthe theimmune immuneresponse, response,followed followedby byMF59 MF59and andaluminium-based aluminium-basedadjuvants. adjuvants. The combination of Quillaja and saponins with CDM in PLGA ISCOMATRIXTM ISCOMATRIXTM andISCOMTM ISCOMTM vaccines, vaccines, using usingtwo twodifferent differentantigens antigensand anddifferent different nanospheres loaded with antigens improved IgA production, Mice Mice delivery deliverysystems systems(intranasal (intranasalor orsubcutaneous), subcutaneous),have havethe thesame same efficacyininreducing reducingH. H. [119]efficacy suggesting better mucosal protection. The same was observed with pylori pylori colonization colonization as as the the gold gold standard standard cholera cholera toxin toxin (CT) (CT) adjuvant adjuvant systemic IgG. Prophylactic Prophylacticvaccination vaccinationwith withadjuvanted adjuvantedTA-CIN TA-CINprotected protectedthe themice micefrom fromviral viral ® Quil-A and QB-90 induced similar immunitywithout regarding humoral oralmost Mice Miceand and challenge, challenge, whereas whereas vaccination vaccination without adjuvant adjuvant was was almost ineffective. ineffective.Moreover, Moreover,GPIGPI[27] cellular responses. monkeys monkeys 0100 0100boosted boostedIFN-γ IFN-γsecreting secretingCD8 CD8+ +TTcell cellresponse responseininmice micecompared comparedtotothe theformulation formulation without without adjuvant. adjuvant. Vaccination Vaccination ofofmacaques macaquesinduced inducedspecific specific cellresponses. responses. Increase in both Th1 andTh2 response. [120] TTcell Miceand and Table 2. Cont. Mice Model Mice and monkeys GPI-0100 GPI-0100; ;Mice sQS-21 sQS-21 GD3-KLH GD3-KLH QS-21 FALVAC-1A FALVAC-1A(different (different epitopes epitopesfrom from Plasmodium Plasmodiumfalciparum) falciparum) GP1-0100 FML FML** AIPO4, AIPO4,QS-21, QS-21,Montanide Montanide ISA-720, ISA-720,or orCRL-1005 CRL-1005 Mice QS-21 QS-21 AD-472 AD-472 or orHSV-2 HSV-2 Quillaja saponins or cross-linked glycoprotein glycoprotein DD dextran microspheres (CDM) GPI-0100 GPI-0100 Rabbits Ovalbumin H9N2 H9N2 ISCOM matrices Aluminium Aluminiumphosphate; phosphate; aluminium aluminiumhydroxide; hydroxide;MF59 MF59 Mice ; ;and andMATRIX-M MATRIX-M# # Tetanus toxoid loaded onto PLGA nanospheres HpaA HpaA and andcatalase catalase Quillaja saponins, CDM TM TM ISCOMATRIX ISCOMATRIXTM ; ;ISCOM ISCOMTM ;; Cholera Choleratoxin; toxin;and andaluminium aluminium Rabbits hydroxide hydroxide Inactivated Bovine herpesvirus-5 Aluminum hydroxide, Quil-A® , or ## TA-CIN QB-90 (fromTA-CIN Quillaja##brasiliensis) ALM (Leishmania major) PLGA nanoparticles containing purified saponin extract Mice HIV-1 gp120 with Q105N mutation Quil-A® or MPL Mice Despite both adjuvants producing specific antibodies, there was no significant neutralizing activity against HIV-1. [121] Tetanus toxoid Quillaja saponins Rabbits Quillaja formulation was more efficient in conferring mucosal protection due to increased levels of IgA. [122] No antigen was used Alhydrogel or MATRIX-MTM formulated with fractions of Q. saponaria Mice Besides stimuli caused by MATRIX-MTM during the immune response (increase in cell recruitment and cytokine secretion), it induced the expression of co-stimulatory molecule CD86, participating in early events of the immune response. [123] Rhoptry Toxoplasma gondii protein Quil-A® Swine The formulation induced high humoral, local, and systemic immune response, partially preventing brains from forming cysts. [124] Mice GPI-0100 GPI-0100 [105] [105 [106] [106 [107] [107 [108] [108 [109] [109 [110] [110 [111] [111 [112] [112 [113] [113 [114] [114 Molecules 2019, 24, 171 14 of 29 Table 2. Cont. Antigen Adjuvant Model Findings Ref. Tetanus toxoid loaded onto chitosan functionalized gold nanoparticles Quillaja saponins Mice When administered orally, the nanoparticles can better deliver the formulation, increasing immune response up to 28-fold compared to control. [125] HIV-1 gp120 QS-21, aluminum hydroxide, MPLA, or ISCOMATRIXTM Mice Each adjuvant stimulated a different cytokine profile secretion, having different properties in the induction of the inflammation. [126] MUC1-KLH (prostate cancer marker) and/or ovalbumin QS-21, synthetic QS-21, or other conjugated saponins Mice Despite QS-21 inducing robust Th1 and Th2 response, conjugated saponins had improved activity and toxicity profiles relative to QS-21. [127] Varicella zoster glycoprotein E and ovalbumin AS01 Mice AS01 can improve adaptive humoral response through the generation of a great number of antigen-presenting cells. [128] Inactivated Poliovirus Quil-A® , QB-90, and Aqueous Extract from Quillaja brasiliensis Mice The humoral enhancements caused by Quil-A® and QB-90 were statistically similar. However, the mucosal immune response was increased with QB-90 by increases in IgA. [26] Tau antigen (associated with Alzheimer’s disease) Quil-A® Mice Animals that received the formulations with Quil-A® had reduced neuroinflammation and tau pathogenesis. [129] MARV-VLP (Marburg virus virus-like particle containing glycoprotein, matrix vp40, and nucleoprotein) QS-21 or polyI:C Cynomolgus macaques Formulations prepared with either of the adjuvants provided full protection in the challenge test. QS-21 produced a lower response to antigens compared to polyI:C. [130] EBOVgp-Fc (Ebola virus glycoprotein fused to Fc fragment of human IgG1) QS-21, aluminum hydroxide, or poly-ICLC Guinea pigs All formulations induced antibody formation, however, despite QS-21 inducing a strong humoral response, in the challenge test only poly-ICLC induced robust protection. [131] Intanza 2013 (Trivalent Influenza Vaccine, Sanofi Pasteur) QS-21 Mice When administered in the skin as a Nanopatch vaccine, a lower dose of QS-21 and adjuvant was required to enhance humoral response compared to intramuscular administration. [132] Ovalbumin or HBsAg (Hepatitis B virus surface antigen) QS-21 liposome Mice The immunoadjuvancy of QS-21 relies on macrophages to induce activation of dendritic cells and stimulate the immune system. [133] FhSAP2-IBs (Fasciola hepatica protein) QS-21 or Montanide ISA720 Mice Increase in both Th1 and Th2 responses with significant increase in Th1 response. [134] Molecules 2019, 24, 171 15 of 29 Table 2. Cont. Antigen Adjuvant Model Findings Ref. QS-21 or QS-21 formulated with HIV-1 gp120 QS-21 Mice QS-21 activates NLRP3 inflammasome. [21] Inactivated bovine viral diarrhea virus Saponins of Q. brasiliensis (QB-90) and Q. brasiliensis aqueous extract (AE) Mice In vaccines adjuvanted with QB-90 and AE higher levels of antibody were detected. Animals that received QB-90 adjuvanted vaccine had enhanced cytokines and IFN-γ production by CD4+andCD8+T lymphocytes whereas AE-adjuvanted preparation stimulated humoral response only. [135] Ovalbumin ISCOMs of Q. brasiliensis saponin fraction (IQB-90) and Quil-A® ISCOMs Mice IQB-90 inoculated subcutaneously induced strong antibody response (IgG1and IgG2a) and increased Th1 response. IQ-90 delivered intranasally induced secretion of serum IgG and IgG1 and mucosal IgA. [25] No antigen was used MATRIX-MTM Swine MATRIX-MTM formulation induced pro-inflammatory cytokines, suggesting enhancement of innate immune response of specific pathogen free pigs exposed to reared pigs. [68] Aβ42 (Alzheimer’s disease pathological hallmark) QS-21 Rhesus monkeys The formulation induced a good humoral response with high titers of IgG and IgA. No inflammatory cellular immune response was observed. [136] Ovalbumin and HBsAg (Hepatitis B virus surface antigen) QS-21 In vitro (THP-1 cell) and mice Human monocyte-derived dendritic cells were directly activated by QS-21, requiring cathepsin B to induce high CD4 and CD8 T cell response. [137] FMP013 (Falsiparum Malaria Protein-013 from Plasmodium falciparum) Molecules Molecules Molecules Molecules 2019, 2019, 2019, 2019, 24, 24, 24, 24, xxxx HBsAg ALF, ALF + aluminum hydroxide, Mice ALFQ + QS-21, or Montanide ISA720 Molecules 2019, 24, x Molecules Molecules2019, 2019,24, 24,xx Molecules Molecules Molecules Molecules Molecules 2019, 2019, 2019, 2019, 2019, 24, 24, 24, 24, x24, xxxx QS-21 liposome Swine The ALF adjuvant conjugated with QS-21 induced the highest antibody titer with the highest IgG2c titers. Also, it augmented the number of activated B-cells compared to Montanide. Molecules Molecules Molecules 2019, 2019, 2019, 24, 24, 24, xxx The study demonstrated that the HBsAg formulated with QS-21 liposomes in dissolvable microneedle arraypatches induced similar Table 2. Cont. immunization to the commercial Table HBsAg Table2.formulation. 2.Cont. Cont. [138] 12 12 12 12 ofofof30 of30 30 30 [139] Table Table Table 2.2.2. Con Co C Table Table Table Table 2.2.2. Cont. 2. Cont. Cont. Cont. Table Table Table Table Table 2.2. 2. Cont. 2. Cont. 2. Cont. Cont. Cont. Fh15 (recombinant 15 Mice immunized with ADAD system with Qs had a surviv The use of Q. brasiliensis saponins in vaccine formulations either Mice15 and Inactivated Bovine Viral QB-90 and IMXQB-90Fh15 (ISCOMs and Fh15 (recombinant (recombinant 15 Fh15 Fh15 (recombinant (recombinant (recombinant 15 15 Mice Miceimmunized immunized with withADAD ADAD system with with Qs Qshad had aMice aMice survival Mice survival immuni immun immu rate rat kDa Fasciola 15 hepatica ADAD SystemFh15 ***** animals without Qs hadsystem a survival rate of 40–50%. Sheep Mice formulated in ISCOM structures or not promoted a satisfactory and [140] Mice Mice and and Mice Mice Mice and and and sheep Fh15 Fh15 Fh15 Fh15 (recombinant (recombinant (recombinant (recombinant 15 15 15 Fh15 Fh15 Fh15 Fh15 Fh15 (recombinant (recombinant (recombinant (recombinant (recombinant Mice Mice Mice Mice 15 15 immunized 15 immunized 15 immunized 15 immunized with with with with ADAD ADAD ADAD ADAD system system system system with with with with Qs Qs Qs Qs had had had had aaADAD survival aADAD survival aADAD survival survival Mice Mice rate Mice rate Mice rate Mice rate immunized of immunized of immunized of immunized 50–62.5% of immunized 50–62.5% 50–62.5% 50–62.5% with while with while with while with while with ADAD ADAD ADAD ADAD ADAD system system system system system with with with with with Qs Qs Qs Qs had Qs had ha h Diarrhea Virus 15 Q. brasiliensis saponins kDa kDaFasciola Fasciolaprotein) hepatica hepatica ADAD ADAD System System ***** ***** kDa kDa kDa Fasciola Fasciola Fasciola hepatica hepatica hepatica animals animals without without System System System Qs Qs had had ***** ***** ***** a a survival survival rate rate of of 40–50%. 40–50%. Sheep animals Sheep animals animals immun immu with wit w promising immune response, similar to Quil-A®. system with Qs showed less hepatic damage compar Mice Mice Mice Mice and and and and Mice Mice Mice Mice Mice and and and and and sheep sheep sheep sheep sheep kDa kDa kDa kDa Fasciola Fasciola Fasciola Fasciola hepatica hepatica hepatica hepatica ADAD ADAD ADAD ADAD System System System System ***** ***** ***** ***** kDa kDa kDa kDa kDa Fasciola Fasciola Fasciola Fasciola Fasciola hepatica hepatica hepatica hepatica animals hepatica animals animals animals without without without without ADAD ADAD ADAD Qs ADAD Qs ADAD Qs Qs had had System had System had System a System a System survival a survival a survival survival ***** ***** ***** ***** ***** rate rate rate rate of of of 40–50%. of 40–50%. 40–50%. 40–50%. Sheep Sheep Sheep Sheep immunized immunized immunized animals immunized animals animals animals animals without without with without with without with without with ADAD ADAD ADAD Qs ADAD Qs Qs Qs had Qs had had had had a a survival a survival a [105] survival a [105] survival [105] survival [105] rate rate rate rate rate of of of 40–50% of 40–50% of 40–50 40–5 40– protein) protein) sheep protein) protein) protein) system system with with Qs Qsshowed showed less lessbeing hepatic hepatic damage damagecompared compared system system system totoc Despite nonspecific sheep sheep sheep sheep sheep sheep sheep sheep * FML: Fucose–Mannose-ligand antigen obtained from Leishmania (L.) donovani Sudan (LD 1S/MHOM/SD/00-strain 1S) antigen. ** LSP PbCS 242–310: long synthetic reactions polypeptide (LSP)observed in the immuniz FML * protein) QS-21 Dogsdamage protein) protein) protein) protein) protein) protein) protein) protein) system system system system with with with with Qs Qs Qs Qs showed showed showed showed less less less less hepatic hepatic hepatic hepatic damage damage damage compared compared compared compared toto to control to control system control system control system system system group. group. with group. with group. with with with Qs Qs Qs Qs showed Qs showed showed showed showed less less less less less hepatic hepatic hepatic hepatic hepatic damage damag damag dama dam Despite Despite nonspecific nonspecific reactions reactions being being observed observed in in the the Despite immunized Despite immunized Despite nonspe nonspe nonsp ani an PbCS 242–310, which represents C-terminus of circumsporozoite protein of Plasmodium berghei. *** HIV-1 DNA prime/protein–Env and Gag: polyvalent DNA prime/protein boost vaccine; veterinary vaccines), a significant decrease was shown w FML FML** QS-21 QS-21 FML FML FML Dogs Dogs *observed *observed *observed QS-21 QS-21 QS-21 Dogs Dogs Dogs Despite Despite Despite Despite nonspecific nonspecific nonspecific reactions reactions reactions reactions being being being being observed ininin the in the the the immunized immunized immunized immunized Despite Despite Despite Despite Despite animals animals animals animals nonspecific nonspecific nonspecific nonspecific nonspecific (similar (similar (similar (similar reactions to reactions to reactions to reactions other to reactions other other other being being being being being observed observed observed observed observed inwith inin the in the in the th **** LT(R192G): Escherichia coli heat-labile enterotoxin trypsin-cleavage site mutant; ***** ADADnonspecific System: includes Q saponaria saponins and/or the hydroalcoholic extract of P. leucotomos veterinary veterinary vaccines), vaccines), a a significant significant decrease decrease was was shown shown veterinary veterinary veterinar with su sit Mice vaccinated with a combination[106] of plant-made LTB-E FML FML FML FML **** QS-21 QS-21 QS-21 QS-21 Dogs Dogs Dogs Dogs FML FML FML FML FML ***** QS-21 QS-21 QS-21 QS-21 QS-21 Dogs Dogs Dogs Dogs Dogs [106] [106] [106] (PAL), emulsified with Montanide ISA763A as a water/oil (30/70) emulsion ;LTB-ESAT-6: Plant-made antigen [transgenic Arabidopsis thaliana plants expressing the immuno-dominant LTB-ESAT-6 and veterinary veterinary veterinary veterinary vaccines), vaccines), vaccines), vaccines), aasignificant asignificant asignificant significant decrease decrease decrease decrease was was was was shown shown shown shown with with with with veterinary subsequent veterinary subsequent veterinary subsequent veterinary subsequent veterinary vaccines), vaccines), doses. vaccines), doses. vaccines), doses. vaccines), doses.of aof asignificant aplant-made significant aplant-made significant asignificant significant decrease decrease decrease decrease decrease was was wa ww Mice Mice vaccinated vaccinated with with aacombination combination Mice LTB-ESAT Mice Mice vaccin vacci vacc adjuvant hadmixture significantly more and IL-10 productionLTB-ESAT-6 when com tuberculosis antigen ESAT-6 fused to the B subunit of the Escherichia. ;;and GPI-0100: semi synthetic analogue of Quillaja Saponin; ;;; sQS-21 (synthetic QS-21): of QS-21-Api LTB-ESAT-6 LTB-ESAT-6 and LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 and and and Bacillus CalmetteQuillaja extract Mice Mice Mice Mice Mice vaccinated vaccinated vaccinated vaccinated with with with with aacombination acombination acombination combination ofofof plant-made of plant-made plant-made plant-made LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 Mice Mice Mice Mice Mice fusion vaccinated fusion vaccinated fusion vaccinated fusion vaccinated vaccinated BCG BCG BCG BCG with oral with oral with oral with oral with aacombination acombination acombination acombination combination ofof of plant-ma of plant-ma of plant-m plant-m plant#significantly adjuvant adjuvant had had significantly more more IL-10 production production when when adjuvant adjuvant adjuvant compared compared had had had sis QS-21-Xyl (65:35); ;;; AD-472 (HSV) typeLTB-ESAT-6 2LTB-ESAT-6 attenuated; MF59: consisting of squalene–oil–water adjuvant; MATRIX-M: mix of IL-10 two without adjuvant. However, nomono protection was shown durin LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 and ; and and and human herpes simplex virus LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 ;;; and ; and ; and and and emulsion Bacillus Bacillus CalmetteCalmetteQuillaja Quillaja extract extract Bacillus Bacillus Bacillus CalmetteMice CalmetteMice CalmetteQuillaja Quillaja Quillaja extract extract extract Mice Mice Mice Guérin (BCG) adjuvant adjuvant adjuvant adjuvant had had had significantly significantly significantly significantly more more more IL-10 IL-10 production production production production when when when when compared compared compared compared adjuvant adjuvant adjuvant adjuvant adjuvant with with with had with had had had mice had mice significantly mice significantly mice significantly significantly significantly vaccinated vaccinated vaccinated vaccinated more more more more more IL-10 IL-10 IL-10 IL-10 IL-10 production production production production production wh w saponin cages to which an antigen is added; ## TA-CIN: recombinant Human papillomavirus typehad 16 (HPV16) L2, E6, more and E7IL-10 inIL-10 a unique protein. without without adjuvant. adjuvant. However, However, no no protection protection was was shown shown without without without during during adjuva adjuv adjuv the the cw oral BCG (with o Bacillus Bacillus Bacillus Bacillus CalmetteCalmetteCalmetteCalmetteQuillaja Quillaja Quillaja Quillaja extract extract extract extract Bacillus Mice Bacillus Mice Bacillus Mice Bacillus Mice Bacillus CalmetteCalmetteCalmetteCalmetteCalmetteQuillaja Quillaja Quillaja Quillaja Quillaja extract extract extract extract extract Mice Mice Mice Mice Mice tuberculosis, in groups treated with [107] [107] [107] [107] Guérin Guérin(BCG) (BCG) Guérin Guérin Guérin (BCG) (BCG) (BCG)was without without without without adjuvant. adjuvant. adjuvant. adjuvant. However, However, However, However, no no no no protection protection protection protection was was was shown shown shown shown during during during during without without without the without the without the the challenge challenge challenge adjuvant. challenge adjuvant. adjuvant. adjuvant. adjuvant. test test test However, test However, with However, with However, with However, with M. M. M. M. no no no no protection no protection protection protection protection was was was was was show show sho sh s tuberculosis, tuberculosis, in in groups groups treated treated with with oral oral BCG BCG (with (with tubercu or tubercu or tuberc with wit GD3-KLH GPI-0100 ; sQS-21 Mice Both synthetic Quillaja saponin molecules produced Guérin Guérin Guérin Guérin (BCG) (BCG) (BCG) (BCG) Guérin Guérin Guérin Guérin Guérin (BCG) (BCG) (BCG) (BCG) (BCG) tuberculosis, tuberculosis, tuberculosis, tuberculosis, in in groups in groups groups groups treated treated treated treated with with with oral oral oral oral BCG BCG BCG BCG (with (with (with (with or or or without or without without without tuberculosis, tuberculosis, tuberculosis, tuberculosis, saponin). tuberculosis, saponin). saponin). saponin). ininin groups in groups in groups groups groups treated treated treated treated treated with with with with with oral oral oral oral oral BCG BC BC B GD3-KLH GD3-KLH (differentGPI-0100 GPI-0100 ; ;sQS-21 sQS-21in GD3-KLH GD3-KLH GD3-KLH Mice Micewith GPI-0100 GPI-0100 Both GPI-0100 Both synthetic synthetic ; ; ; sQS-21 sQS-21 sQS-21 Quillaja Quillaja saponin saponin molecules Mice molecules Mice Mice produced produced Both Both antibo Both antib sy s FALVAC-1A QS-21 was the second adjuvant that induced the most signi AIPO4, QS-21, Montanide GD3-KLH GD3-KLH GD3-KLH GD3-KLH GPI-0100 GPI-0100 GPI-0100 GPI-0100 ; ; ; ; sQS-21 sQS-21 sQS-21 sQS-21 Mice Mice Mice Mice GD3-KLH GD3-KLH GD3-KLH GD3-KLH GD3-KLH Both Both Both Both synthetic synthetic GPI-0100 synthetic GPI-0100 synthetic GPI-0100 GPI-0100 GPI-0100 Quillaja Quillaja ; Quillaja ; Quillaja ; ; sQS-21 ; sQS-21 sQS-21 sQS-21 saponin sQS-21 saponin saponin saponin molecules molecules molecules molecules Mice Mice Mice produced Mice produced Mice produced produced antibodies antibodies antibodies antibodies Both Both Both Both in Both in synthetic in synthetic mice. in synthetic mice. synthetic mice. synthetic mice. Quillaja Quillaja Quillaja Quillaja Quillaja [108] saponin [108] saponin [108] saponin [108] saponin saponin molecules molecules molecules molecules molecule pr FALVAC-1A FALVAC-1A (different (different FALVAC-1A FALVAC-1A FALVAC-1A (different (different (different QS-21 QS-21 was wasthe thepredominantly second secondadjuvant adjuvant that thatinduced induced the theinduced most most QS-21 QS-21 QS-21 significant significant was was was the thtp epitopes from Mice inducing IgG2c. QS-21 also the high AIPO4, AIPO4, QS-21, QS-21, Montanide Montanide AIPO4, AIPO4, AIPO4, QS-21, QS-21, QS-21, Montanide Montanide Montanide ISA-720, or CRL-1005 FALVAC-1A FALVAC-1A FALVAC-1A FALVAC-1A (different (different (different (different FALVAC-1A FALVAC-1A FALVAC-1A FALVAC-1A FALVAC-1A (different (different (different QS-21 (different QS-21 (different QS-21 QS-21 was was was was the the the the second second second second adjuvant adjuvant adjuvant adjuvant that that that that induced induced induced induced the the the the most most most most significant significant significant significant QS-21 QS-21 QS-21 QS-21 QS-21 levels levels was levels was levels was was was the of the of the of the antibodies, of second the antibodies, second antibodies, second antibodies, second second adjuvant adjuvant adjuvant adjuvant adjuvant that that that that that induced induced induced induced induced the the the the m th epitopes epitopes from from epitopes epitopes epitopes Mice Mice from from from inducing inducing predominantly predominantly IgG2c. IgG2c. QS-21 also also Mice Mice Mice induced induced inducing the inducing the inducing highest highest predo pred pre lev lem Plasmodium falciparum) ISA-720, to the otherQS-21 adjuvants, indicating Th1 and Th AIPO4, AIPO4, AIPO4, AIPO4, QS-21, QS-21, QS-21, QS-21, Montanide Montanide Montanide Montanide AIPO4, AIPO4, AIPO4, AIPO4, AIPO4, QS-21, QS-21, QS-21, QS-21, QS-21, Montanide Montanide Montanide Montanide Montanide ISA-720, or or CRL-1005 CRL-1005 ISA-720, ISA-720, ISA-720, or or or CRL-1005 CRL-1005 CRL-1005 epitopes epitopes epitopes epitopes from from from from Mice Mice epitopes Mice epitopes Mice epitopes epitopes epitopes from from inducing from inducing from inducing from inducing predominantly predominantly predominantly predominantly IgG2c. IgG2c. IgG2c. IgG2c. QS-21 QS-21 QS-21 QS-21 also also also also induced induced induced induced Mice Mice Mice Mice Mice the the the the highest highest highest highest inducing inducing inducing inducing inducing levels levels levels levels predominantly of predominantly of predominantly of predominantly IL-4 of predominantly IL-4 IL-4 IL-4 compared compared compared compared IgG2c. IgG2c. IgG2c. IgG2c. IgG2c. [109] [109] QS-21 [109] QS-21 [109] QS-21 QS-21 QS-21 also also also also also induced induced induce induc indu Plasmodium Plasmodiumfalciparum) falciparum) Plasmodium Plasmodium Plasmodium falciparum) falciparum) falciparum) totothe theother otherrobust adjuvants, adjuvants, indicating indicatingTh1 Th1 and and Th2 Th2levels resp resp The vaccine induced immunogenicity with high ISA-720, ISA-720, ISA-720, ISA-720, or or or CRL-1005 or CRL-1005 CRL-1005 CRL-1005 ISA-720, ISA-720, ISA-720, ISA-720, ISA-720, or or or CRL-1005 or CRL-1005 or CRL-1005 CRL-1005 CRL-1005 FML * QS-21 Dogs Plasmodium Plasmodium Plasmodium Plasmodium falciparum) falciparum) falciparum) falciparum) Plasmodium Plasmodium Plasmodium Plasmodium Plasmodium falciparum) falciparum) falciparum) falciparum) falciparum) to toto the to the the the other other other other adjuvants, adjuvants, adjuvants, adjuvants, indicating indicating indicating indicating Th1 Th1 Th1 Th1 and and and and Th2 Th2 Th2 Th2 responses. responses. responses. responses.tototo the to the to the the other the other other other other adjuvants, adjuvants, adjuvants, adjuvants, adjuvants, indicating indicating indicating indicating indicating Th1 Th TT Molecules 2019, 24, 171 16 of 29 According to Table 2, saponins can be formulated with a variety of antigens, either by pathogens or human proteins, and elicit a strong immune response, generally geared towards a humoral response [137]. Interestingly, over time there has been an increase in the number of studies using QS-21 rather than Quil-A® or Quillaja extracts, probably due to its reduced toxicity [141]. Meanwhile, the chemical instability of QS-21 is reflected by acyl side chain hydrolysis, which occurs spontaneously in aqueous solutions at room temperature [142], so studies of the structure-activity relationship have been performed to enhance its adjuvant activity and reduce the undesirable effects [90,103,127]. QS-21 deacylation at Fuc-C4 was critical for Th1-type response but it was not so important for Th2-type response [90,142]. The combination of Quillaja saponins with other immunostimulants and formulation with carriers can take advantage of different delivery systems, such as nanoparticles [124], ISCOMs [25,86,91,96,101,113,126], ISCOM Matrix [88,113,123] and liposomes [133] for intranasal [117] or subcutaneous injections. In Table 2 over 60 pre-clinical studies for infectious diseases, degenerative disorders, and cancer are reported. For human infectious diseases the most tested antigen was Fucose-Mannose-Ligand (FML) antigen from Leishmania (L.) donovani [90,95,97,103,106,110]. Other vaccines for infectious diseases have also been well studied, such as hepatitis B [133,137,139], malaria [113,142], and HIV-1 [21,121,126] For veterinary use, pre-clinical trials have focused on Bovine Herpes Virus-5 [27] and Bovine Viral Diarrhea Virus [25,143] Fewer studies have been reported for degenerative disorders (Alzheimer’s disease) [129,136] and oncology [116,127]. In view of the amount of studies and the promising findings, many clinical trials have been developed, as mentioned in Table 4. Most clinical trials test the safety and efficacy of the formulations. Although Quil-A® and other Quillaja saponins have been used in pre-clinical studies, only one study reported Quillaja saponins for human use [144]. In all other studies, only QS-21 has been evaluated for human use [142,144–148]. QS-21 has been tested in prophylactic vaccines for treatment of malaria. A phase III clinical trial was conducted to determine the efficacy, safety, and immunogenicity of the RTS/S ASO1 vaccine and received positive regulatory approval from WHO, which recommended more evaluations before high level introduction in Africa, where the clinical study was conducted. Prophylactic vaccines have been tested using gp120 protein from HIV-I [149] and hepatitis B virus surface antigen [150]. QS-21 has been used in therapeutic vaccines to treat cancer patients. In these cases, instead of being administered in healthy people, the vaccine formulation needs to induce immune responses against existing tumors [83,145,146,151,152]. A more important experiment using QS-21 in therapeutic vaccines occurred after a surgical procedure in patients in stage II melanoma. The vaccine was well tolerated, and robust antibody responses were obtained [142,145]. Molecules 2019, 24, 171 17 of 29 Table 3. Clinical trials using Quillaja saponins as an adjuvant in vaccine formulations. Antigen Adjuvant CHO-derived gp120 protein from HIV-1 QS-21 or Al(OH)3 Molecules 2019, 24, x Objective Findings Ref. Evaluate safety and assess kinetics of immune response Improved T cell response inducing CTL activation with T helper cells was observed in formulations with QS-21. [149] 12 of 30 Determine the formulation toxicity against 12 12ofof30 30 Increase in specific antibody production and increased cancerous cells, its immune response, and if cytotoxicity either from complement system or [145] the conjugation of Globo H with KLH would Table 2. Cont. antibody signaling. affect the immune response Table Table2.2.Cont. Cont. Fh15 (recombinant 15 Mice immunized with ADAD system with Qs had a survival rate of 50–62.5% while Mice and PolySA and NP-polySA both Fh15 Fh15 (recombinant (recombinant 15 15 Mice immunized immunized with with ADAD system system with with Qs Qs had hadantibody aasurvival survival rate rateofof50–62.5% 50–62.5% while while kDa Fasciola hepatica ADAD System the *****immune response after Mice animals without QsADAD had a survival rate 40–50%. Sheep immunized with ADAD [105] Determine NP-polySA vaccination resulted in of higher Mice Mice and and Molecules Molecules 2019, 2019, 2019, 24, 24, 24, xxx 12 12 12ofofof30 30 3 conjugated with KLH (protein Molecules sheep kDa kDaFasciola Fasciola hepatica hepatica ADAD ADAD System System ***** ***** animals animals without without Qs Qs had had aasurvival survival rate rate ofof40–50%. 40–50%. Sheep Sheep immunized with with ADAD ADAD [105] [105] QS-21 vaccination and assess the impact of polySA titers with IgM response. The IgM was reactive to immunized [146] protein) system with Qs showed less hepatic damage compared to control group. that in adults is associated with sheep sheep chemical manipulation small cell lung cancer. protein) protein) system system with withQs Qs showed showed less less hepatic hepatic damage damage compared totocontrol controlgroup. group. Despite nonspecific reactions being observed in thecompared immunized animals (similar to other small cell lung cancer) FML * QS-21 Dogs [106] Table Table Table 2.2. 2. Cont. Cont. Cont. abeing Despite Despiteveterinary nonspecific nonspecific reactions reactions beingobserved observed ininthe the immunized immunized animals animals (similar (similar totoother other vaccines), significant decrease was shown with subsequent doses. FML FML** QS-21 QS-21 Dogs Dogs [106] [106] GPI-0100 (5000 µg) and QS-21 (100 µg) produced veterinary vaccines), vaccines), aasignificant significant decrease decrease was wasshown shown with withsubsequent subsequent doses. doses. Present the use of GPI-0100 in humans and veterinary Mice vaccinated with awith combination of plant-made LTB-ESAT-6 fusion BCG oral while Fh15 Fh15 Fh15 (recombinant (recombinant (recombinant 15 15 15 Mice Mice Mice immunized immunized immunized with with ADAD ADAD ADAD system system system with with with Qs Qs Qs had had had aasurvival asurvival survival rate rate rate ofofof 50–62.5% 50–62.5% 50–62.5% while while GPI-0100 ; GPI-0100-P comparable antibody titers. All adjuvanted vaccine Mice Mice Mice and and and MUC-2G/Globo H-KLH LTB-ESAT-6 ; and determine the safety and immunogenicity of a vaccinated [83] Mice Mice vaccinated with withaacombination combination of ofplant-made plant-made LTB-ESAT-6 LTB-ESAT-6 fusion fusion BCG BCGvaccinated oral oral adjuvant had significantly more IL-10 production when compared with mice kDa kDa kDa Fasciola Fasciola Fasciola hepatica hepatica hepatica ADAD ADAD ADAD System System System ***** ***** ***** animals animals animals without without Qs Qs Qs had had had aasurvival asurvival survival rate rate rate ofofof 40–50%. 40–50%. 40–50%. Sheep Sheep Sheep immunized immunized immunized with with with ADAD ADAD ADAD[107][105] [105] [105] QS-21 doses without were well tolerated and antigen-specific LTB-ESAT-6 LTB-ESAT-6 ;;;and and Bacillus CalmetteQuillaja extract Mice vaccine with different doses of GPI-0100. sheep sheep sheep adjuvant adjuvant had significantly significantly more more IL-10 IL-10 production production when when compared compared with mice micevaccinated vaccinated withouthad adjuvant. However, no protection was shown during thewith challenge test with M. antibody titers matched increasing dose levels. protein) protein) protein) system system system with with with Qs Qs Qs showed showed showed less less less hepatic hepatic hepatic damage damage damage compared compared compared tototo control control control group. group. group. Bacillus Bacillus CalmetteCalmetteQuillaja Quillajaextract extract Mice Mice [107] [107] Guérin (BCG) without withoutadjuvant. adjuvant. However, However, no noprotection protection was was shown shown during during the the challenge challenge test testwith withM. M. tuberculosis, in groups treated with oral BCG (with or without saponin). Despite Despite Despite nonspecific nonspecific nonspecific reactions reactions reactions being being being observed observed observed ininin the the the immunized immunized immunized animals animals animals (similar (similar (similar tototo other other other Guérin Guérin(BCG) (BCG) The adjuvanted vaccine induced more significative FML FML FML *** QS-21 QS-21 QS-21 Dogs Dogs [106] [106] [106] tuberculosis, tuberculosis, iningroups groups treated treated with with oral oralBCG BCG (with (withor orwithout withoutsaponin). saponin). GD3-KLH GPI-0100 ; sQS-21 MiceDogs Both synthetic Quillaja saponin molecules produced antibodies in mice. veterinary veterinary veterinary vaccines), vaccines), aasignificant asignificant significant decrease decrease decrease was was was shown shown shown with with with subsequent subsequent subsequent doses. doses. doses. [108] humoral and vaccines), Th1 immune responses compared with Evaluate the potential of an AS02A adjuvanted GD3-KLH GD3-KLH GPI-0100 GPI-0100; ;sQS-21 sQS-21 Mice Mice Both Both synthetic synthetic Quillaja Quillaja saponin saponin molecules molecules produced produced antibodies antibodies ininmice. mice. [108] [108] FALVAC-1A (different QS-21 was the second adjuvant that induced the most significant levels of antibodies, the non-adjuvanted formulation. Despite the AS02A Mice Mice Mice vaccinated vaccinated vaccinated with with with aacombination acombination combination ofof of plant-made plant-made plant-made LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 fusion fusion fusion BCG BCG BCG oral oral oral QS-21, Montanide SL * AS02A in healthy individuals [153]of LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 and and and AIPO4,formulation FALVAC-1A FALVAC-1A (different (different QS-21 was was the thesecond second adjuvant adjuvant that that induced induced the themost most significant significant levels levels ofofIL-4 antibodies, antibodies, epitopes from ;;; Mice andQS-21 inducing predominantly IgG2c. QS-21 alsoproduction induced the highest levels compared [109] recipients reporting local and general reactions more adjuvant adjuvant adjuvant had had had significantly significantly significantly more more more IL-10 IL-10 IL-10 production production when when when compared compared compared with with with mice mice mice vaccinated vaccinated vaccinated AIPO4, AIPO4, QS-21, QS-21, Montanide Montanide ISA-720, or CRL-1005 compare it toextract aextract non-adjuvanted Bacillus Bacillus Bacillus CalmetteCalmetteCalmetteQuillaja Quillaja Quillaja extract Mice Mice Mice [107] [107] epitopes epitopes from from Mice Mice vaccine. inducing inducingfrequently predominantly predominantly IgG2c. IgG2c. QS-21 QS-21also also induced induced the theand highest highest levels levelsofofIL-4 IL-4compared compared [109] [109] [107] Plasmodium falciparum) to than the other adjuvants, indicating Th1 Th2 responses. the non-adjuvanted group, safety without without without adjuvant. adjuvant. adjuvant. However, However, However, no no no protection protection protection was was was shown shown shown during during during the the the challenge challenge challenge test test test with with with M. M. M. ISA-720, ISA-720,or orCRL-1005 CRL-1005 Guérin Guérin Guérin (BCG) (BCG) (BCG) Plasmodium Plasmodium falciparum) falciparum) totothe theother other adjuvants, adjuvants, indicating indicating Th1 Th1 and andlevels Th2 Th2responses. responses. profile was acceptable. The vaccine tuberculosis, induced robust immunogenicity with high of FML-seroconversion, as tuberculosis, tuberculosis, ininin groups groups groups treated treated treated with with with oral oral oral BCG BCG BCG (with (with (with or or or without without without saponin). saponin). saponin). FML * QS-21 Dogs [110] The Thevaccine vaccine induced induced robust robust immunogenicity immunogenicity with with high high levels ofofFML-seroconversion, FML-seroconversion,as as demonstrated by CD8+ and CD4+ Tlevels cell populations. AS02A (oil-in-water FMP1 formulated with AS02A was well tolerated and GD3-KLH GPI-0100 GPI-0100 GPI-0100 ; ; sQS-21 sQS-21 sQS-21 Dogs Mice Mice Both Both Both synthetic synthetic synthetic Quillaja Quillaja Quillaja saponin saponin saponin molecules molecules molecules produced produced produced antibodies antibodies antibodies ininin mice. mice. mice. [110] [108] [108] FML FML *GD3-KLH *GD3-KLH QS-21 QS-21 ; Dogs Mice [110] [108] demonstrated demonstrated by by CD8+ CD8+ and and CD4+ CD4+ T T cell cell populations. populations. AD-472 or HSV-2 Guinea Both formulations reduced clinical disease; however, GPI-0100 improved formulation with Evaluate the safety and immunogenicity of showed high immunogenicity with specific FALVAC-1A FALVAC-1A FALVAC-1A (different (different (different QS-21 QS-21 QS-21 was was was the the the second second second adjuvant adjuvant adjuvant that that that induced induced induced the the the most most most significant significant significant levels levels levels ofofthe of antibodies, antibodies, antibodies, [147] FMP1 ** GPI-0100 [111] AIPO4, AIPO4, AIPO4, QS-21, QS-21, QS-21, Montanide Montanide Montanide AD-472 AD-472 or orHSV-2 HSV-2 Guinea Guinea Both Both formulations formulations reduced reduced clinical clinical disease; disease; however, however, GPI-0100 GPI-0100improved improvedthe the anti-MSP-142 antibody titers boosted and prolonged monophosphoryl malaria vaccine FMP1/AS02A inMice adults glycoprotein Dfrom pigs glycoprotein-D formulation only. epitopes epitopes epitopes from from Mice Mice inducing inducing inducing predominantly predominantly predominantly IgG2c. IgG2c. IgG2c. QS-21 QS-21 QS-21 also also also induced induced induced the the the highest highest highest levels levels levels ofofof IL-4 IL-4 IL-4 compared compared compared [109] [109] GPI-0100 GPI-0100 [111] [111] [109] ISA-720, ISA-720, ISA-720, or or or CRL-1005 CRL-1005 CRL-1005 pigs due toimmune the vaccination. lipid ADand glycoprotein glycoprotein D QS-21 pigs glycoprotein-D glycoprotein-D formulation formulation only. only.response (towards a Th1 MATRIX-M shifted the response to an IgG2a Plasmodium Plasmodium Plasmodium falciparum) falciparum) falciparum) Aluminium phosphate; tototo the the the other other other adjuvants, adjuvants, adjuvants, indicating indicating indicating Th1 Th1 Th1 and and and Th2 Th2 Th2 responses. responses. responses. + T-cell MATRIX-M MATRIX-M shifted shifted the theimmune immune response response totoan anresponse IgG2a IgG2a response response (towards (towards aaTh1 Th1 response). On the other hand,immunogenicity the CD8 could be improved using Local pain and post vaccination myalgias were greater The The The vaccine vaccine vaccine induced induced induced robust robust robust immunogenicity immunogenicity with with with high high high levels levels levels ofofof FML-seroconversion, FML-seroconversion, FML-seroconversion, as as as Aluminium Aluminium phosphate; phosphate; H9N2 aluminium hydroxide; MF59 MiceDogs [112] + +T-cell FML FML FML *** QS-21 QS-21 QS-21 Dogs DogsMATRIX-M [110] [110] [110] response). response). On OnMF59. the theother other hand, hand, the the CD8 CD8 T-cellresponse response could couldbe beimproved improved using using in Evaluate QS21 as an adjuvant and compare it in individuals that received QS-21. Despite increased or Among all tested molecules, MATRIX-M was the most effective demonstrated demonstrated demonstrated by by by CD8+ CD8+ CD8+ and and and CD4+ CD4+ CD4+ TTT cell cell cell populations. populations. populations. # H9N2 H9N2 QS-21 aluminium aluminium hydroxide; MF59 MF59 Mice Mice [112] [112] Influenza [148] ; hydroxide; and MATRIX-M with standard trivalent inactivated influenza serum antibodies, the mean titers for formulations MATRIX-M MATRIX-M or or MF59. MF59. Among Among all all tested tested molecules, molecules, MATRIX-M MATRIX-M was was the the most most effective effective in in the immune response, followed bydisease; MF59 and aluminium-based adjuvants. AD-472 AD-472 AD-472 or or or HSV-2 HSV-2 HSV-2 Guinea Guinea Guinea inducingBoth Both Both formulations formulations formulations reduced reduced reduced clinical clinical clinical disease; disease; however, however, however, GPI-0100 GPI-0100 GPI-0100 improved improved improved the the the ; ;and andMATRIX-M MATRIX-M# # (with or without QS-21) were by not different. GPI-0100 GPI-0100 GPI-0100 [111] [111] [111] TM TM inducing inducingthe the immune immune response, response, followed followed byMF59 MF59 and andaluminium-based aluminium-based adjuvants. vaccines, using two different antigensadjuvants. and different glycoprotein glycoprotein glycoprotein DDD ISCOMATRIX ; ISCOM ; pigs pigs pigs ISCOMATRIXTM and ISCOMTM glycoprotein-D glycoprotein-D glycoprotein-D formulation formulation formulation only. only. only. TM TM TM ISCOMATRIX ISCOMATRIX ; ;ISCOM ISCOM ;; ISCOMATRIXTM ISCOMATRIXTM and and ISCOMTM ISCOMTM vaccines, vaccines, using using two twodifferent different antigens antigens and and different different HpaA and catalase Cholera toxin;TM and aluminium Mice delivery systems (intranasal or subcutaneous), have the same efficacy in reducing H. [113] Lyophilized formulation is as efficient and safe as the MATRIX-M MATRIX-M MATRIX-M shifted shifted shifted the the the immune immune immune response response response tototo an an an IgG2a IgG2a IgG2a response response response (towards (towards (towards aaTh1 aTh1 Th1 RTS,S (recombinant proteins Evaluate the immunogenicity of RTS,S/AS02D Aluminium Aluminium Aluminium phosphate; phosphate; phosphate; Mice HpaA HpaAand andcatalase catalase Cholera Choleratoxin; toxin; and andaluminium aluminium Mice delivery deliverysystems systems (intranasal (intranasal or or subcutaneous), have the thesame same efficacy efficacy ininreducing reducingH. H. [113] [113] AS02D liquid formulation, promoting satisfactory [154] hydroxide pylori colonization assubcutaneous), the gold standard cholera toxin (CT) adjuvant +have +T-cell +T-cell response). response). response). On On On the the the other other other hand, hand, hand, the the the CD8 CD8 CD8 T-cell response response response could could could be be be improved improved improved using using using from P. falciparum) formulation (liquid and lyophilized) H9N2 H9N2 H9N2 aluminium aluminium aluminium hydroxide; hydroxide; hydroxide; MF59 MF59 MF59 Mice Mice Mice [112] [112] [112] immunization. hydroxide hydroxide pylori pyloricolonization colonization as aswith the thegold gold standard standard cholera choleraprotected toxin toxin(CT) (CT) adjuvant adjuvant Prophylactic vaccination adjuvanted TA-CIN the mice from viral MATRIX-M MATRIX-M MATRIX-M or or or MF59. MF59. MF59. Among Among Among all all all tested tested tested molecules, molecules, molecules, MATRIX-M MATRIX-M MATRIX-M was was was the the the most most most effective effective effective ininin ### ; ; ; and and and MATRIX-M MATRIX-M MATRIX-M Prophylactic Prophylactic vaccination vaccination with withwithout adjuvanted adjuvanted TA-CIN TA-CIN protected protected the themice micefrom from viral viral GPIMice and challenge, whereas vaccination adjuvant was almost ineffective. Moreover, inducing inducing inducing the the the immune immune immune response, response, response, followed followed followed by by by MF59 MF59 MF59 and and and aluminium-based aluminium-based aluminium-based adjuvants. adjuvants. adjuvants. TA-CIN ## GPI-0100 [114] + T adjuvant Mice Mice and and challenge, challenge, whereas whereas vaccination vaccination without without adjuvant was wasin almost almost ineffective. ineffective.to Moreover, Moreover, GPIGPI0100 boosted IFN-γ secreting CD8 cell response mice compared the formulation TM TM TM TM TM ISCOMATRIX ISCOMATRIX ISCOMATRIX ; ;ISCOM ;ISCOM ISCOM ; ;monkeys ; ISCOMATRIXTM ISCOMATRIXTM ISCOMATRIXTM and and and ISCOMTM ISCOMTM ISCOMTM vaccines, vaccines, vaccines, using using using two two two different different different antigens antigens antigens and and and different different different TA-CIN TA-CIN#### GPI-0100 GPI-0100 TM [114] [114] + + monkeys monkeys 0100 0100boosted boosted IFN-γ IFN-γ secreting secreting CD8 CD8 TTcell cell response responsein inmice micecompared compared to the the formulation formulation without adjuvant. Vaccination of macaques induced specific Ttoefficacy cell responses. HpaA HpaA HpaA and and and catalase catalase catalase Cholera Cholera Cholera toxin; toxin; toxin; and and and aluminium aluminium aluminium Mice Mice Mice delivery delivery delivery systems systems systems (intranasal (intranasal (intranasal or or or subcutaneous), subcutaneous), subcutaneous), have have have the the the same same same efficacy efficacy ininin reducing reducing reducing H. H. H. [113] [113] [113] without withoutadjuvant. adjuvant.Vaccination Vaccinationofofmacaques macaquesinduced inducedspecific specificTTcell cellresponses. responses. hydroxide hydroxide hydroxide pylori pylori pylori colonization colonization colonization as as as the the the gold gold gold standard standard standard cholera cholera cholera toxin toxin toxin (CT) (CT) (CT) adjuvant adjuvant adjuvant Prophylactic Prophylactic Prophylactic vaccination vaccination vaccination with with with adjuvanted adjuvanted adjuvanted TA-CIN TA-CIN TA-CIN protected protected protected the the the mice mice mice from from from viral viral viral Mice Mice Mice and and and challenge, challenge, challenge, whereas whereas whereas vaccination vaccination vaccination without without without adjuvant adjuvant adjuvant was was was almost almost almost ineffective. ineffective. ineffective. Moreover, Moreover, Moreover, GPIGPIGPI###### TA-CIN TA-CIN TA-CIN GPI-0100 GPI-0100 GPI-0100 [114] [114] [114] + +T +TT monkeys monkeys monkeys 0100 0100 0100 boosted boosted boosted IFN-γ IFN-γ IFN-γ secreting secreting secreting CD8 CD8 CD8 cell cell cell response response response ininin mice mice mice compared compared compared tototo the the the formulation formulation formulation without without without adjuvant. adjuvant. adjuvant. Vaccination Vaccination Vaccination ofofof macaques macaques macaques induced induced induced specific specific specific TTT cell cell cell responses. responses. responses. Molecules Molecules2019, 2019,24, 24,xx Globo H-KLH (carbohydrate antigen found in most breast QS-21 cancer cells) Molecules 2019, 24, 171 18 of 29 Table 4. Clinical trials using Quillaja saponins as an adjuvant in vaccine formulations. Antigen Objective Findings Ref. AlhydrogelTM , Montanide ISA720 and AS02 Adjuvant System Evaluate the immunogenicity and safety of Pf AMA1 antigen (two different doses with three different adjuvants) All formulations caused different reactogenicity with no serious reported adverse effects. All formulations tested induced antibody production, with AS02 being the most pronounced. [155] RTS,S **** AS02A Determine the safety of the RTS,S antigen formulated with AS02A adjuvant. The formulation was well tolerated in children, with a good safety profile within the number of doses. The AS02A formulation caused fewer serious adverse events compared to the control group. [156] FMP2.1 ***** AS02A Evaluate the reactogenicity, safety, and immunogenicity of the malaria vaccine FMP2.1/AS02A in adults. The formulation was well tolerated and showed good safety. Also, it was highly immunogenic. [157] No antigen was used Quillaja saponins Determine if dietary QS can modify macrophage activity and investigate its effects on liver function and inflammatory response An increase in chemotactic and phagocytosis activities were observed. Furthermore, no adverse effects were seen since no significant changes in immunoglobulin, transaminase, IL-1α, and TNF- α were observed. [144] NP-polySA-KLH (Polysialic acid conjugated to keyhole limpet hemocyanin (KLH)) QS-21 Confirm the safety profile and determine the optimal dose The lowest optimal immunogenic dose was 10 µg, which resulted in consistent high-titer antibody responses. [151] MAGE-A3 (tumor-specific protein usually expressed in melanoma) AS02B or AS15 Discover which adjuvant would cause a more robust and persistent immune response AS15 provided a more robust immune response by activating more dendritic and B cells. [152] AS02B, AS02V, or AS01B Molecules Molecules 2019, 2019,24, 24,xx Evaluate the duration of humoral and cellular responses Molecules Molecules Molecules 2019, 2019, 2019, 24, 24, 24, xxx QS-21 Evaluate safety and immunogenicity of the Table 2. Cont. pentavalent synthetic vaccine Pf AMA1 *** HBsAg (Hepatitis B virus surface antigen) Molecules 2019, 24, x Unimolecular conjugated Globo-H, GM2, sTn, TF, and Tf (markers usually expressed on cancer cell-surface) Fh15 (recombinantovarian 15 Adjuvant All formulations induced persistent T CD4+ and CD8+ [150] specific response, as well as B-cell response, indicating 12 of 30 immunological memory. 83% of individuals responded to at least three antigens Table Table 2.2.Cont. Cont. Table Table Table 2.2.2. Cont. Cont. Cont. [158] with satisfactory immune response. Fh15 Fh15(recombinant (recombinant15 15 Mice immunized Fh15 Fh15 Fh15 (recombinant (recombinant (recombinant 15 15 15 system with Qs had aMice Mice immunized immunized with withADAD ADAD Mice system Mice system Mice immunized immunized immunized with withQs Qshad had with with with aasurvival ADAD survival ADAD ADAD system rate system rate system ofofwith 50–62.5 with 50–62 with QQ with ADAD survival rate of 50–62.5% while Mice Miceand and Mice Mice Mice and and and Mice and kDaFasciola Fasciolahepatica hepatica animals kDa ADAD kDa ADAD kDa Fasciola Fasciola Fasciola System System hepatica hepatica hepatica ***** ***** ADAD ADAD ADAD System System System ***** animals ***** animals *****immunized without withoutQs Qs had had aasurvival survival animals animals animals rate rate without without ofwithout of40–50%. 40–50%. Qs Qs Qs had Sheep had Sheep had aasurvival aimmunized survival immunized survival rate rate rate with of wit ofof 40 4 kDa Fasciola hepatica ADAD System ***** kDa without Qs had a survival rate of 40–50%. Sheep with ADAD [105] HBsAg (surface antigen of sheep sheep sheep sheep sheep sheep Investigate how combining AS01 triggers innate response, such as NK-cells, and protein) protein) protein) protein) system system with with Qs Qs showed showed less less hepatic hepatic system system system damage damage with with with Qs Qs Qs compared compared showed showed showed less less to less tocontrol hepatic control hepatic hepatic gro da gr da d protein) Hepatitis B virus), ovalbumin, system protein) with Qs showed less hepatic damage compared to control group. CSP (P. falciparum AS01 immune-stimulants results in innate immune activates CD8 T-cells in the lymph nodes, depending [159] Despitenonspecific nonspecific reactionsto being Despite being Despite Despite observed observed nonspecific nonspecific nonspecific ininthe the reactions reactions immunized reactions immunized being being being animals animals observed observed observed (simi (sim ini Despite nonspecific reactions being observed in the Despite immunized animalsreactions (similar other and response on macrophage, IL-12,Dogs and IL-18. FML FML ** FML QS-21 FML QS-21 FML *** Dogs DogsQS-21 QS-21 QS-21 Dogs Dogs FML * circumsporozoite protein), QS-21 Dogs [106] veterinary veterinary vaccines), vaccines), a a significant significant veterinary veterinary veterinary decrease decrease vaccines), vaccines), vaccines), was was shown shown a a significant a significant significant with with subsequent subsequent decrease decreas decrea veterinary vaccines), a significant decrease was shown with subsequent doses. Varicella zoster glycoprotein E Mice Micevaccinated vaccinated with with aacombination combination Mice Mice vaccinated vaccinated of vaccinated ofplant-made plant-made with with with LTB-ESAT-6 aLTB-ESAT-6 acombination acombination combination fusion fusion ofofof plan pla pl BC B Mice vaccinated with a combination of plant-made LTB-ESAT-6 fusion BCG oral Mice GPI-0100: Semi synthetic analogue of Quillaja saponin; ;;and GPI-0100P: GPI-0100 purified; ;;; AS02A: LTB-ESAT-6 LTB-ESAT-6 and LTB-ESAT-6 LTB-ESAT-6 LTB-ESAT-6 and and and oil-in-water formulation containing monophosphoryl lipid A and QS-21; * SL: LTB-ESAT-6 ; and adjuvant adjuvant had had significantly significantly more more adjuvant adjuvant adjuvant IL-10 IL-10 production production had had had significantly significantly significantly when when compared more compared more more IL-10 IL-10 IL-10 with with producti product produc mice mice adjuvant had significantly more IL-10 production when compared with mice vaccinated recombinant hepatitis B protein containing the small protein (S) and the modified large (L) protein of the hepatitis B viral envelope, containing pre-Sl and pre-S2 sequences in addition to Bacillus BacillusCalmetteCalmetteBacillus Bacillus Quillaja Bacillus Quillaja CalmetteCalmetteextract Calmetteextract Mice Mice Quillaja Quillaja Quillaja extract extract extract Mice Mice Mice Bacillus CalmetteQuillaja extract Mice [107] the entire S sequence. ** FMP1 (Falciparum malaria protein 1): recombinant based on the carboxy-terminal merozoite surface (MSP-142) from 3D7 clone of P. during without without adjuvant. adjuvant. However, However, no no without without without protection protection adjuvant. adjuvant. was wasshown shown However, However, However, during no no no protection the protection the protection challenge challenge was wa wt without protein adjuvant. However, no protectionend wasofshown during theprotein-1 challenge test with M.theadjuvant. Guérin (BCG) (BCG) Antigen; **** RTS,S: Guérin Guérin Guérin (BCG) (BCG) (BCG) Guérin (BCG) falciparum; *** Pf AMA1: Plasmodium falciparum Guérin Apical Membrane Plasmodium falciparum circumsporozoite surface antigen; ***** FMP2.1: recombinant protein (FMP2.1) tuberculosis, iningroups groupstreated treated tuberculosis, tuberculosis, with tuberculosis, withoral oralBCG BCG ininin groups (with groups (with groups or or treated treated without treated without with with with sapon sapon ora or o tuberculosis, in groups treated with oral BCG (withtuberculosis, or without saponin). based on Apical Membrane Antigen-1 (AMA-1) from the 3D7 clone of P. falciparum. GD3-KLH GD3-KLH GPI-0100 GPI-0100 GD3-KLH GD3-KLH GD3-KLH ; ;sQS-21 sQS-21 saponin GPI-0100 GPI-0100 GPI-0100 Mice Mice ; ; ; sQS-21 sQS-21 sQS-21 Both Both synthetic synthetic Mice Mice Mice Quillaja Quillajasaponin saponin Both Both molecules Both molecules synthetic synthetic synthetic produced produced Quillaja Quillaja Quillaja antibodies saponin antibodies saponin saponin molecu molec in mole inmi m GD3-KLH GPI-0100 ; sQS-21 Mice Both synthetic Quillaja molecules produced antibodies in mice. [108] FALVAC-1A FALVAC-1A(different (different QS-21 FALVAC-1A FALVAC-1A FALVAC-1A (different (different (different QS-21 QS-21 was wasthe the second second adjuvant QS-21 QS-21 that QS-21 thatinduced was induced was was the the the second the second the second most most adjuvant adjuvant significant adjuvant significant that that that levels levels induced induced induced ofofaat FALVAC-1A (different was the second adjuvant that induced the most significant levels of adjuvant antibodies, AIPO4, AIPO4,QS-21, QS-21,Montanide Montanide AIPO4, AIPO4, AIPO4, QS-21, QS-21, QS-21, Montanide Montanide Montanide AIPO4, QS-21, Montanide epitopes epitopes from from epitopes epitopes epitopes from from from Mice Mice inducing predominantly predominantly Mice Mice Mice IgG2c. inducing inducing inducing QS-21 QS-21also also predominantly predominantly predominantly induced inducedthe theIgG2c. highest IgG2c. highest IgG2c. QS-21 QS-21 levels QS-21 levels also also of also ofIL-4 ind ILinin epitopes from Mice inducing predominantly IgG2c. QS-21 also inducedinducing the highest levels of IL-4 IgG2c. compared [109] ISA-720, ISA-720,or orCRL-1005 CRL-1005 ISA-720, ISA-720, ISA-720, or or or CRL-1005 CRL-1005 CRL-1005 ISA-720, or CRL-1005 Plasmodium Plasmodiumfalciparum) falciparum) Plasmodium Plasmodium Plasmodium falciparum) falciparum) falciparum) totothe theother otheradjuvants, adjuvants,indicating indicating tototo the the Th1 the Th1 other other and other andadjuvants, Th2 adjuvants, Th2 adjuvants, responses. responses. indicatin indicati indicat Plasmodium falciparum) to the other adjuvants, indicating Th1 and Th2 responses. The Thelevels vaccine vaccine inducedrobust robustimmunogenicity immunogenicity The The The vaccine vaccine vaccine induced induced induced with withhigh robust high robust robust levels levels immunogenicity immunogenicity immunogenicity ofofFML-serocon FML-seroco ww The vaccine induced robust immunogenicity with high ofinduced FML-seroconversion, as FML FML ** FML QS-21 FML QS-21 FML *** Dogs DogsQS-21 QS-21 QS-21 Dogs Dogs Dogs FML * QS-21 Dogs [110] Molecules 2019, 24, 171 19 of 29 4. Toxicity of Quillaja Saponins Regarding the toxicity of Q. saponaria and Q. brasiliensis, there are few studies in the literature. The studies available focus on the preclinical toxicity evaluation. One of the first studies to address the toxicity of Q. saponaria and its fractions was published in 1995, by Rönnberg et al. [160]. This study evaluated the toxicity of crude Q. saponaria extract and QH-A, QH-B, and QH-C purified triterpenoid components in WEHI cells (immature B-lymphocytes of Mus musculus). The evaluation of the lytic activities of Q. saponaria, responsible for the local reactions after injection, demonstrated that QH-A was safer than QH-B and QH-C, causing hemolysis only at a concentration ten-times higher. In addition, QH-B and QH-C did not affect RNA or protein synthesis, suggesting that they are not the primary toxicity targets. Similarly, hydrolyzed saponins of Q. saponaria, resulting in deacylated forms, have significantly reduced the toxicity of the substance. This supports the role of fatty acids in the toxic activity of saponins [161]. Thought-provokingly, none of the Quillaja saponins presented hemolytic activity after incorporation into the ISCOM matrix [160]. Although QH-B has a strong adjuvant effect, it represents the most toxic triterpenoid saponin, thus limiting its application in vitro and in vivo. Rönnberg et al. [162] demonstrated that a 10-fold higher QH-B concentration treated with sodium periodate reduced the enzymatic activities of the cells exposed to QH-B without sodium periodate by a factor of ten. The QH-B toxicity reduction is possibly the result of changes in the sugars’ chemical structure, such as xylose and galactose [160,162]. Similarly, hydrolyzed saponins of Q. saponaria, resulting in deacylated forms, have significantly reduced the toxicity of the substance. This supports the role of fatty acids in the toxic activity of saponins [161]. Arabski et al. [46] also studied the in vitro toxicity of Q. saponaria, but in the CHO-K1 cell line (cells from the ovary of Cricetulus griseus). They demonstrated that the saponins significantly increased death in this cell line by early apoptosis, at doses ranging from 12 to 50 µg/mL. Minor doses were not able to lyse the exposed erythrocytes, elucidating the minimal concentration required to intersperse with cell membranes in order to lyse them. Quillaja saponin toxicity has also been studied in alternative mammalian models and in vivo models. Abdula et al. [163] evaluated the effects of Q. saponaria saponins regarding lethality and alterations in Danio rerio morphology during the first 3 days. This invertebrate, also known as zebrafish, is an important alternative to mammalian tests and is effective for studying toxicity mechanisms. The results showed that Q. saponaria presented both harmful and beneficial effects on the embryos of zebrafish, since a 5 µg/mL concentration promoted growth and a 10 µg/mL concentration was mortal to zebrafish. These findings suggest that the toxic effects of Q. saponaria saponins could affect the semipermeable protective chorion expressed by the embryo in development. Alternatively, it could affect cells in the embryo bodies, by permeating the noncellular chorion. The toxicity of Quillaja extracts and their saponin derivatives was also studied in neonates of the aquatic crustacean D. magna and in D. rerio zebrafish embryos [164]. The authors demonstrated that Quillaja extract and Q. saponaria fractions QS-18 (35.1 ± 4.1% of the total Quillaja saponins) and QS-21 (3.7 ± 0.3%) have similar toxicity; nevertheless, with a toxicity potential three to eight times higher than non-saponins. Thus, Quillaja saponins contribute to the majority of the toxicity (85.1–93.6%), with QS-21 being slightly more toxic than QS-18. The non-saponins contribute to only 14 and 6%. The higher lipophilicity of QS-18 and QS-21 is probably responsible for their higher toxicity, since lipophilic substances can easily cross the embryo chorion, resulting in a higher uptake in zebrafish embryos [165]. In vivo Quillaja extract acute toxicity was studied by Tam and Roner [8]. They orally treated newborn mice with 50 µL of saponin extract. The concentrations ranged from 0 to 0.5 mg/mouse. The animals were treated for seven days and observed for two months. The 0.0375 mg/mouse dose presented more than 50% mortality, thus the LD50 of the extract was established at 0.0325 mg/mouse. The weight evaluation demonstrated that there was no short-term health impact in mice that survived treatment with saponins. On the other hand, the high concentrations of the extract killed the mice Molecules 2019, 24, 171 20 of 29 within 24–36 h, suggesting an important alteration in the membrane environment of the gastrointestinal tract, leading cells to burst. Finally, Vinai et al. [166] studied the safe dose of vaccine adjuvants, such as Quillaja saponins, for intra-peritoneal vaccination. Flounder fingerlings were acutely treated and lethality and toxicity signs, such as body color, feeding, and swimming behavior, were evaluated. The group treated with saponins showed different toxicity levels, according to the concentrations tested. The toxicity signs observed were fish lethargy, body color alterations, and alterations in feeding behavior; the liver and intestine presented important hemorrhages, the fish showed pale gills, and the peritoneal cavity was filled with red ascites, suggesting high toxicity of the highest dose tested. In this study, Quillaja saponins presented a LD50 of 105 µg/fish (22.4 mg/kg), reinforcing the high toxicity. In addition, they caused severe histological damage, as well as liver damage, observed by the increase in AST level at doses above 16 µg/fish and of ALT at 160 µg/fish. In clinical studies only QS-21 toxicity have been studied. For most patients the dose-limiting toxicity is about 50 µg/dose, except for cancer patients (100–200 µg per vaccination) [142]. Therefore, the safety of Quillaja saponins is still not well established and more studies are necessary. 5. Conclusions Quillaja saponins have been employed in the food industry as emulsifiers for beverages and food additives. In the cosmetic industry, they have been used as antidandruff, cleansing, emulsifying, foaming, masking, moisturizing, skin conditioning, and surfactant agents. However, these are not the only applications of Quillaja saponins. The particular chemical characteristics of these metabolites, along with their biological activities, show potential for drug discovery and vaccines development. Most of these saponins are bidesmosides and contain a triterpenic aglycone belonging to the b-amyrin series, most frequently quillaic acid. Vaccine adjuvation is the main propriety studied for Quillaja saponins, which have shown potent immunogenic activity and the ability to modulate immunity towards a cell-mediated response or to enhance antibody production. In addition to improving immunoadjuvant activity and minimizing their toxicity, these saponins are been evaluated in different formulations, such as ISCOMs, oil-in-water formulations, and liposomes, among others. Furthermore, studies have demonstrated antiviral (herpes simplex virus type 1, human immunodeficiency viruses 1 and 2, varicella zoster virus, vaccinia virus, rhesus rotavirus, and reovirus), antifungal (Penicillium roguefortii, Aspergillus ochraceus, Alternaria solani, Fusarium oxysporum, Verticillium dahlia, and Pythium ultimum), and antibacterial (Staphylococcus aureus, Salmonella typhimurium, and Asaia spp.) activities for Quillaja saponins. 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