Regulate structure and properties of κ-carrageenan/konjac glucomannan composite hydrogel by filling effects of Quillaja saponin-stabilized solid lipid nanostructure.

κ-Carrageenan/konjac glucomannan (κ-CA/KGM) composite hydrogels often fail to meet industrial requirements due to their low gel strength and poor mechanical properties, while solid lipid nanoparticles are potential materials to address this challenge due to their good biocompatibility. In the study, we propose using Quillaja saponin-stabilized solid lipid nanoparticle (QSLN) as nanofillers to enhance properties of κ-carrageenan/konjac glucan (κ-CA/KGM) composite hydrogels, and with emphasis on the effect of QSLN filling concentration on the structure and properties of composite hydrogels and the possible mechanisms were investigated. The best performance of QSLN-filled composite hydrogels was achieved at the QSLN concentration of 2.4 %. QSLN was uniformly distributed in the hydrogel matrix and formed electrostatic interactions and hydrogen bonding interactions with the matrix at an appropriate filling level, which enhanced the textural and rheological properties of the hydrogel greatly. In addition, the results of low-field NMR experiments showed that the filling of QSLN reduced the water mobility by enhancing the entanglement of polymer chains in the hydrogel matrix, which improved the freeze-thaw stability and regulated the swelling and deswelling behavior of the composite hydrogel. However, with the increasing of QSLN filling concentration, the above improvements were weakened by the depletion of van der Waals interactions due to the large amount of QSLN aggregation and the weakening of electrostatic interaction. In turn, the hydrogel was found to modulate the crystalline behavior of QSLN by X-ray diffraction and differential scanning calorimeter monitoring. Overall, the optimal synergistic effect between structure and properties could be achieved when the QSLN filling concentration was 2.4 %. These results provide a basis for the development of products that require excellent gel properties and structure.

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.

Surface-active natural saponins. Properties, safety, and efficacy.

In the future, cleaning products must fulfil the principles of green chemistry while maintaining efficacy against bacteria. This study aims to evaluate the detergent properties, ecotoxicity, and anti-biofilm potential of natural saponins compared to synthetic surfactants. We tested sodium dodecyl sulphate, quillaja saponin, escin, and sapogenin for emulsifying capacity, critical micelle concentration, ecotoxicity to yeast, and antibacterial and anti-biofilm potential against bacteria. The results show that the emulsifying capacities of quillaja saponin and sodium dodecyl sulphate are similar, while the critical micelle concentration for quillaja saponin is much lower . Furthermore, the antibacterial and antibiofilm potentials are much higher for quillaja saponin than for synthetic sodium dodecyl sulphate . Moreover, we have shown that natural saponins are less toxic to the S. cerevisiae than synthetic saponin is. All these facts indicate that quillaja is a suitable candidate to replace synthetic products as it meets the requirements of efficacy and safety.

Evaluation of Quil-A, E. coli DNA and Montanide™ ISA 206 adjuvant combination on the antibody response to foot-and-mouth disease vaccine in sheep.

Vaccination is one of the basic strategies in the fight against foot-and-mouth disease (FMD) in endemic regions. Today, commercially available FMD vaccines are prepared with inactive whole virion, which has low immunogenicity. Therefore, considerable effort has been devoted to finding novel adjuvants. Although mineral oils are among the most common adjuvants, it is still difficult to provide a long-term and robust immune response. Combined adjuvant systems are currently being studied to solve the problem. Saponins and CpG-ODNs have been shown to increase the immune response to vaccines individually in various studies. In this study, the effect of different adjuvants and their combinations (Quil-A, E. coli DNA, and MontanideTM ISA 206) on total and neutralizing antibody response in sheep was investigated. According to the results, the Quil-A group induced the highest antibody level, followed by the combination of Quil-A and the E. coli DNA group. The group containing E. coli DNA also caused a higher antibody response than the group containing only MontanideTM ISA 206 for certain days of sampling. These affordable alternatives of saponin and CpG sources can be used individually to increase the potency of the FMD vaccine for mass vaccinations of sheep. Keywords: foot-and-mouth disease; vaccine; adjuvant; Quil-A; E. coli DNA; combination of adjuvants.

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.

Aeromonas veronii infection remarkably increases expression of lysozymes in grass carp (Ctenopharyngodon idellus) and injection of lysozyme expression cassette along with QCDC adjuvant significantly upregulates immune factors and decreases cumulative mortality.

Aeromonas veronii AvX005 is a pathogenic bacterium with high toxicity to grass carp (Ctenopharyngodon idellus). The expression levels of g-type (goose-type lysozyme, Lys-g) and c-type lysozyme (chicken-type lysozyme, Lys-c) in the spleen of grass carp infected with AvX005 were significantly increased by approximately 4.5 times and 27 times, respectively. The recombinant proteins rLys-g and rLys-c produced in a recombinant expression system of Escherichia coli showed significant antibacterial activity against the pathogenic bacteria AvX005. A challenge test was conducted after rLys-g and rLys-c were expressed in grass carp L8824 liver cells, and compared with the survival rate of the control cells (46.3%), the survival rate of the experimental cells (77.6% for rLys-g and 68.6% for rLys-c) was significantly increased. Grass carp were infected with AvX005 on the second day after delivering pcDNA3.1-lys-g and pcDNA-lys-c with the Quil A/cholesterol/DDA/Carbopol (QCDC) adjuvant, and both pcDNA3.1-lys-g and pcDNA-lys-c provided 70% relative protection for grass carp. The activity of lysozyme and alkaline phosphatase in the serum of grass carp was significantly increased after injection of DNA. The expression of the immune factors IgM, C3 and IL8 in the kidney was upregulated to varying degrees for pcDNA3.1-lys-g and immune factors C3 and IgM was upregulated for pcDNA-lys-c. The results indicated that pcDNA3.1-lys-g and pcDNA-lys-c may be used as immunostimulants to protect grass carp from the pathogenic bacterium AvX005.

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.

Formulation and physicochemical stability of oil-in-water nanoemulsion loaded with α-terpineol as flavor oil using Quillaja saponins as natural emulsifier.

Alpha-terpineol (α-TOH) is a promising monoterpenoid detaining several biological activities. However, as a volatile molecule, the incorporation of α-TOH within formulated products poses several challenges related to its stability. In this sense, nanoencapsulation works as a key technology to protect the bioactivity of low molecular weight oils, like α-TOH, against environmental stresses (heat, light, and moisture), mitigating their susceptibility to degradation (oxidation and volatilization). Physical properties of encapsulated flavor/essential oil have been extensively reported, whereas there is a lack in the literature regarding their chemical stability, which is usually the main purpose of encapsulation. Thus, in this study, the physicochemical stability of the formulated oil-in-water nanoemulsion loaded with α-TOH stabilized with Quillaja saponins (QSs) as a natural emulsifier (α-TOH-QSs-NE) were tracked in a long-term (up to 280th day). Along with time, mean droplet diameter (MDD) and turbidity were used as a reference for physical parameters; while the chemical stability was monitored using gas chromatography analysis to quantify the mark content of α-TOH into the NE. Results indicated that α-TOH-QSs-NE was successfully formulated with a high-load amount of α-TOH (90 mg mL-1). α-TOH-QSs-NE showed great physicochemical stability regardless the storage-temperature (5 °C or 25 °C) up to 280th day, with no significant alterations in the MDD or turbidity, where c.a. 79% of the initial amount of the nanoemulsified α-TOH remained detectable in α-TOH-QSs-NEs, with no finding of degradation products. Thus, the data here disclosure may be useful for innovative application of α-TOH in foodstuffs.

Matrix M Adjuvanted H5N1 Vaccine Elicits Broadly Neutralizing Antibodies and Neuraminidase Inhibiting Antibodies in Humans That Correlate With In Vivo Protection.

The highly pathogenic avian influenza H5N1 viruses constantly evolve and give rise to novel variants that have caused widespread zoonotic outbreaks and sporadic human infections. Therefore, vaccines capable of eliciting broadly protective antibody responses are desired and under development. We here investigated the magnitude, kinetics and protective efficacy of the multi-faceted humoral immunity induced by vaccination in healthy adult volunteers with a Matrix M adjuvanted virosomal H5N1 vaccine. Vaccinees were given escalating doses of adjuvanted vaccine (1.5µg, 7.5µg, or 30µg), or a non-adjuvanted vaccine (30µg). An evaluation of sera from vaccinees against pseudotyped viruses covering all (sub)clades isolated from human H5N1 infections demonstrated that the adjuvanted vaccines (7.5µg and 30µg) could elicit rapid and robust increases of broadly cross-neutralizing antibodies against all clades. In addition, the adjuvanted vaccines also induced multifaceted antibody responses including hemagglutinin stalk domain specific, neuraminidase inhibiting, and antibody-dependent cellular cytotoxicity inducing antibodies. The lower adjuvanted dose (1.5µg) showed delayed kinetics, whilst the non-adjuvanted vaccine induced overall lower levels of antibody responses. Importantly, we demonstrate that human sera post vaccination with the adjuvanted (30µg) vaccine provided full protection against a lethal homologous virus challenge in mice. Of note, when combining our data from mice and humans we identified the neutralizing and neuraminidase inhibiting antibody titers as correlates of in vivo protection.

Recent Advances and Applications of Plant-Based Bioactive Saponins in Colloidal Multiphase Food Systems.

The naturally occurring saponins exhibit remarkable interfacial activity and also possess many biological activities linking to human health benefits, which make them particularly attractive as bifunctional building blocks for formulation of colloidal multiphase food systems. This review focuses on two commonly used food-grade saponins, Quillaja saponins (QS) and glycyrrhizic acid (GA), with the aim of clarifying the relationship between the structural features of saponin molecules and their subsequent self-assembly and interfacial properties. The recent applications of these two saponins in various colloidal multiphase systems, including liquid emulsions, gel emulsions, aqueous foams and complex emulsion foams, are then discussed. A particular emphasis is on the unique use of GA and GA nanofibrils as sole stabilizers for fabricating various multiphase food systems with many advanced qualities including simplicity, ultrastability, stimulability, structural viscoelasticity and processability. These natural saponin and saponin-based colloids are expected to be used as sustainable, plant-based ingredients for designing future foods, cosmetics and pharmaceuticals.

Discrepancies in the German Pharmacopoeia procedure for quality control of Quillaja saponin extracts.

This study focused on the evaluation of Quillaja saponin extracts with the additional quality designation DAB-which means the abbreviation of the German Pharmacopoeia (Deutsches Arzneibuch). This label suggests that Quillaja saponin extracts marked in this way are of pharmacopoeial quality and thus stand out from other Quillaja saponin extracts. The DAB ninth edition listed Quillaia saponin as a reagent. According to DAB, its quality must be checked by thin-layer chromatography (TLC), and three closely spaced zones in a defined retention factor (Rf) interval specify the saponin reagent. All the Quillaja saponin extracts obtained from different manufacturers and labeled as DAB quality complied with the TLC test. However, the analysis with high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (HPLC-Q-ToF-MS) clearly showed additionally an intense peak pattern of Madhuca saponins in all measured samples. The TLC test for Mahua seed cake, which is the press residue from Madhuca longifolia, surprisingly showed the same three closely spaced zones in the defined Rf interval. The three zones could be identified as Mi-saponins from Madhuca after scraping and extracting them from the stationary phase of the TLC plate and subsequent measurement by HPLC-Q-ToF-MS. Therefore, the specification of the saponin reagent in DAB characterizes erroneously Madhuca saponins that are not listed as a saponin plant source for the saponin reagent.

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.

Polymeric matrix hydrophobicity governs saponin packing-density on nanoparticle surface and the subsequent biological interactions.

This study investigated the loading behavior of Quillaja saponin as a model surface-active cargo on (NP) nanoparticles prepared with various hydrophobic polymers and using different organic solvents through emulsification/solvent evaporation, and the impact of NP surface hydrophobicity upon the cytotoxic and hemolytic properties of the loaded entity. A superficial monolayered arrangement of saponins on NP was established (R2 > 0.9) for all NP, as the saponin loading values complied with the Langmuir adsorption isotherm over the entire concentration range. Next, based on the measurement of interfacial tension between formulation phases, and the subsequent use of Gibb's adsorption isotherm, the packing density (Гexc) and loading of saponins on various nanospheres could be predicted with good correlation with the actual values (R2 > 0.95). The results demonstrated that the hydrophobicity of the polymeric matrix was the major determinant of saponin packing density on the nanospheres. Finally, the impact of NP surface properties upon saponin biological interactions was investigated, where a linear correlation was found between the NP surface hydrophobicity and their hemolytic properties (R2 â‰… 0.79), and cytotoxicity against two cancer cell lines (R2 > 0.76). The surface hydrophobicity of the polymeric NP seemingly governed the NP-cell membrane binding, which in turn determined the amount of membrane-bound saponins per unit NP surface area. As the saponins exert their cytotoxicity mainly through strong permeabilization of the cell membrane, a higher amount of NP-membrane association governed by a more hydrophobic matrix can lead to higher levels of cytotoxicity. These findings highlight the importance of a detailed characterization of NP surface properties, particularly in case of surface-active cargos, for these dictate the side effects and biological interactions of the delivery system.

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.

Oil-Water Interfacial-Directed Spontaneous Self-Assembly of Natural Quillaja Saponin for Controlling Interface Permeability in Colloidal Emulsions.

Assembly of amphiphiles at the interface of two immiscible fluids is of great scientific and technological interest in offering efficient routes to smart vehicles for functional deliveries. Natural Quillaja saponin (QS) has gathered widespread interest within the scientific community as a result of its unique interfacial properties. Herein, spontaneously interface-driven self-assembly (SIDSA) of QS at the oil-water interface was systematically studied by morphology and spectroscopy. It was found to self-assemble into a micrometer-scale network in helical fibers by combined intermolecular π-π stacking and hydrogen bonding among saponins at the liquid-liquid interface. From SIDSA, multilayer films on the surfaces of dispersed droplets were formed and enhanced emulsion stability. Interfacial QS-based films on droplet surfaces were also shown to confine interfacial diffusion processes by serving as transport barriers. Furthermore, they can be exploited to control the release of volatiles from the dispersed liquid phase by regulating the interface film, which is shown by molecular dynamics to occur through a hydrogen-bonded mechanism. These results provide new insight into the interfacial assembly structure that can enable unique controllable release in a broad range of applications in food, beverages, pharmaceuticals, and cosmetics.

Formation and stability of emulsions stabilized by Quillaja saponin-egg lecithin mixtures.

Knowledge of binary emulsifiers' influence on the formation and stability of emulsion-based products is still limited. The aim of this study was to investigate the emulsifying properties of Quillaja saponin-egg lecithin mixtures at different concentration ratios (r = 5:0, 4:1, 3:2, 2:3, 1:4, and 0:5) with total emulsifier concentration set to 0.5% or 1.0% (w/w). For this, oil-in-water emulsions (10% oil, pH 7) were prepared via high-pressure homogenization. Furthermore, emulsion stability against different environmental stresses was tested. All the binary emulsifier mixtures formed submicron sized emulsions upon homogenization. The most stable emulsions among the mixed emulsifiers were obtained at low Quillaja saponin concentration at r = 1:4 that showed similar physical stability over time to emulsions stabilized by Quillaja saponins and egg lecithin alone. The data suggested that the mixtures of Quillaja saponins and egg lecithins built mixed interfacial layers that were prone to changes over time. Emulsions stabilized by the binary mixtures were in general less stable against changes in pH and ionic strength than the emulsions stabilized by the individual emulsifiers. An exception were the emulsions at r = 1:4 that showed improved stability at pH 2 over the phase separated Quillaja saponin-stabilized emulsions at the same pH. Moreover, all the emulsions were heat stable up to 90 °C. On the other hand, none of the emulsions were stable upon freeze-thawing. These results increase our understanding of technofunctionality of binary emulsifier systems. PRACTICAL APPLICATION: Food-grade and natural emulsifier mixtures composed of Quillaja saponins and egg lecithin may be used in selected emulsion-based food or personal care product applications to replace synthetic surfactants due to issues with consumer acceptance and regulatory restrictions.

Increased susceptibility to Haemonchus contortus infection by interleukin-5 modulation of eosinophil responses in sheep.

Eosinophils are prominent effector cells in immune responses against gastrointestinal nematode infections in ruminants, but their in vivo role has been hard to establish in large animals. Interleukin-5 is a key cytokine in the induction and stimulation of anti-parasitic eosinophil responses. This study attempted to modulate the eosinophil response in sheep through vaccination with recombinant interleukin-5 (rIL-5) and determine the effect on subsequent Haemonchus contortus infection. Nematode-resistant Canaria Hair Breed (CHB) sheep vaccinated with rIL-5 in Quil-A adjuvant, had lower blood eosinophil counts and higher mean worm burdens than control sheep vaccinated with Quil-A adjuvant alone. In addition, adult worms in IL-5-vaccinated sheep were significantly longer with higher eggs in utero in female worms, supporting an active role of eosinophils against adult parasites in CHB sheep. These results confirm that eosinophils can play a direct role in effective control of H contortus infection in sheep and offer a new approach to study immune responses in ruminants.

Construction and immunological evaluation of recombinant adenovirus vaccines co-expressing GP3 and GP5 of EU-type porcine reproductive and respiratory syndrome virus in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) keeps causing economic damages in the swine sector across the globe. There has been emergence of the European (EU) genotype of porcine reproductive and respiratory syndrome virus (Genotype-I PRRSV) in China in recent years. The presently available vaccines cannot unable to provide safeguard against PRRSV infection completely. This study was aimed to construct recombinant adenovirus expressing the ORF3 and ORF5 genes of the EU-type PRRSV strain. Then, the recombinant adenovirus vaccines for EU-type PRRSV (rAd-E3518, rAd-E35, rAd-E3 and rAd-E5) which we constructed and evaluated were constructed and identified by western blot and PCR. All recombinant adenovirus vaccines were evaluated for humoral and cellular responses and EU-type PRRSV challenge in pigs. The results showed that the group of rAd-E3518+Quil A developed higher GP3 and GP5 specific antibody responses compared to the group of rAd-E3518. The majority of the neutralizing antibody titers were higher than 1:16 (P<0.05), the fusion of IL-18 has increased significantly PRRSV-stimulated secretion of IFN-γ and IL-4 in porcine serum, the group of rAd-E3518+Quil A produced highest T-lymphocytes (CD3+CD4+ and CD3+CD8+ T cells) proliferative in peripheral blood of pigs. The animals were challenged with the EU-type PRRSV strain and the viral load was detected in the several tissues, the viral load of rAd-E3518 and rAd-E3518+Quil A were lower than the wild-type adenovirus group. Our findings provide evidence to confirm that the recombinant adenovirus vaccine can protect pigs from EU-PRRSV infection.

Fabrication and characterization of nanostructured lipid carriers (NLC) using a plant-based emulsifier: Quillaja saponin.

Nanostructured lipid carriers (NLCs) are a type of colloidal delivery system that was developed in the pharmaceutical industry to combine the advantages and eliminate the shortcomings of oil-in-water (O/W) nanoemulsions and solid lipid nanoparticles (SLNs). The hydrophobic core of the particles within NLCs consists of a solidified fat phase with a partially disorganized structure, which inhibits morphological changes and bioactive expulsion. In the present study, we formulated NLCs using a hot-homogenization approach using fully hydrogenated soybean oil (HSO) as the lipid phase and quillaja saponins as a natural surfactant. The NLCs formed had a low viscosity and milky white appearance similar to that of O/W nanoemulsions. The fabrication conditions were optimized, including the number of passes through the microfluidizer, stirring conditions, cooling rate, and emulsifier level. Unlike bulk HSO, the emulsified form had to be supercooled substantially to promote crystallization of the lipid droplets, which was attributed to differences in nucleation behavior. The crystallization temperature decreased with increasing saponin concentration, which was probably because smaller droplets were formed at higher emulsifier levels. For instance, at 3, 6, 9, and 12 wt% saponin, the degree of supercooling was 10, 15, 18, and 18 °C, while the mean particle diameter was 0.82, 0.53, 0.41, and 0.44 µm, respectively. The melting and crystallization behavior of the NLCs was characterized using an optical microscope and differential scanning calorimetry (DSC), while the morphology of the NLCs was characterized using transmission electron microscopy (TEM). This analysis showed that the NLCs contained spherical particles with a crystallization temperature around 31 °C. This information may be useful for formulating NLC from natural ingredients for application in the food and beverage industry.

QuilA-Adjuvanted T. gondii Lysate Antigens Trigger Robust Antibody and IFNγ+ T Cell Responses in Pigs Leading to Reduction in Parasite DNA in Tissues Upon Challenge Infection.

Toxoplasma gondii is an intracellular parasite of all mammals and birds, responsible for toxoplasmosis. In healthy individuals T. gondii infections mostly remain asymptomatic, however this parasite causes severe morbidity and mortality in immunocompromised patients and congenital toxoplasmosis in pregnant women. The consumption of raw or undercooked pork is considered as an important risk factor to develop toxoplasmosis in humans. Since effective therapeutic interventions to treat toxoplasmosis are scarce, vaccination of meat producing animals may prevent T. gondii transmission to humans. Here, we evaluated the elicited immune responses and the efficacy of a potential vaccine candidate, generated by size fractionation of T. gondii lysate proteins, to reduce the parasite burden in tissues from experimentally T. gondii infected pigs as compared to vaccination with total lysate antigens (TLA). Our results show that both the vaccine candidate and the TLA immunization elicited strong serum IgG responses and elevated percentages of CD4+CD8+IFNγ+ T cells in T. gondii infected pigs. However, the TLA vaccine induced the strongest immune response and reduced the parasite DNA load below the detection limit in brain and skeletal muscle tissue in most animals. These findings might inform the development of novel vaccines to prevent T. gondii infections in livestock species and humans.