Saponin and Ribosome-Inactivating Protein Synergistically Trigger Lysosome-Dependent Apoptosis by Inhibiting Lysophagy: Potential to Become a New Antitumor Strategy.

The susceptibility of lysosomal membranes in tumor cells to cationic amphiphilic drugs (CADs) enables CADs to induce lysosomal membrane permeabilization (LMP) and trigger lysosome-dependent cell death (LDCD), suggesting a potential antitumor therapeutic approach. However, the existence of intrinsic lysosomal damage response mechanisms limits the display of the pharmacological activity of CADs. In this study, we report that low concentrations of QS-21, a saponin with cationic amphiphilicity extracted from Quillaja Saponaria tree, can induce LMP but has nontoxicity to tumor cells. QS-21 and MAP30, a type I ribosome-inactivating protein, synergistically induce apoptosis in tumor cells at low concentrations of both. Mechanistically, QS-21-induced LMP helps MAP30 escape from endosomes or lysosomes and subsequently enter the endoplasmic reticulum, where MAP30 downregulates the expression of autophagy-associated LC3 proteins, thereby inhibiting lysophagy. The inhibition of lysophagy results in the impaired clearance of damaged lysosomes, leading to the leakage of massive lysosomal contents such as cathepsins into the cytoplasm, ultimately triggering LDCD. In summary, our study showed that coadministration of QS-21 and MAP30 amplified the lysosomal disruption and can be a new synergistic LDCD-based antitumor therapy.

The predominant Quillaja Saponaria fraction, QS-18, is safe and effective when formulated in a liposomal murine cancer peptide vaccine.

Extracts of the Chilean soapbark tree, Quillaja Saponaria (QS) are the source of potent immune-stimulatory saponin compounds. This study compared the adjuvanticity and toxicity of QS-18 and QS-21, assessing the potential to substitute QS-18 in place of QS-21 for vaccine development. QS-18, the most abundant QS saponin fraction, has been largely overlooked due to safety concerns. We found that QS-18 spontaneously inserted into liposomes, thereby neutralizing hemolytic activity, and following administration did not induce local reactogenicity in a footpad swelling test in mice. With high-dose intramuscular administration, transient weight loss was minor, and QS-18 did not induce significantly more weight loss compared to a liposome vaccine adjuvant system lacking it. Two days after administration, no elevation of inflammatory cytokines was detected in murine serum. In a formulation including cobalt-porphyrin-phospholipid (CoPoP) for short peptide sequestration, QS-18 did not impact the formation of peptide nanoparticles. With immunization, QS-18 peptide particles induced higher levels of cancer neoepitope-specific and tumor-associated antigen-specific CD8+ T cells compared to QS-21 particles, without indication of greater toxicity based on mouse body weight. T cell receptor sequencing of antigen-specific CD8+ T cells showed that QS-18 induced significantly more T cell transcripts. In two murine cancer models, vaccination with QS-18 peptide particles induced a similar therapeutic effect as QS-21 particles, without indication of increased toxicity. Antigen-specific CD8+ T cells in the tumor microenvironment were found to express the exhaustion marker PD-1, pointing to the rationale for exploring combination therapy. Taken together, these data demonstrate that QS-18, when formulated in liposomes, can be a safe and effective adjuvant to induce tumor-inhibiting cellular responses in murine models with potential to facilitate or diminish costs of production for vaccine adjuvant systems. Further studies are warranted to assess liposomal QS-18 immunogic, reactogenic and toxicological profiles in mice and other animal species.

Ação antifúngica de extratos de plantas isolados sobre espécies de Candida albicans e Candida não-albicans / Antifungal action of isolated plant extracts on Candida albicans and Candida non-albicans species

As espécies de Candida spp. apresentam-se como o principal patógeno fúngico humano, podendo causar infecções superficiais e invasivas. A emergência de novas espécies em infecções, apresentando alta resistência aos antifúngicos utilizados desafia pesquisadores a propor novas terapias no controle desta infecção, entre as quais podemos citar a fitoterapia realizando o uso de extratos de plantas para propor novos protocolos. Por isto, este trabalho objetiva avaliar a ação antifúngica dos extratos isolados de Quilaia (Quillaja saponaria) e Alcachofra (Cynara scolymus) sobre C. albicans, C. glabrata, C. krusei, C. tropicalis e C. dubliniensis em formas planctônica e biofilmes monotípicos. Inicialmente foram feitas análises da ação antifúngica dos extratos de Quilaia e Alcachofra por meio do teste de microdiluição em caldo (CLSI ­ Protocolo M27-S4), para determinar as Concentrações Inibitórias Mínimas (CIM) e as Concentrações Fungicidas Mínimas (CFM) de espécies. Os biofilmes foram formados por 48 h em poços de microplacas, os quais receberam tratamentos de concentrações dos extratos (100 mg/mL, 50 mg/mL, 25 mg/mL, 12,5 mg/mL e 6,25 mg/mL), assim como foram testados os grupos controles positivo e negativo, para determinação da viabilidade celular por meio do teste MTT. Os dados foram analisados estatísticamente pelos testes ANOVA e Tukey, com significância de 5%. Os resultados da CIM e CFM para as espécies C. albicans, C. krusei e C. glabrata foram de 12,5mg/mL para ambos os extratos, os valores para C tropicalis foi 12,5 mg/mL para o extrato de Quilaia e 25 mg/mL para Alcachofra, ambos os extratos apresentaram o mesmo valor de 6,25 mg/mL para a espécie C. dubliniensis. A ação antibiofilme do extrato de Quilaia apresentou redução fúngica do biofilme principalmente nas duas maiores concentrações (100 mg/mL e 50 mg/mL) do extrato para ambos os tempos (5 min e 24 h) quando comparados com o grupo controle negativo que não recebeu tratamento, apresentando diferenças estatísticas significativas (p<0.001). A ação antibiofilme do extrato de Alcachofra apresentou reduções dos biofilmes significativas nas cinco concentrações (100 mg/mL, 50 mg/mL, 25 mg/mL, 12,5 mg/mL e 6,25 mg/mL) em ambos os tempos, na maioria das espécies, apresentando diferenças significativas (p<0.001). Diante disso, concluímos que os extratos glicólicos de Q. saponaria e C. scolymus apresentam ação antifúngica em todas as espécies de Candida spp. analisadas, sendo um potencial antifúngico para C. albicans e as espécies C. não-albicans, mas na espécie de C. krusei as reduções de biofilme só ocorrem nas maiores concentrações. Os resultados da ação antibiofilme manteve um padrão de ação, quanto maior a concentração do extrato, maior a redução, isto para ambos os extratos e para a maioria das espécies analisadas (AU)

Candida spp. They are the main human fungal pathogen and can cause superficial and invasive infections. The emergence of new species in infections, presenting high resistance to the antifungals used, challenges researchers to propose new therapies to control this infection, among which we can mention phytotherapy using plant extracts to propose new protocols. Therefore, this work aims to evaluate the antifungal action of extracts isolated from Quilaia (Quillaja saponaria) and Artichoke (Cynara scolymus) on C. albicans, C. glabrata, C. krusei, C. tropicalis and C. dubliniensis in planktonic forms and biofilms monotypic. Initially, analyzes of the antifungal action of Quilaia and Artichoke extracts were carried out using the broth microdilution test (CLSI ­ Protocol M27-S4), to determine the Minimum Inhibitory Concentrations (MICs) and Minimum Fungicide Concentrations (MFCs) of species. Biofilms were formed for 48 h in microplate wells, which received extract concentration treatments (100 mg/mL, 50 mg/mL, 25 mg/mL, 12.5 mg/mL and 6.25 mg/mL), as well as the positive and negative control groups were tested to determine cell viability using the MTT test. The data were statistically analyzed using the ANOVA and Tukey tests, with a significance of 5%. The MIC and CFM results for the species C. albicans, C. krusei and C. glabrata were 12.5 mg/mL for both extracts, the values for C tropicalis were 12.5 mg/mL for the Quilaia extract and 25 mg/mL for Artichoke, both extracts presented the same value of 6.25 mg/mL for the species C. dubliniensis. The antibiofilm action of the Quilaia extract showed a fungal reduction of the biofilm mainly at the two highest concentrations (100 mg/mL and 50 mg/mL) of the extract for both times (5 min and 24 h) when compared with the negative control group that did not receive treatment, showing significant statistical differences (p<0.001). The antibiofilm action of Artichoke extract showed significant reductions in biofilms at the five concentrations (100 mg/mL, 50 mg/mL, 25 mg/mL, 12.5 mg/mL and 6.25 mg/mL) at both times, in most species, showing significant differences (p<0.001). Therefore, we conclude that glycolic extracts of Q. saponaria and C. scolymus have antifungal action on all species of Candida spp. analyzed, with antifungal potential for C. albicans and non-albicans C. species, but in the C. krusei species, biofilm reductions only occur at higher concentrations. The results of the antibiofilm action maintained a pattern of action, the higher the concentration of the extract, the greater the reduction, this for both extracts and for the majority of species analyzed(AU)

Quillaja brasiliensis nanoparticle adjuvant formulation improves the efficacy of an inactivated trivalent influenza vaccine in mice.

The threat of viral influenza infections has sparked research efforts to develop vaccines that can induce broadly protective immunity with safe adjuvants that trigger robust immune responses. Here, we demonstrate that subcutaneous or intranasal delivery of a seasonal trivalent influenza vaccine (TIV) adjuvanted with the Quillaja brasiliensis saponin-based nanoparticle (IMXQB) increases the potency of TIV. The adjuvanted vaccine (TIV-IMXQB) elicited high levels of IgG2a and IgG1 antibodies with virus-neutralizing capacity and improved serum hemagglutination inhibition titers. The cellular immune response induced by TIV-IMXQB suggests the presence of a mixed Th1/Th2 cytokine profile, antibody-secreting cells (ASCs) skewed toward an IgG2a phenotype, a positive delayed-type hypersensitivity (DTH) response, and effector CD4+ and CD8+ T cells. After challenge, viral titers in the lungs were significantly lower in animals receiving TIV-IMXQB than in those inoculated with TIV alone. Most notably, mice vaccinated intranasally with TIV-IMXQB and challenged with a lethal dose of influenza virus were fully protected against weight loss and lung virus replication, with no mortality, whereas, among animals vaccinated with TIV alone, the mortality rate was 75%. These findings demonstrate that TIV-IMXQB improved the immune responses to TIV, and, unlike the commercial vaccine, conferred full protection against influenza challenge.

Elucidation of the pathway for biosynthesis of saponin adjuvants from the soapbark tree.

The Chilean soapbark tree (Quillaja saponaria) produces soap-like molecules called QS saponins that are important vaccine adjuvants. These highly valuable compounds are sourced by extraction from the bark, and their biosynthetic pathway is unknown. Here, we sequenced the Q. saponaria genome. Through genome mining and combinatorial expression in tobacco, we identified 16 pathway enzymes that together enable the production of advanced QS pathway intermediates that represent a bridgehead for adjuvant bioengineering. We further identified the enzymes needed to make QS-7, a saponin with excellent therapeutic properties and low toxicity that is present in low abundance in Q. saponaria bark extract. Our results enable the production of Q. saponaria vaccine adjuvants in tobacco and open the way for new routes to access and engineer natural and new-to-nature immunostimulants.

A booster for vaccines from plants.

Reconstituting a plant biosynthetic pathway enables a sustainable supply of vaccine adjuvants.

Antiviral Effects of Quillaja saponaria Extracts Against Human Noroviral Surrogates.

Aqueous extracts of Quillaja saponaria Molina are US FDA approved as food additives in beverages with known antiviral activity. Due to lack of commercially available vaccines against human noroviruses (HNoVs), alternate methods to prevent their spread and the subsequent emergence of variant strains are being researched. Furthermore, HNoVs are not yet culturable at high enough titers to determine inactivation, therefore surrogates continue to be used. This research analyzed the effect of aqueous Quillaja saponaria extracts (QE) against HNoV surrogates, Tulane virus (TV), murine norovirus (MNV-1), and feline calicivirus (FCV-F9) at room temperature (RT) and 37 °C. Viruses (~ 5 log PFU/mL) were individually treated with 1:1 or 1:5 (v/v) diluted QE (pH ~ 3.75), malic acid control (pH 3.0) or phosphate-buffered saline (pH 7.2, as control) at 37 °C or RT for up to 6 h. Individual treatments were replicated three times using duplicate plaque assays for each treatment. FCV-F9 at ~ 5 log PFU/mL was not detectable after 15 min by 1:1 QE at 37 °C and RT. At RT, 1:5 QE lowered FCV-F9 titers by 2.05, 2.14 and 2.74 log PFU/mL after 0.5 h, 1 h and 2 h, respectively. MNV-1 showed marginal reduction of < 1 log PFU/mL after 15 min with 1:1 or 1:5 QE at 37 °C without any significant reduction at RT, while TV titers decreased by 2.2 log PFU/mL after 30 min and were undetectable after 3 h at 37 °C. Longer incubation with higher QE concentrations may be required for improved antiviral activity against MNV-1 and TV.

Emulsification by vitamin E TPGS or Quillaja extract enhanced absorption of berberine without affecting its metabolism in humans.

Berberine is widely used for the prevention of cancers and diabetes. However, the absorption rate of berberine is less than 1% in humans. The objective of this research was to determine whether emulsification improves the absorption and affects the metabolism of orally ingested berberine. Twelve healthy subjects, both men and women, received 800 mg berberine in a powder or emulsified form by vitamin E TPGS or Quillaja extract using a randomized crossover design. Blood samples were collected 12 hours after a dose. Berberine and its metabolites in plasma were analyzed with and without hydrolysis by glucuronidase and sulfatase on UHPLC-MS/MS. The area under the curve (AUC0-12 h) and peak plasma concentration (Cmax) of berberine was 6.7 nM h and 0.9 nM in participants who received berberine powder. They were increased to 12.6 nM h and 2.0 nM by TPGS emulsification and 28.0 nM h and 5.1 nM by Quillaja extract emulsification, respectively. Berberrubine and demethyleneberberine were detected as major phase-1 metabolites of berberine. The AUC0-12 of both free and total berberrubine was significantly increased by TPGS and Quillaja extract. Emulsification by Quillaja extract was more effective than TPGS to increase the plasma concentrations of free and total demethyleneberberine. However, the ratios of phase-1 metabolites and ratios of phase-2 conjugates were not affected by emulsification. Absorption increases of berberine by TPGS or Quillaja extract emulsification may lead to enhanced bioactivity in humans.

Effects of N-acylation on the immune adjuvanticity of analogs of the Quillaja saponins derivative GPI-0100.

Modification of the 3-glucuronic acid (GlcA) residue from the Quillaja saponin (QS) adjuvants by N-acylation, yields derivatives with linear alkylamides that show structural and functional changes. Structural, since the relatively unreactive added hydrophobic alkyl chains may modify these glycosides' conformation and micellar structure. Functional, because altering the availability of proposed pharmacophores, like fucose (Fucp) and aldehyde groups, to interact with their cellular receptors, may change these glycosides' adjuvanticity. While deacylated QS (DS-QS) adjuvants bias the response toward a sole anti-inflammatory Th2 immunity against an antigen, their N-alkylated derivatives carrying octyl to dodecylamide residues, modify that response to a pro-inflammatory Th1 immunity. As shown by their IgG2a/IgG1 titer ratios, which are higher than those for Th2 immunity. A result of the fact that in mice, the IgG2a levels are dependent on the direct influence of secreted interferon-γ (IFN-γ), a crucial Th1 cytokine. But addition of the longer and more lipophilic tetradecylamide group, yields derivatives that like DS-QS induce Th2 immunity, as shown by their low IgG2a/IgG1 ratio. Results that imply that changes in these analogs' conformation and micellar structure, would affect the immunomodulatory properties or adjuvanticity of N-acylated DS-QS. Physical changes that may alter the availability of groups like Fucp, to bind to its presumed dendritic cells' lectin receptor DC-SIGN; an essential step in the stimulation of Th2 immunity. Structural properties that in an aqueous environment, would depend on these glycosides' balance of their hydrophilic and lipophilic moieties (HLB), and the interactions of the newly introduced alkyl chain with the native QS' lipophilic triterpene aglycone and hydrophilic oligosaccharide chains. A situation that would explain these new derivatives' qualitative and quantitative changes in adjuvanticity.

A Bioinformatics Tool for Efficient Retrieval of High-Confidence Terpene Synthases (TPS) and Application to the Identification of TPS in Coffea and Quillaja.

Terpenoids are a class of compounds that are found in all living organisms. In plants, some terpenoids are part of primary metabolism, but most terpenes found in plants are classified as specialized metabolites, encoded by terpene synthases (TPS). It is not obvious how to assign the putative product of a given TPS using bioinformatics tools. Phylogenetic analyses easily assign TPS into families; however members of the same TPS family can synthetize more than one terpenoid-and, in many biotechnological applications, researchers are more interested in the product of a given TPS rather than its phylogenetic profile. Automated protein annotation can be used to classify TPS based on their products, despite the family they belong to. Here, we implement an automated bioinformatics method, search_TPS, to identify TPS proteins that synthesize mono, sesqui and diterpenes in Angiosperms. We verified the applicability of the method by classifying wet lab validated TPS and applying it to find TPS proteins in Coffea arabica, C. canephora, C. eugenioides, and Quillaja saponaria. Search_TPS is a computational tool based on PERL scripts that carries out a series of HMMER searches against a curated database of TPS profile hidden Markov models. The tool is freely available at https://github.com/liliane-sntn/TPS .

Soapbark Triterpenes: Quillaja brasiliensis Cell Culture Sapogenin and Free Sterol Analysis by GCMS.

Triterpene saponins of the genus Quillaja (Quillajaceae) are known for their immunoadjuvant, hypocholesterolemic, and anti-inflammatory activity. Plant cell cultures are useful for the study of saponin metabolism and industrial production of these bioactive compounds. While structurally related phytosterols are primary metabolites essential to growth and development, saponins are responsive to pathogen and abiotic stress, fulfilling roles in plant specialized metabolism. For cell culture production of saponins, phytosterols may be considered a competing pathway which relies on a common pool of cytosolic isoprenoid precursors.Understanding the metabolic allocation of resources between these two related pathways is key to maximizing saponin production in in vitro production systems. Sterols and saponins naturally occur in multiple conjugated forms, which complicate separation and quantification. The acid hydrolysis of conjugated sterols and saponins to their free forms is a useful technique to simplify their analysis by gas chromatography. Here we provide the workflow for the quantification of free sterols and sapogenins in cell cultures of Quillaja brasiliensis .

Formulation of IMXQB: Nanoparticles Based on Quillaja brasiliensis Saponins to be Used as Vaccine Adjuvants.

Adjuvants are essential components of subunit, recombinant, nonreplicating and killed vaccines, as they are substances that boost, shape, and/or enhance the immune response triggered by vaccination. Saponins obtained from the Chilean Q. saponaria tree are used as vaccine adjuvants in commercial vaccines, although they are scarce and difficult to obtain. In addition, tree felling is needed during its extraction, which has ecological impact. Q. brasiliensis leaf-extracted saponins arise as a more sustainable alternative, although its use is still limited to preclinical studies. Despite the remarkable immunostimulating properties of saponins, they are toxic to mammalian cells, due to their intrinsic characteristics. For these reasons they are mostly used in veterinary vaccines, although recently the Q. saponaria purified saponin QS-21 has been included in adjuvant systems for human vaccines, such as Mosquirix and Shingrix (GSK). In order to abrogate the toxicity of the saponins fractions, they can be formulated as immunostimulating complexes (ISCOMs). ISCOM-matrices are cage-like nanoparticles of approximately 40 nm, formulated combining saponins and lipids, without antigen, and are great adjuvants able to promote Th1-biased immune responses in a safe manner. Herein we describe how to formulate ISCOM-matrices nanoparticles using Q. brasiliensis purified saponin fractions (IMXQB) by the dialysis method. In addition, we indicate how to verify the appropriate size and homogeneity of the formulated nanoparticles.

Structure Elucidation of Triterpenoid Saponins Found in an Immunoadjuvant Preparation of Quillaja brasiliensis Using Mass Spectrometry and 1H and 13C NMR Spectroscopy.

An immunoadjuvant preparation (named Fraction B) was obtained from the aqueous extract of Quillaja brasiliensis leaves, and further fractionated by consecutive separations with silica flash MPLC and reverse phase HPLC. Two compounds were isolated, and their structures elucidated using a combination of NMR spectroscopy and mass spectrometry. One of these compounds is a previously undescribed triterpene saponin (Qb1), which is an isomer of QS-21, the unique adjuvant saponin employed in human vaccines. The other compound is a triterpene saponin previously isolated from Quillaja saponaria bark, known as S13. The structure of Qb1 consists of a quillaic acid residue substituted with a ß-d-Galp-(1→2)-[ß-d-Xylp-(1→3)]-ß-d-GlcpA trisaccharide at C3, and a ß-d-Xylp-(1→4)-α-l-Rhap-(1→2)-[α-l-Arap-(1→3)]-ß-d-Fucp moiety at C28. The oligosaccharide at C28 was further substituted at O4 of the fucosyl residue with an acyl group capped with a ß-d-Xylp residue.

The effects of a quillaja and yucca combination on performance and carcass traits of coccidia-vaccinated broilers exposed to an enteric disease challenge.

A series of 6 floor pen trials was conducted to determine the effects of a quillaja and yucca combination product on the performance and carcass traits of growing broiler chickens vaccinated for coccidiosis at the hatchery. In each of the trials graded levels (0, 250, and 500 ppm) of a quillaja and yucca combination (QY) were fed to Ross 708 broilers for the duration of each 42 d test. Trials were arranged in completely randomized block designs involving a minimum of 11 blocks per trial. At the start of each trial, pens contained 55 broilers. In order to provide each bird with an enteric disease challenge, 5 kg commercial broiler litter containing 104 CFU Clostridium perfringens per gram was placed in each pen. In addition, the sporulated oocysts of Eimeria acervulina and E. maxima were added to each pen at the outset of each test. At d 21 of the trials, coccidial lesion scores, mortality and performance were determined; final performance and total mortality were assessed at 42 d. At the completion of each test, 10 birds of average body weight per pen were selected for carcass evaluations; whole and chilled carcass yield were determined, and pre- and post-chill breast measurements were made. A combined analysis of the results of the 6 trials (75 replications per treatment) was used to determine treatment effects and each variable was assessed by linear regression analysis. Results indicated that QY significantly reduced mortality and coccidial lesions scores at d 21 (P < 0.05). Performance was significantly improved by both levels of QY at 21 and 42 d, and significant linear effects were observed for these variables (P < 0.05). All carcass characteristics were significantly improved by QY administration and significant linear responses were observed for each carcass variable (P < 0.05). These results indicate that by reducing intestinal disease challenge, QY provided linear improvements in performance. In addition, QY positively affected carcass parameters as each variable responded linearly to QY feeding (P < 0.05).

Off-line two-dimensional liquid chromatography separation for the quality control of saponins samples from Quillaja Saponaria.

Quil-A is a purified extract of saponins with strong immunoadjuvant activity. While specific molecules have been identified and tested in clinical trials, Quil-A is mostly used as a totum of the Quillaja Saponaria bark extract. Quality control of the extract stability is usually based on the monitoring of specific saponins, whereas the comparison of samples with an initial chromatogram seems more appropriate. A reference fingerprint based on comprehensive two-dimensional liquid chromatography offers a rapid detection of nonconform samples. To fulfill quality control constraints, off-line configuration using basic instrumentation was promoted. Hence, reversed-phase liquid chromatography × reversed-phase liquid chromatography and hydrophilic interaction chromatography × reversed-phase liquid chromatography methods with ultraviolet and single-quadrupole mass spectrometry detection were kinetically optimized. The reversed-phase liquid chromatography × reversed-phase liquid chromatography method used a pH switch between dimensions to maximize orthogonality. Despite diagonalization, it led to a high peak capacity of 831 in 2 h. On the other hand, the combination of hydrophilic interaction chromatography and reversed-phase liquid chromatography offered a larger orthogonality but a lower, yet satisfactory peak capacity of 673. The advantages of both methods were illustrated on degraded samples, where the reversed-phase liquid chromatography × reversed-phase liquid chromatography contour plot highlighted the loss of fatty acid chains, while the hydrophilic interaction chromatography × reversed-phase liquid chromatography method was found useful to evidence enzymatic loss of sugar moieties.

Zika Virus Envelope Domain III Recombinant Protein Delivered With Saponin-Based Nanoadjuvant From Quillaja brasiliensis Enhances Anti-Zika Immune Responses, Including Neutralizing Antibodies and Splenocyte Proliferation.

Nanoadjuvants that combine immunostimulatory properties and delivery systems reportedly bestow major improvements on the efficacy of recombinant, protein-based vaccines. Among these, self-assembled micellar formulations named ISCOMs (immune stimulating complexes) show a great ability to trigger powerful immunological responses against infectious pathogens. Here, a nanoadjuvant preparation, based on saponins from Quillaja brasiliensis, was evaluated together with an experimental Zika virus (ZIKV) vaccine (IQB80-zEDIII) and compared to an equivalent vaccine with alum as the standard adjuvant. The preparations were administered to mice in two doses (on days zero and 14) and immune responses were evaluated on day 28 post-priming. Serum levels of anti-Zika virus IgG, IgG1, IgG2b, IgG2c, IgG3 were significantly increased by the nanoadjuvant vaccine, compared to the mice that received the alum-adjuvanted vaccine or the unadjuvanted vaccine. In addition, a robust production of neutralizing antibodies and in vitro splenocyte proliferative responses were observed in mice immunized with IQB80-zEDIII nanoformulated vaccine. Therefore, the IQB80-zEDIII recombinant preparation seems to be a suitable candidate vaccine for ZIKV. Overall, this study identified saponin-based delivery systems as an adequate adjuvant for recombinant ZIKV vaccines and has important implications for recombinant protein-based vaccine formulations against other flaviviruses and possibly enveloped viruses.

The combination of quillaja and yucca saponins in broilers: effects on performance, nutrient digestibility and ileal morphometrics.

1. Two series of studies were conducted to determine the effects of a combination of ground plant material derived from Quillaja saponaria trees and Yucca schidigera plants (QY) as sources of saponin, on performance, productivity, nutrient digestibility and ileal morphology of growing broilers. In each trial, 480 Cobb male birds were allocated equally to 24 pens at one-day-of-age according to body weight2. The studies consisted of two identical floor pen trials in which performance and nutrient digestibility were assessed and two trials where performance and ileal morphology were determined. In each trial, 0, 250 or 500 ppm QY were included in feed given to the broilers from 1-35 or 1-42 d of age, respectively. Eight (digestibility) or 12 (morphology) randomised replicate pens were used.3. In the digestibility trials, two birds per pen were moved to metabolism cages at d 21. Excreta was collected for a five-day period (d 21 to 25) for the determination of apparent total tract digestibility of dry and organic matter, fat and ash and nitrogen retention. For intestinal morphology, ileal segments were collected from four birds/pen on d 21 to determine villus height and crypt depth. Performance data were collected in each trial series.4. Results showed that feeding graded levels of QY produced significant linear improvements in performance and productivity at d 35, and similar linear effects were observed for N retention and all apparent digestibility measurements. Morphology data showed that birds receiving 250 and 500 ppm QY had significantly increased villus height5. These results indicated that QY exerted a positive influence on the intestinal tract by increasing the absorptive surface and improving nutrient digestibility. These effects were considered to be associated with the performance improvements recorded in both experiments.

Concurrent use of saponins and live coccidiosis vaccines: the influence of a quillaja and yucca combination on anticoccidial effects and performance results of coccidia-vaccinated broilers.

A series of studies was conducted to determine the effects of a quillaja and yucca (saponin) combination (QY) product on postvaccination oocyst production, development of coccidial immunity, and final bird performance of broilers administered live coccidiosis vaccines. In all, 3 groups of tests were carried out. Study 1 evaluated the effects of QY (0 and 250 ppm) on oocyst per gram of feces (OPG) following vaccination at day-of-age; OPG were measured from 5 to 12 d postvaccination. Study 2 determined the effects of QY (250 ppm) in the presence of 3 commercial coccidiosis vaccines in floor pens. OPG were measured weekly for birds receiving each vaccine and for each corresponding vaccine group fed QY. To determine whether QY influenced the development of coccidial immunity induced by the 3 vaccines, 5 birds were removed from each pen at 28 d and challenged with pathogenic levels of Eimeria spp. At 6 d post challenge, lesion scores were used to evaluate the effects of QY on immune protection provided by each vaccine. In addition, comparisons of final bird performance were made between birds given each vaccine and their corresponding vaccinates fed QY. Study 3 comprised a meta-analysis of 15 floor pen trials in which 21- and 42-d body weight, feed conversions, and total mortality were compared between coccidiosis-vaccinated broilers and similarly vaccinated broilers fed QY (250 ppm). Results of these experiments indicated that feeding QY to vaccinated broilers did not significantly affect OPG from days 5 through 12 postvaccination (P > 0.05). For each vaccine tested in study 2, OPG values were the highest at 14 and 21 d postvaccination. QY significantly reduced OPG at 14 d postvaccination for 2 of the vaccines tested, and produced a similar effect in 1 vaccine at 21 d postvaccination. The remaining vaccine was not affected by QY in the postvaccination OPG results. Despite these changes in OPG, significant differences in lesion scores following the Eimeria challenge were not observed for any vaccinated groups receiving QY. Irrespective of the vaccine, both interim and final feed conversion values were significantly improved when QY was fed (P < 0.01). Similarly, results of a 15-trial meta-analysis indicated that QY-fed vaccinated broilers had higher body weights, improved feed conversions, and lower mortality than their vaccinated controls. Results show that while QY may induce changes in OPG following vaccination, coccidia-vaccinated broilers fed QY develop immunity equivalent to that of controls and show significant improvements in performance and mortality.

IMXQB-80: A Quillaja brasiliensis saponin-based nanoadjuvant enhances Zika virus specific immune responses in mice.

Vaccine adjuvants are compounds that enhance/prolong the immune response to a co-administered antigen. Saponins have been widely used as adjuvants for many years in several vaccines - especially for intracellular pathogens - including the recent and somewhat revolutionary malaria and shingles vaccines. In view of the immunoadjuvant potential of Q. brasiliensis saponins, the present study aimed to characterize the QB-80 saponin-rich fraction and a nanoadjuvant prepared with QB-80 and lipids (IMXQB-80). In addition, the performance of such adjuvants was examined in experimental inactivated vaccines against Zika virus (ZIKV). Analysis of QB-80 by DI-ESI-ToF by negative ion electrospray revealed over 29 saponins that could be assigned to known structures existing in their congener Q. saponaria, including the well-studied QS-21 and QS-7. The QB-80 saponins were a micrOTOF able to self-assembly with lipids in ISCOM-like nanoparticles with diameters of approximately 43 nm, here named IMXQB-80. Toxicity assays revealed that QB-80 saponins did present some haemolytical and cytotoxic potentials; however, these were abrogated in IMXQB-80 nanoparticles. Regarding the adjuvant activity, QB-80 and IMXQB-80 significantly enhanced serum levels of anti-Zika virus IgG and subtypes (IgG1, IgG2b, IgG2c) as well as neutralized antibodies when compared to an unadjuvanted vaccine. Furthermore, the nanoadjuvant IMXQB-80 was as effective as QB-80 in stimulating immune responses, yet requiring fourfold less saponins to induce the equivalent stimuli, and with less toxicity. These findings reveal that the saponin fraction QB-80, and particularly the IMXQB-80 nanoadjuvant, are safe and capable of potentializing immune responses when used as adjuvants in experimental ZIKV vaccines.

A Two-Step Orthogonal Chromatographic Process for Purifying the Molecular Adjuvant QS-21 with High Purity and Yield.

QS-21 is a triterpene glycoside saponin found in the bark of the Chilean soap bark tree Quillaja saponaria. It is a highly potent vaccine adjuvant that is included in two approved vaccines and has shown promise in numerous other vaccine candidates in the research and clinical pipelines. One major hurdle to the widespread use of this adjuvant is the difficulty of obtaining it in high yield and purity. Previously reported purification approaches either showed suboptimal purity and/or yield, lacked efficiency, or had strict requirement on the composition of the starting material. Here, we report the development of a new two-step orthogonal chromatographic process, consisting of a polar reversed-phase (RP) chromatography step followed by a hydrophilic interaction chromatography (HILIC) step, for purifying QS-21 from a commercially available Quillaja saponaria bark extract with high yield and > 97% purity. This process makes available a simple and efficient method for obtaining highly pure QS-21 from saponin-enriched bark extract.