Differentially Expressed Genes in Response to a Squalene-Supplemented Diet Are Accurate Discriminants of Porcine Non-Alcoholic Steatohepatitis.

Squalene is the major unsaponifiable component of virgin olive oil, the fat source of the Mediterranean diet. To evaluate its effect on the hepatic transcriptome, RNA sequencing was carried out in two groups of male Large White x Landrace pigs developing nonalcoholic steatohepatitis by feeding them a high fat/cholesterol/fructose and methionine and choline-deficient steatotic diet or the same diet with 0.5% squalene. Hepatic lipids, squalene content, steatosis, activity (ballooning + inflammation), and SAF (steatosis + activity + fibrosis) scores were analyzed. Pigs receiving the latter diet showed hepatic squalene accumulation and twelve significantly differentially expressed hepatic genes (log2 fold change < 1.5 or <1.5) correlating in a gene network. These pigs also had lower hepatic triglycerides and lipid droplet areas and higher cellular ballooning. Glutamyl aminopeptidase (ENPEP) was correlated with triglyceride content, while alpha-fetoprotein (AFP), neutralized E3 ubiquitin protein ligase 3 (NEURL3), 2'-5'-oligoadenylate synthase-like protein (OASL), and protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B) were correlated with activity reflecting inflammation and ballooning, and NEURL3 with the SAF score. AFP, ENPEP, and PPP1R1B exhibited a remarkably strong discriminant power compared to those pathological parameters in both experimental groups. Moreover, the expression of PPP1R1B, TMEM45B, AFP, and ENPEP followed the same pattern in vitro using human hepatoma (HEPG2) and mouse liver 12 (AML12) cell lines incubated with squalene, indicating a direct effect of squalene on these expressions. These findings suggest that squalene accumulated in the liver is able to modulate gene expression changes that may influence the progression of non-alcoholic steatohepatitis.

Analysis of Antidiabetic Activity of Squalene via In Silico and In Vivo Assay.

Squalene has been tested widely in pharmacological activity including anticancer, antiinflammatory, antioxidant, and antidiabetic properties. This study aims to examine antidiabetic activity of squalene in silico and in vivo models. In the in silico model, the PASS server was used to evaluate squalene antidiabetic properties. Meanwhile, the in vivo model was conducted on a Type 2 Diabetes Mellitus (T2DM) with the rats separated into three groups. These include squalene (160 mg/kgbw), metformin (45 mg/kgbw), and diabetic control (DC) (aquades 10 mL/kgbw) administered once daily for 14 days. Fasting Blood Glucose Level (FBGL), Dipeptidyl Peptidase IV (DPPIV), leptin, and Superoxide Dismutase (SOD) activity were measured to analysis antidiabetic and antioxidant activity. Additionally, the pancreas was analysed through histopathology to examine the islet cell. The results showed that in silico analysis supported squalene antidiabetic potential. In vivo experiment demonstrated that squalene decreased FBGL levels to 134.40 ± 16.95 mg/dL. The highest DPPIV level was in diabetic control- (61.26 ± 15.06 ng/mL), while squalene group showed the lowest level (44.09 ± 5.29 ng/mL). Both metformin and squalene groups showed minor pancreatic rupture on histopathology. Leptin levels were significantly higher (p < 0.05) in diabetic control group (15.39 ± 1.77 ng/mL) than both squalene- (13.86 ± 0.47 ng/mL) and metformin-treated groups (9.22 ± 0.84 ng/mL). SOD activity were higher in both squalene- and metformin-treated group, particularly 22.42 ± 0.27 U/mL and 22.81 ± 0.08 U/mL than in diabetic control (21.88 ± 0.97 U/mL). In conclusion, in silico and in vivo experiments provide evidence of squalene antidiabetic and antioxidant properties.

A-910823, a squalene-based emulsion adjuvant, induces T follicular helper cells and humoral immune responses via α-tocopherol component.

Background: Adjuvants are chemical or biological materials that enhance the efficacy of vaccines. A-910823 is a squalene-based emulsion adjuvant used for S-268019-b, a novel vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is currently in clinical development. Published evidence has demonstrated that A-910823 can enhance the induction of neutralizing antibodies against SARS-CoV-2 in humans and animal models. However, the characteristics and mechanisms of the immune responses induced by A-910823 are not yet known. Methods and Results: To characterize A-910823, we compared the adaptive immune response profile enhanced by A-910823 with that of other adjuvants (AddaVax, QS21, aluminum salt-based adjuvants, and empty lipid nanoparticle [eLNP]) in a murine model. Compared with other adjuvants, A-910823 enhanced humoral immune responses to an equal or greater extent following potent T follicular helper (Tfh) and germinal center B (GCB) cell induction, without inducing a strong systemic inflammatory cytokine response. Furthermore, S-268019-b containing A-910823 adjuvant produced similar results even when given as a booster dose following primary administration of a lipid nanoparticle-encapsulated messenger RNA (mRNA-LNP) vaccine. Preparation of modified A-910823 adjuvants to identify which components of A-910823 play a role in driving the adjuvant effect and detailed evaluation of the immunological characteristics induced by each adjuvant showed that the induction of humoral immunity and Tfh and GCB cell induction in A-910823 were dependent on α-tocopherol. Finally, we revealed that the recruitment of inflammatory cells to the draining lymph nodes and induction of serum cytokines and chemokines by A-910823 were also dependent on the α-tocopherol component. Conclusions: This study demonstrates that the novel adjuvant A-910823 is capable of robust Tfh cell induction and humoral immune responses, even when given as a booster dose. The findings also emphasize that α-tocopherol drives the potent Tfh-inducing adjuvant function of A-910823. Overall, our data provide key information that may inform the future production of improved adjuvants.

Dietary squalene supplementation decreases triglyceride species and modifies phospholipid lipidomic profile in the liver of a porcine model of non-alcoholic steatohepatitis.

Squalene is a key minor component of virgin olive oil, the main source of fat in the Mediterranean diet, and had shown to improve the liver metabolism in rabbits and mice. The present research was carried out to find out whether this effect was conserved in a porcine model of hepatic steatohepatitis and to search for the lipidomic changes involved. The current study revealed that a 0.5% squalene supplementation to a steatotic diet for a month led to hepatic accumulation of squalene and decreased triglyceride content as well as area of hepatic lipid droplets without influencing cholesterol content or fiber areas. However, ballooning score was increased and associated with the hepatic squalene content. Of forty hepatic transcripts related to lipid metabolism and hepatic steatosis, only citrate synthase and a non-coding RNA showed decreased expressions. The hepatic lipidome, assessed by liquid chromatography-mass spectrometry in a platform able to analyze 467 lipids, revealed that squalene supplementation increased ceramide, Cer(36:2), and phosphatidylcholine (PC[32:0], PC[33:0] and PC[34:0]) species and decreased cardiolipin, CL(69:5), and triglyceride (TG[54:2], TG[55:0] and TG[55:2]) species. Plasma levels of interleukin 12p40 increased in pigs receiving the squalene diet. The latter also modified plasma lipidome by increasing TG(58:12) and decreasing non-esterified fatty acid (FA 14:0, FA 16:1 and FA 18:0) species without changes in total NEFA levels. Together this shows that squalene-induced changes in hepatic and plasma lipidomic profiles, non-coding RNA and anti-inflammatory interleukin are suggestive of an alleviation of the disease despite the increase in the ballooning score.

Media Supplementation with Mannitol and Biotin Enhances Squalene Production of Thraustochytrium ATCC 26185 through Increased Glucose Uptake and Antioxidative Mechanisms.

Media supplementation with exogenous chemicals is known to stimulate the accumulation of important lipids produced by microalgae and thraustochytrids. However, the roles of exogenous chemicals in promoting and preserving the terpenoids pool of thraustochytrids have been rarely investigated. Here, we realized the effects of two media supplements-mannitol and biotin-on the biomass and squalene production by a thraustochytrid strain (Thraustochytrium sp. ATCC 26185) and elucidated their mechanism of action. A significant change in the biomass was not evident with the exogenous addition of these supplements. However, with mannitol (1 g/L) supplementation, the ATCC 26185 culture achieved the best concentration (642 ± 13.6 mg/L) and yield (72.9 ± 9.6 mg/g) of squalene, which were 1.5-fold that of the control culture (non-supplemented). Similarly, with biotin supplementation (0.15 mg/L), the culture showed 459 ± 2.9 g/L and 55.7 ± 3.2 mg/g of squalene concentration and yield, respectively. The glucose uptake rate at 24 h of fermentation increased markedly with mannitol (0.31 g/Lh-1) or biotin (0.26 g/Lh-1) supplemented culture compared with non-supplemented culture (0.09 g/Lh-1). In addition, the reactive oxygen species (ROS) level of culture supplemented with mannitol remained alleviated during the entire period of fermentation while it alleviated after 24 h with biotin supplementation. The ∆ROS with mannitol was better compared with biotin supplementation. The total antioxidant capacity (T-AOC) of the supplemented culture was more than 50% during the late stage (72-96 h) of fermentation. Our study provides the potential of mannitol and biotin to enhance squalene yield and the first lines of experimental evidence for their protective role against oxidative stress during the culture of thraustochytrids.

Antiplasmodial and cytotoxic effects of the methanol extract, canthinone alkaloids, squalene- and protolimonoid-type triterpenes from Homalolepis suffruticosa roots.

ETHNOPHARMACOLOGICAL RELEVANCE: Different species of the Simaroubaceae family are used in traditional medicine to treat malaria. Among these is Homalolepis suffruticosa (syn. Simaba suffruticosa and Quassia suffruticosa), which is native to Central Brazil and popularly known as calunga. However, there is a lack of investigation concerning its antimalarial effects. AIM OF THE STUDY: To investigate the antiplasmodial and cytotoxic effects of the isolated metabolites and methanol extract from H. suffruticosa roots as well as to conduct the dereplication of this extract aiming to characterize its metabolic profile by UPLC-DAD-ESI-MS/MS. MATERIALS AND METHODS: Methanol extract of the H. suffruticosa roots and six isolated compounds were evaluated against chloroquine-resistant Plasmodium falciparum W2 strain by the PfLDH method and cytotoxicity in HepG2 cells by the MTT assay. Dereplication of the extract was performed by UPLC-DAD-ESI-MS/MS. RESULTS: The six isolated compounds disclosed high to moderate antiplasmodial activity (IC50 0.0548 ± 0.0083 µg/mL to 26.65 ± 2.40 µg/mL) and cytotoxicity was in the range of CC50 0.62 ± 0.33 µg/mL to 56.43 ± 2.54 µg/mL, while 5-metoxycantin-6-one proved to be the most potent constituent of the six assayed ones. The methanol extract of the roots showed high in vitro antiplasmodial activity (IC50 1.88 ± 0.56 µg/mL), moderate cytotoxicity (CC50 41.93 ± 2.30 µg/mL), and good selectivity index (SI = 22.30). Finally, C20 quassinoids and canthin-6-one alkaloids were putatively identified in the H. suffruticosa methanol extract by LC-MS. CONCLUSIONS: Taken together, the isolated compounds, mainly the 5-metoxycantin-6-one and the methanol extract from H. suffruticosa roots, disclose good antiplasmodial activity, supporting the ethnopharmacological history of the Simaroubaceae species as traditional antimalarial drugs.

The single-cell epigenomic and transcriptional landscape of immunity to influenza vaccination.

Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we mapped the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza induced persistently diminished H3K27ac in monocytes and myeloid dendritic cells (mDCs), which was associated with impaired cytokine responses to Toll-like receptor stimulation. Single-cell ATAC-seq analysis revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs. This was associated with elevated expression of antiviral genes and heightened resistance to the unrelated Zika and Dengue viruses. These results demonstrate that vaccination stimulates persistent epigenomic remodeling of the innate immune system and reveal AS03's potential as an epigenetic adjuvant.

Comparative Study on Functional Components, Physicochemical Properties and Antioxidant Activity of Amaranthus Caudatus L. Oils Obtained by Different Solvents Extraction.

Functional compositions, physicochemical properties and antioxidant activities of Amaranthus caudatus L. oils (ACO) obtained by different solvents were comparatively investigated. All the resulted ACO were enrich in 75% unsaturated fatty acid and in squalene of about 4 g/100 g. Different solvents showed varying in oil extraction, where acetone results a highest yield of 6.80 g/100 g. ACO extracted by ethanol showed a highest tocopherol (1351.26 mg/kg), polyphenols (211.28 mg/kg) and squalene (42519.13 mg/kg). However, phytosterols in ACO extracted by hexane (27571.20 mg/kg) was higher than that by acetone (19789.91 mg/kg), ethanol (22015.73 mg/kg) and petroleum ether (24763.30 mg/kg). Furthermore, antioxidant activity of ACO was also measured by DPPH, ABTS and FRAP assay. According to principal component and correlation analysis, squalene was correlated with the DPPH scavenging ability, but phytosterols and tocopherols was correlated with the ABTS and ferric reducing ability of the oils, respectively. This study provides a promising excellent source of functional oil for food industries.

Effects of graded levels of dietary squalene supplementation on the growth performance, plasma biochemical parameters, antioxidant capacity, and meat quality in broiler chickens.

This study was conducted to evaluate the effects of dietary squalene supplementation on the growth performance, plasma biochemical indices, antioxidant status, and meat quality in broilers. Two hundred and forty 0-day-old male chicks were allocated into 5 groups of 6 replicates and were fed a basal diet supplemented with 0 (Control group), 250, 500, 1,000, or 2,000 mg/kg squalene for 42 d. Dietary squalene supplementation linearly increased weight gain and feed efficiency of broilers during the grower and overall periods (P < 0.05). Squalene linearly decreased 21-d malondialdehyde (MDA) level and 42-d glutathione peroxidase (GSH-Px) activity, and both linearly and quadratically decreased 42-d MDA level in plasma (P < 0.05). In contrast, squalene linearly increased plasma reduced form of glutathione (GSH) level on 21 and 42 d and superoxide dismutase activity on 42 d (P < 0.05). Squalene supplementation linearly decreased 21-d MDA accumulation but linearly increased GSH level on 21 d and 42 d and both linearly and quadratically increased 21-d GSH-Px activity in liver (P < 0.05). Supplementing squalene linearly increased pH value at 48 h and linearly decreased lightness at 48 h and 24-h drip loss of breast muscle (P < 0.05). The lightness at 24 h and cooking loss of breast muscle were both linearly and quadratically reduced by squalene (P < 0.05). Dietary squalene administration linearly decreased MDA accumulation but linearly increased GSH level and GSH-Px activity of breast muscle (P < 0.05). Compared with the control group, aforementioned growth performance, antioxidant-related parameters (except 42-d GSH-Px in plasma and breast and hepatic GSH), and meat quality were improved by squalene when its level was 1,000 and 2,000 mg/kg (P < 0.05), with their results being similar between these 2 groups (P > 0.05). It was concluded that squalene administration especially at a level of 1,000 mg/kg can improve growth performance, antioxidant status, and meat quality in broilers, providing insights into its application as a potential feed additive in broiler production.

Dietary Squalene Induces Cytochromes Cyp2b10 and Cyp2c55 Independently of Sex, Dose, and Diet in Several Mouse Models.

SCOPE: To investigate the effects of squalene, the main hydrocarbon present in extra virgin olive oil, on liver transcriptome in different animal models and to test the influence of sex on this action and its relationship with hepatic lipids. METHODS AND RESULTS: To this purpose, male C57BL/6J Apoe-deficient mice are fed a purified Western diet with or without squalene during 11 weeks and hepatic squalene content is assessed, so are hepatic lipids and lipid droplets. Hepatic transcriptomic changes are studied and confirmed by RT-qPCR. Dietary characteristics and influence of squalene doses are tested in Apoe-deficient on purified chow diets with or without squalene. These diets are also given to Apoa1 and wild-type mice on C57BL/6J background and to C57BL/6J xOla129 Apoe-deficient mice. Squalene supplementation increases its hepatic content without differences among sexes and hormonal status. The Cyp2b10 and Cyp2c55 gene expressions are significantly up-regulated by the squalene intake in all models, with independence of sex, sexual hormones, dietary fat content, genetic background and dose, and in Apoe-deficient mice consuming extra-virgin olive oil. CONCLUSION: Hepatic squalene increases the expression of these cytochromes and their changes in virgin olive oil diets may be due to their squalene content.

In vitro estimation of superfluid critical extracts of some plants for their antimicrobial potential, phytochemistry, and GC-MS analyses.

BACKGROUND: Along with swift economic evolution and continuous amelioration of lifestyle, people at present are paying more attention to health issues. Synthetic drugs will be compensated with other natural ones that belong to natural origin. Plants have always been considered as sources of several compounds that are used in many fields, especially human and animal health, starting from boosting immunity to the treatment of infectious diseases caused by some pathogenic microbes such as bacteria, fungi as well as viruses. This study aimed to incorporate some types of plants within the antimicrobial portfolio through the examination of different six plants which were Cichorium intybus, Cinnamomum camphora, Commiphora myrrha, Foeniculum vulgare, Nerium oleander, and Spartium junceum. As well, attempting to identify the active constituents of their extracts using GC-MS. MATERIALS AND METHODS: All selected plants were analyzed to determine their phytochemical composition such as phenolics, alkaloids, flavonoids, terpenoids, and so on. The extraction step was done by sophisticated equipment called supercritical fluid extractor SFE through adjustment of specific conditions include temperature, time, flow rate and pressure to change the behavior of CO2. Testing the antimicrobial activity of each plant extract via agar well diffusion method through the formation of clear zones against a wide range of test microorganisms including both Gram-positive and Gram-negative bacteria as well as yeasts. Finally, attempting to primarily identify the constituents of each plant extract using GC-MS. RESULTS AND DISCUSSION: The crude extract of F. vulgare showed the highest potency against C. albicans, E. faecalis and S. typhimurium, it contains some unique compounds such as squalene, eugenol and isoeugenol while, Extract of C. intybus showed a moderate activity especially against C. lipolytica and MRSA and it includes Vitamin A like compound which indicates antioxidant property. CONCLUSION: Conclusively, fennel gave a promising result as a good wide spectrum antimicrobial agent because it contains some compounds act as antimicrobial agents such as eugenol which was used as food preservatives in addition to squalene which acts as an antioxidant and antimycotic agent so, it will be useful especially while it was used in highly purified form excluding all undesirable subcomponents.

Investigation of the Effects of Squalene and Squalene Epoxides on the Homeostasis of Coenzyme Q10 in Rats by UPLC-Orbitrap MS.

Squalene has been used as a dietary supplement for a long history due to its potential cancer-preventive function. However, the mechanism has not been investigated in detail yet. Therefore, the aim of this study is to see if the plasma coenzyme Q10 (CoQ10) level will be altered by gavage of squalene and oxidosqualenes to rats. In the present work, a sensitive and simple high-performance analytical method based on ultra-high-performance liquid chromatography coupled with an Orbitrap mass spectrometry (UPLC-Orbitrap-MS) was developed for the quantification of CoQ10 in rat plasma. Coenzyme Q9 (CoQ9) was employed as the internal standard. CoQ10 was determined after acetonitrile-mediated plasma protein precipitation using UPLC-Orbitrap-MS in negative ion mode. Intragastric administration of squalene and the two squalene epoxides into rats once daily for several days elevated the level of CoQ10 in their plasma, but there was no significant difference between high-dose (286 mg/kg) and low-dose (143 mg/kg) groups. Intragastric administration of squalene once a day for 5 consecutive days and oxidosqualenes once a day for 3 consecutive days is necessary for reaching the steady-state level of CoQ10. Our present findings indicate that squalene and oxidosqualenes may be useful for stimulating the synthesis of CoQ10 in rats.

Vaccine adjuvant activity of emulsified oils from species of the Pinaceae family.

BACKGROUND: Next to aluminum salts, squalene nanoemulsions comprise the most widely employed class of adjuvants in approved vaccines. Despite their importance, the mechanisms of action of squalene nanoemulsions are not completely understood, nor are the structure/function requirements of the oil composition. PURPOSE: In this study, we build on previous work that compared the adjuvant properties of nanoemulsions made with different classes of oil structures to squalene nanoemulsion. Here, we introduce nanoemulsions made with polyprenols derived from species of the Pinaceae family as novel vaccine adjuvant compositions. In contrast with long-chain triglycerides that do not efficiently enhance an immune response, both polyprenols and squalene are comprised of multimeric isoprene units, which may represent an important structural property of oils in nanoemulsions with adjuvant properties. STUDY DESIGN: Oils derived from species of the Pinaceae family were formulated in nanoemulsions, with or without a synthetic Toll-like receptor 4 (TLR4) ligand, and characterized regarding physicochemical and biological activity properties in comparison to squalene nanoemulsions. METHODS: Oils were extracted from species of the Pinaceae family and used to prepare oil-in-water nanoemulsions by microfluidization. Emulsion droplet diameter stability was characterized by dynamic light scattering. Nanoemulsions were evaluated for in vitro biological activity using human whole blood, and in vivo biological activity in mouse, pig, and ferret models when combined with pandemic influenza vaccine antigens. RESULTS: Nanoemulsions comprised of Pinaceae-derived polyprenol oils demonstrated long-term physical stability, stimulated cytokine production from human cells in vitro, and promoted antigen-specific immune responses in various animal models, particularly when formulated with the TLR4 ligand glucopyranosyl lipid adjuvant (GLA). CONCLUSION: Pinaceae-derived nanoemulsions are compatible with inclusion of a synthetic TLR4 ligand and promote antigen-specific immune responses to pandemic influenza antigens in mouse, pig, and ferret models.

Evaluation of Oxasqualenoids from the Red Alga Laurencia viridis against Acanthamoeba.

Acanthamoeba genus is a widely distributed and opportunistic parasite with increasing importance worldwide as an emerging pathogen in the past decades. This protozoan has an active trophozoite stage, a cyst stage, and is dormant and very resistant. It can cause Acanthamoeba keratitis, an ocular sight-threatening disease, and granulomatous amoebic encephalitis, a chronic, very fatal brain pathology. In this study, the amoebicidal activity of sixteen Laurencia oxasqualenoid metabolites and semisynthetic derivatives were tested against Acanthamoeba castellanii Neff. The results obtained point out that iubol (3) and dehydrothyrsiferol (1) possess potent activities, with IC50 values of 5.30 and 12.83 µM, respectively. The hydroxylated congeners thyrsiferol (2) and 22-hydroxydehydrothyrsiferol (4), active in the same value range at IC50 13.97 and 17.00 µM, are not toxic against murine macrophages; thus, they are solid candidates for the development of new amoebicidal therapies.

Modulation of the neurotransmitter systems through the anti-inflammatory and antidepressant-like effects of squalene from Aurantiochytrium sp.

Although algae have been the focal point of biofuel research, studies on their biological activities have been limited. In recent years, however, the importance of algae as sources of functional ingredients has been recognized due to their health beneficial effects. In this study, we evaluated the antidepressant-like activities of ethanol extract of Aurantiochytrium sp. (EEA) in the forced swimming test (FST)-induced depression in ICR mice. Imipramine, a commercially available tricyclic antidepressant drug, was used as positive control. Animals were administered EEA orally for 14 consecutive days and were subjected to the locomotor activity testing. Additionally, changes in gene expression in mice brain were assessed by real-time PCR and microarray assays to understand the molecular mechanisms underlying the effect of EEA. We found that the immobility time in FST was significantly reduced in the EEA-treated mice compared to that of in the control mice. Microarray and real-time PCR results revealed that EEA treatment induced changes in several genes in mice brain associated with pro-inflammation and dopaminergic, cholinergic, glutamatergic, and serotonergic synapses. It has previously been reported that several cytokines, such as IL-6 and TNF-α, which mediate neuroinflammation, are also responsible for indirectly altering brain neurotransmitter levels in neuropsychiatric disorders. Therefore, the regulation of the expression of pro-inflammatory genes in EEA-administered mice brain is considered to contribute to the enhancement of neurotransmitter systems-related gene expression in our study. Moreover, our in vitro study suggested that squalene, a component produced by Aurantiochytrium, was one of the active substances in EEA. In conclusion, our study provides the first evidence that Aurantiochytrium sp. can reduce neuroinflammation that may contribute to the modulation of the neurotransmitter systems, which could underlie its antistress and antidepressant effects.

Efficacy and safety of a non-mineral oil adjuvanted injectable vaccine for the protection of Atlantic salmon (Salmo salar L.) against Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the causative agent of Rainbow Trout Fry Syndrome which has had a major impact on global salmonid aquaculture. Recent outbreaks in Atlantic salmon in Scotland and Chile have added to the need for a vaccine to protect both salmon and trout. At present no licensed vaccines are available in Europe, leaving antibiotics as the only course of action to contain disease outbreaks. Outbreaks generally occur in fry at temperatures between 10 and 15 °C. Recently outbreaks in larger fish have given added impetus to the development of a vaccine which can provide long term protection from this highly heterogeneous pathogen. Most fish injectable vaccines are formulated with oil emulsion adjuvants to induce strong and long lasting immunity, but which are known to cause side effects. Alternative adjuvants are currently sought to minimise these adverse effects. The current study was performed to assess the efficacy of a polyvalent, whole cell vaccine containing formalin-inactivated F. psychrophilum to induce protective immunity in Atlantic salmon. The vaccine was formulated with an adjuvant containing squalene and aluminium hydroxide, and was compared to a vaccine formulated with a traditional oil adjuvant, Montanide ISA 760VG, and a non-adjuvanted vaccine. Duplicate groups of salmon (23.5 ± 6.8 g) were vaccinated with each of the vaccine formulations or phosphate buffered saline by intraperitoneal injection. Fish were challenged by intramuscular injection with F. psychrophilum six weeks post-vaccination to test the efficacy of the vaccines. Cumulative mortality reached 70% in the control salmon, while the groups of salmon that received vaccine had significantly lower mortality than the controls (p = 0.0001), with no significant difference in survival between vaccinated groups. The squalene/alum adjuvant was safe, more readily metabolised by the fish and induced less histopathological changes than the traditional oil adjuvant.

Characterization of squalene-induced PgCYP736B involved in salt tolerance by modulating key genes of abscisic acid biosynthesis.

Cytochrome P450 genes as the one of the largest superfamily genes mediate a wide range of plant biochemical pathways. In this study, a full-length cytochrome P450 monooxygenase (CYP736B) cDNA was isolated and characterized from Panax ginseng. It was revealed that the deduced amino acid of PgCYP736B shares a high degree of sequence homology with CYP736A12 encoded by P. ginseng. Expression of PgCYP736B was differentially induced not only during a Pseudomonas syringae infection (7.7-fold) and wounding (47.3-fold) but also after exposure to salt (7.4-fold), cold (8.3-fold), and drought stress (3.24-fold). The gene transcription was highly affected by methyl jasmonate (476-fold) in the ginseng, suggesting that PgCYP736B was elicitor-responsive. Furthermore, we overexpressed the PgCYP736B gene in Arabidopsis and found that PgCYP736B is a transmembrane protein. Overexpression of PgCYP736B in Arabidopsis conferred enhanced resistance to salt stress via decreased H2O2 accumulation, increased carotenoid levels, and through abscisic acid biosynthesis gene expression. Our results suggest that the induction of ginsenoside biosynthetic pathway genes along with PgCYP736B by an exogenous supply of 10-100 µM of squalene most likely affects the metabolite profile of ginsenoside triterpenoid. Overall, our findings indicate that PgCYP736B protects ginseng from salt stress and may contribute to triterpenoid biosynthesis.

Systems analysis of human vaccine adjuvants.

The discovery and wide spread use of vaccines have saved millions of lives in the past few decades. Vaccine adjuvants represent an integral part of the modern vaccines. Despite numerous efforts, however, only a handful of vaccine adjuvants is currently available for human use. A comprehensive understanding of the mechanisms of action of adjuvants is pivotal to harness the potential of existing and new adjuvants in mounting desirable immune responses to counter human pathogens. Decomposing the host response to vaccines and its components at systems level has recently been made possible owing to the recent advancements in Omics technology and cutting edge immunological assays powered by systems biology approaches. This approach has begun to shed light on the molecular signatures of several human vaccines and adjuvants. This review is an attempt to provide an overview of the recent efforts in systems analysis of vaccine adjuvants that are currently in clinic.

Correlates of adjuvanticity: A review on adjuvants in licensed vaccines.

After decades of slow progress, the last years have seen a rapid acceleration of the development of adjuvanted vaccines which have lately been approved for human use. These adjuvants consist of different components, e.g. aluminium salts, emulsions such as MF59 and AS03, Toll-like receptor (TLR) agonists (CpG ormonophosphoryl lipid A (MPL) adsorbed on aluminium salts as in AS04) or combination of immunopotentiators (QS-21 and MPL in AS01). Despite their distinctive features, most of these adjuvants share some key characteristics. For example, they induce early activation (although at different levels) of innate immunity which then translates into higher antibody and cellular responses to the vaccine antigens. In addition, most of these adjuvants (e.g. MF59, AS03, AS04) clearly induce a wider breadth of adaptive responses able to confer protection against, for example, heterovariants of the influenza viruses (MF59, AS03) or against human papillomavirus strains not contained in the vaccine (AS04). Finally, the use of some of these adjuvants has contributed to significantly enhance the immune response and the efficacy and effectiveness of vaccines in the elderly who experience a waning of the immune responsiveness to infection and vaccination, as shown for MF59- or AS03-adjuvanted influenza vaccines and AS01-adjuvanted herpes zoster vaccine. These results, together with the track record of acceptable safety profiles of the adjuvanted vaccines, pave the way for the development of novel vaccines at the extremes of age and against infections with a high toll of morbidity and mortality. Here, we review the mechanisms associated with the performance of those adjuvanted vaccines in animal models and in humans through recent advances in systems vaccinology and biomarker discovery. We also provide some perspectives on remaining knowledge gaps but also on opportunities that could accelerate the development of new vaccines.

Antioxidative Protection of Squalene Adjuvant and Rabies Vaccine with Adjuvant.

The authors verified the possibility of antioxidative protection of squalene adjuvant emulsions by the antioxidants α-tocopherol and ß-carotene. They determined the influence of ß-carotene on the stability and antigenic effectiveness of adjuvant emulsion in combination with rabies vaccine. The composition of the adjuvant emulsions or vaccines was: 2.5% squalene; 6% detergents; 0.5% antioxidant; 91% water phase. The oxidative injury after UV-irradiation was followed by the detection of the peroxide value of the emulsions. The stability of the emulsions was evaluated by the determination of the emulsion's particle size. The level of rabies antibodies (RAB) in mice sera until day 90 after vaccination, was determined by the rapid fluorescent focus inhibition test. In the in vitro system of squalene adjuvant, α-tocopherol acted as a prooxidant, while ß-carotene effectively reduced the oxidative injury. The homogenization of the squalene adjuvant during a prolonged period from 8 to 10 min did not change the particle size. The oxidation processes were efficiently reduced by ß-carotene during the preparation process and also during the 70-d storage. The vaccine with ß-carotene induced a gradual increase in the RAB levels with the highest value on day 28. While the inactivated rabies vaccine with adjuvant without ß-carotene developed a rapid formation of RAB, the application of the vaccine with ß-carotene induced a slower but more uniform production of RAB. The level of RAB was significantly higher after the application of the vaccine with ß-carotene and reached the protective value of 0.5 IU/mL, in contrast to the vaccine without ß-carotene.