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In this study, a preparation of saponins (QB-90U) extracted from leaves of Quillaja brasiliensis collected in Uruguay was evaluated as a vaccine adjuvant by comparison with alum and the well known saponin-based adjuvant, Quil A. The haemolytic activity and cellular toxicity of the saponin preparations were also evaluated. QB-90U was only slightly haemolytic and showed a low cytotoxicity when compared to Quil A. The adjuvant properties of QB-90U were assayed by sub-cutaneous immunization of mice with a preparation of inactivated bovine herpesvirus 5 (BoHV-5) either with no adjuvant or adjuvanted with QB-90U, Quil A or alum. Serum levels of anti-BoHV-5 IgG, IgG1, IgG2a, IgG2b and also IgG3 were significantly increased by QB-90U and were of the same order as those elicited by Quil A. Furthermore, high titres of neutralizing antibodies were found to be present in the serum of immunized animals from both groups. The cellular response induced by QB-90U did also reproduce the one elicited by Quil A. In fact, a robust DTH response was observed in mice immunized with both saponin preparations; as well as increased splenocytes levels of Th1-type cytokines, namely IFN-γ and IL-2. Taken together, the above results confirm and extend our previous observation regarding the similarity of the responses elicited by Quil A and the saponin preparation from Q. brasiliensis (Fleck et al., 2006) and indicate that QB-90U is worth of further studies as a safe and potent vaccine adjuvant., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The haemolytic activities and adjuvant potentials of Platycodon grandiflorum saponin (PGS) and its fractions on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were evaluated. PGS was subjected to silica gel column chromatography to afford four fractions, and two fractions PGSC and PGSD selected for testing for activities because of containing dominant saponin peaks. PGS, PGSC, and PGSD showed a slight haemolytic effect, with their HD50 value being 37.91+/-2.24, 21.30+/-1.22, 37.58+/-1.86 microg/ml against 0.5% rabbit red blood cell, respectively. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing Alum (200 microg), Quil A (10 microg), PGS (50, 100 or 200 microg), PGSC, or PGSD (25, 50 or 100 microg) on days 1 and 15. Two weeks later (day 28), concanavalin A (Con A)-, pokeweed (PWM)-, and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. PGS and PGSC significantly enhanced the Con A-, PWM-, and OVA-induced splenocyte proliferation in OVA-immunized mice at three doses (P<0.01 or P<0.001). However, no significant differences (P>0.05) were observed among the OVA group, OVA/Alum group and OVA/PGSD group. OVA-specific IgG, IgG1, and IgG2b antibody levels in serum were significantly enhanced by PGS, PGSC, and PGSD compared with OVA control group (P<0.05, P<0.01, or P<0.001). Moreover, the adjuvant effects of PGSC (50 or 100 microg) on the OVA-specific IgG, IgG1, and IgG2b antibody responses to OVA in mice were more significant than those of Alum. In conclusion, PGS seem to be a promising balanced Th1 and Th2 directing immunological adjuvants which can enhance the immunogenicity of vaccine.

The chemical characterization of aqueous extracts (AE) of barks, leaves and branches and the saponin fraction denominated QB-90 obtained from Quillaja brasiliensis, a native species from Southern Brazil, show remarkable similarities to Quillaja saponaria saponins which are known as adjuvants in vaccine formulations. In vivo toxicity assays of AE and QB-90 showed not to be lethal for mice in doses ranging from 50 to 1600 microg and 50-400 microg, respectively. Experimental vaccines prepared with bovine herpesvirus type 1 (BHV-1) antigen and either AE (barks 100 microg, leaves 400 microg, branches 400 microg) or QB-90 (100 microg) were able to enhance the immune responses of mice in a comparable manner to saponins from Q. saponaria (QuilA, 100 microg). BHV-1 specific IgG, IgG1 and IgG2a antibody levels in serum were also significantly enhanced by AE, QB-90 and QuilA compared to control group (p<0.05). These results showed that AE and QB-90 from Q. brasiliensis are potential candidates as adjuvants in vaccines.

The adjuvant of the FML-vaccine against murine and canine visceral leishmaniasis, the Riedel de Haen saponin mixture, was fractionated by ion exchange chromatography on DEAE-cellulose to afford one TLC homogeneous Quillaja saponaria Molina QS21 saponin fraction (18.0%), a mixture of two deacylsaponins (19.4%), sucrose (39.9%), sucrose and glucose (19.7%), rutin (0.8%) and quercetin (2.2%), that were identified by comparison of 1H and 13C NMR spectroscopy. The QS21 shows the typical aldehyde group in C-23 (65% equatorial) and a normonoterpene moiety acylated in C-28. The deacylsaponins show the aldehyde group but do not have the normonoterpene moiety. Balb/c mice were vaccinated with 150 microg of FML antigen of Leishmania donovani and 100 microg of each obtained fraction and further challenged by infection with 10(8) amastigotes of Leishmania chagasi. The safety analysis and the effect on humoral and cellular immune responses and in clinical signs showed that the QS21 saponin and the deacylsaponins are the most active adjuvant compounds of the Riedel the Haen saponin mixture. Both induced the highest and non-significantly different increases in DTH, CD4+ T lymphocytes in spleen, IFN-gamma in vitro, body weight gain and the most pronounced reduction of parasite burden in liver (95% for QS21 and 86% for deacylsaponins; p>0.05). While the QS21 showed mild toxicity, significant adjuvant effect on the anti-FML humoral response before and after infection, and decrease in liver relative weight, the deacylsaponins showed no toxicity, less haemolysis and antibody and DTH responses increased mainly after infection, still inducing a stronger Leishmania-specific in vitro splenocyte proliferation. Our results confirm in the Riedel de Haen saponin extract the presence of deacylsaponins normonoterpene-deprivated which are non-toxic and capable of inducing a specific and strong immunoprotective response in vaccination against murine visceral leishmaniasis.

In this study, the haemolytic activities of Glycyrrhiza uralensis saponins (GLS) and its adjuvant potentials on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were evaluated. We determined the haemolytic activity of GLS using 0.5% rabbit red blood cell. Haemolytic percents of GLS-treated red blood cell were 11.20 and 5.54% at the concentrations of 500 and 250 microg/ml, respectively. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing Alum (200 microg), QuilA (10 and 20 microg), or GLS (50, 100, or 200 microg) on Days 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)-, and OVA-stimulated splenocyte proliferation and OVA-specific serum antibodies were measured. GLS significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice at a dose of 100 microg (P<0.025). OVA-specific IgG, IgG1, and IgG2b antibody titers in serum were also significantly enhanced by GLS compared with OVA control group (P<0.025). Moreover, no significant differences (P>0.05) were observed between enhancing effect of GLS and QuilA on the OVA-specific IgG, IgG1, and IgG2b antibody responses to OVA in mice. The results suggest that GLS showed a slight haemolytic effect and enhanced significantly a specific antibody and cellular response against OVA in mice, and deserved further researches to be developed as immunological adjuvant.

In this study, the haemolytic activities of Bupleurum chinense saponins (BCS) and its adjuvant potentials on the immune responses of ICR mice against ovalbumin (OVA) were evaluated. BCS showed a slight haemolytic effect, with its haemolytic percents being 3.32% and 1.19% at the concentrations of 500 and 250 microg/ml, respectively. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing aluminium hydroxide gel (Alum, 200 microg), QuilA (10 and 20 microg) or BCS (50, 100 or 200 microg) on Days 1 and 15. Two weeks later (Day 28), concanavalin A (Con A)-, lipopolysaccharide (LPS)- and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. BCS significantly enhanced the Con A-, LPS-, and OVA-induced splenocyte proliferation in the OVA-immunized mice especially at a dose of 100 microg (P<0.05 or P<0.001). OVA-specific IgG, IgG1 and IgG2b antibody levels in serum were also significantly enhanced by BCS compared with OVA control group (P<0.01 or P<0.001). Moreover, no significant differences (P>0.05) were observed between enhancing effect of BCS and QuilA on the OVA-specific IgG2b antibody responses to OVA in mice. In conclusion, the results suggest that BCS could be safely used as adjuvant with low or non-haemolytic effect.

In this study, the haemolytic activities of Panax notoginseng saponins (PNS) and its adjuvant potentials on the cellular and humoral immune responses of ICR mice against ovalbumin (OVA) were evaluated. We determined the haemolytic activity of PNS using 0.5% rabbit red blood cell. PNS showed a slight haemolytic effect, with its haemolytic percents being 11.59 and 3.60% at the concentrations of 500 and 250 microg/ml, respectively. Furthermore, the adjuvant potential of PNS at three dose levels on the cellular and humoral immune responses of ICR mice against ovalbumin were investigated. ICR mice were immunized subcutaneously with OVA 100 microg alone or with OVA 100 microg dissolved in saline containing aluminum hydroxide gel (Alum) (200 microg), Quil A (10 and 50 microg) or PNS (50, 100 or 200 microg) on days 1 and 15. Two weeks later (day 28), concanavalin A (Con A)-, pokeweed (PWM)- and OVA-stimulated splenocyte proliferation and OVA-specific antibodies in serum were measured. PNS significantly enhanced the Con A-, PWM-, and OVA-induced splenocyte proliferation in the OVA-immunized mice at a dose of 100 microg (P < 0.05 or P < 0.025). OVA-specific IgG, IgG1 and IgG2b antibody levels in serum were significantly enhanced by PNS compared with OVA control group (P < 0.025). Moreover, enhancing effect of PNS on the OVA-specific IgG2b antibody responses to OVA in mice were more significant than that of Quil A (P < 0.025). In conclusion, the results suggest that PNS could be safely used as adjuvant with low or non-haemolytic effect.

An 87.7% (P < 0.01) and 84% (P < 0.001) of protection against visceral leishmaniasis was achieved in CB hamsters and Balb/c mice, respectively, with saponin combined to the fucose-mannose ligand of Leishmania donovani (FML). However, an undesirable haemolytic effect was described for several saponins. Aiming to improve the formulation with FML/saponin, we comparatively analysed the haemolytic potential of recently characterized plant saponins and currently used adjuvants. The haemolytic activity of steroidic saponins from Agave sisalana; Smilax officinalis as well as commercial saponin (Riedel De Haën's), was higher than that of triterpenoid ones (Bredemeyera floribunda; Periandra mediterranea) and the Freund's complete adjuvant. The concentration resulting in 50% haemolysis was 500 micrograms ml-1 for aluminum hydroxide. The low haemolytic effect of P. mediterranea saponin was abolished by removal of its glycidic moiety and its sapogenin fraction as well as the Freund's Incomplete Adjuvant were non-haemolytic within this range. Furthermore, the adjuvant effect of three doses of P. mediterranea saponin injected with the FML antigen of L. donovani, was assayed in mice, either by the intraperitoneal (i.p.) or the subcutaneous (s.c.) route. The anti-FML IgG antibody levels increased and detectable levels were observed up to 3 months in the s.c. group. The response was expanded in both groups after an injection with a fourth vaccine dose. The IgG response showed increased levels of IgG2a only in the i.p. group, while IgG2b and IgG1 but not IgG3 antibodies were higher than controls in both groups. In conclusion, the results suggest that the recently described triterpenoid fractions of P. mediterranea can be safely used as adjuvant with low or non-haemolytic effect.

It is well established that ISCOMs function efficiently as an antigen-presenting system and protective immunity has been evoked against a variety of infectious agents. The built-in saponin adjuvant from Quillaja saponaria Molina is responsible for the strong immunoenhancing activity displayed by the ISCOM. However, to allow the use of ISCOMs in human vaccines it is necessary to determine the immunological properties and toxicity of chemically defined Quillaja components. Thus, the present study was carried out in a mouse model to determine the adjuvant activity and toxicity of "free", isolated Quillaja components, as well as formulated into particles, i.e. ISCOM matrix. The purified Quillaja components and the ISCOM matrix formulations were examined for their adjuvant activity in a model system consisting of purified influenza virus antigen and Quillaja saponins. It was demonstrated that a Quillaja component, designated QH-C, either as a "free" component or in an ISCOM matrix, has a strong adjuvant activity, but little or no toxicity in the doses tested. In addition, QH-C in the form of ISCOM matrix does not induce any local reactions at the site of injection. Thus, ISCOMs containing the QH-C component, devoid of toxicity, but with strong adjuvant activity, can prove to be useful in adjuvant formulations for human use.