Investigation and Regulation of DNA Nanostructures on Activating cGAS-STING Signaling.

DNA nanostructures have shown great potential in biomedical fields. However, the immune responses, especially the activation of the cGAS-STING signaling (A-cGSs), induced by DNA nanostructures, remain incompletely understood. Here, the ability of various DNA nanostructures from double-stranded DNA (dsDNA), single-stranded tiles (SSTs) to DNA origami is investigated on A-cGSs. Unlike natural dsDNA which triggers potent A-cGSs, the structural interconnectivity of various DNA configurations can substantially reduce the occurrence of A-cGSs, irrespective of their form, dimensions, and conformation. However, wireframe DNA nanostructures can activate the cGAS-STING signaling, suggesting that decreasing A-cGSs is dsDNA compactness-dependent. Based on this, a reconfigurable DNA Origami Domino Array (DODA) is used to systematically interrogate how dsDNA influences the A-cGSs and demonstrates that the length, number, and space of dsDNA array coordinately influence the activation level of cGAS-STING signaling, realizing a regulation of innate immune response. The above data and findings enhance the understanding of how DNA nanostructures affect cellular innate immune responses and new insights into the modulation of innate immune responses by DNA nanomedicine.

Assessment of dietary yeast-based additives for cultured catfish and tilapia health.

Channel catfish (Ictalurus punctatus) and Nile tilapia (Oreochromis niloticus) are two aquaculture species of great importance. Intensive production is often hindered by poor growth performance and disease mortality. The aim of this study was to evaluate the potential of a commercial fermented yeast product, DVAQUA, on channel catfish and Nile tilapia growth performance metrics and disease resistance. Channel catfish and Nile tilapia were fed practical diets supplemented with 0%, 0.1% or 0.4% of DVAQUA over approximately 2-month feeding periods in recirculation aquaculture systems. To assess the potential of the postbiotic against common aquaculture pathogens, juvenile catfish were subsequently challenged by immersion with Edwardsiella ictaluri S97-773 or virulent Aeromonas hydrophila ML09-119. Nile tilapia juveniles were challenged by injection with Streptococcus iniae ARS-98-60. Serum lysozyme activity, blood chemistry and growth metrics were measured at the end of the feeding period, but no differences were observed across the different metrics, except for survival. For the pathogen challenges, there were no differences in endpoint mortality for channel catfish with either pathogen (p > .05). In contrast, Nile tilapia survivability to S. iniae infection increased proportionally to the inclusion of DVAQUA (p = .005). Changes to sera lysozyme activity were also noted in the tilapia trial, with a reduction of activity in the fish fed the 0.4% DVAQUA diet compared to the control diet (p = .031). Expression profiles of proinflammatory genes and antibodies were also found to be modulated in channel catfish fed the postbiotic, indicating some degree of protective response. These results suggest that this postbiotic may be beneficial in protecting Nile tilapia against S. iniae infection by influencing immune parameters and additional research is needed to evaluate the potential of this DVAQUA for improving catfish health and disease control.

Exploring the Interactive Impacts of Citronellol, Thymol, and Trans-Cinnamaldehyde in Broilers: Moving towards an Improved Performance, Immunity, Gastrointestinal Integrity, and Clostridium perfringens Resistance.

AIMS: This study aimed to explore the effectiveness of dietary citronellol, thymol, and trans-cinnamaldehyde (CTC) essential oils blend on broilers` growth performance, immunity, intestinal microbial count, gut integrity, and resistance against Clostridium perfringens utilizing the necrotic enteritis (NE) challenge model. METHODS AND RESULTS: A total of 200 Ross 308 male broiler chicks received either a control diet or diet supplemented with three graded levels of CTC blend including 300, 600, and 900 mg of CTC blend/Kg diet and experimentally infected with C. perfringens strain at 23 days of age. Herein, dietary CTC blend fortifications significantly improved the broilers` growth performance, which was supported by upregulating the expression levels of MUC-2, occludin, and JAM-2 genes. Moreover, dietary CTC blend inclusion significantly enhanced the levels of blood phagocytic percentage and serum IgA, IgG, and MPO, and reduced the values of serum CRP, and NO at 5 days pre-infection, 10-, and 15 days post-infection (dpi) with C. perfringens. At 15 dpi, CTC blend inclusion significantly reduced the intestinal digesta pH, coliforms and C. perfringens loads, and the expression levels of genes related to C. perfringens virulence (cpe, cnaA, and nanI), proinflammatory cytokines (IL-1ß and TNF-α), and chemokines (CCL20), in addition to increasing the count of beneficial total Lactobacillus and total aerobic bacteria, and the expression levels of genes related to anti-inflammatory cytokines (IL-10) and chemokines (AvBD6, and AvBD612). CONCLUSION: Our results point to the growth-provoking, immunostimulant, antibacterial, anti-inflammatory, and antivirulence characteristics of the CTC blend, which improves the broilers' resistance to C. perfringens and ameliorates the negative impacts of NE.

Development of semisynthetic saponin immunostimulants.

Many natural saponins demonstrate immunostimulatory adjuvant activities, but they also have some inherent drawbacks that limit their clinical use. To overcome these limitations, extensive structure-activity-relationship (SAR) studies have been conducted. The SAR studies of QS-21 and related saponins reveal that their respective fatty side chains are crucial for potentiating a strong cellular immune response. Replacing the hydrolytically unstable ester side chain in the C28 oligosaccharide domain with an amide side chain in the same domain or in the C3 branched trisaccharide domain is a viable approach for generating robust semisynthetic saponin immunostimulants. Given the striking resemblance of natural momordica saponins (MS) I and II to the deacylated Quillaja Saponaria (QS) saponins (e.g., QS-17, QS-18, and QS-21), incorporating an amide side chain into the more sustainable MS, instead of deacylated QS saponins, led to the discovery of MS-derived semisynthetic immunostimulatory adjuvants VSA-1 and VSA-2. This review focuses on the authors' previous work on SAR studies of QS and MS saponins.

Endogenous capsid-forming protein ARC for self-assembling nanoparticle vaccines.

The application of nanoscale scaffolds has become a promising strategy in vaccine design, with protein-based nanoparticles offering desirable avenues for the biocompatible and efficient delivery of antigens. Here, we presented a novel endogenous capsid-forming protein, activated-regulated cytoskeleton-associated protein (ARC), which could be engineered through the plug-and-play strategy (SpyCatcher3/SpyTag3) for multivalent display of antigens. Combined with the self-assembly capacity and flexible modularity of ARC, ARC-based vaccines elicited robust immune responses against Mpox or SARS-CoV-2, comparable to those induced by ferritin-based vaccines. Additionally, ARC-based nanoparticles functioned as immunostimulants, efficiently stimulating dendritic cells and facilitating germinal center responses. Even without adjuvants, ARC-based vaccines generated protective immune responses in a lethal challenge model. Hence, this study showed the feasibility of ARC as a novel protein-based nanocarrier for multivalent surface display of pathogenic antigens and demonstrated the potential of exploiting recombinant mammalian retrovirus-like protein as a delivery vehicle for bioactive molecules.

Unveiling the Immunostimulatory Potential of Rhus Toxicodendron in Immunocompromised Balb/C Mice Induced with Cyclophosphamide.

This study investigated how Rhus toxicodendron (RT) (6C, 30C, and 200C) can boost the immune system of BALB/c mice that were given cyclophosphamide (CPM), which is an anticancer drug that weakens the immune system. RT, known for its historical use in traditional homeopathic remedies, has demonstrated immunomodulatory and anti-inflammatory effects in various experimental models. To test the immune-boosting effects of RT, CPM (80 mg/kg) was given intraperitoneally to mice on days 4, 8, and 12 of the study but not to the normal control group. CPM-induced immunosuppression led to significant decreases in red blood cell (RBC), white blood cell (WBC), and hemoglobin (Hb) levels, and reduced spleen and thymus indices. Phagocytic activity, cytokine concentrations, and spleen architecture were also adversely affected. RT treatment, particularly at 200C, significantly ameliorated these effects, improving RBC, WBC, and Hb levels. Furthermore, RT partially prevented CPM-induced atrophy of immune organs. Treatment positively influenced cytokine production at both the protein and mRNA levels, restoring immune balance. Histopathological results confirmed that RT stimulated the immune system. The cells were more stable, and the white pulp in the spleen was arranged in a regular pattern. These findings suggest that RT may serve as an adjunctive immunostimulant therapy for conditions characterized by immunosuppression. However, further investigations in other immunocompromised states must validate these results before considering human clinical trials.

Impact of liposomal hesperetin in broilers: prospects for improving performance, antioxidant potential, immunity, and resistance against Listeria monocytogenes.

Liposomal encapsulated phytogenics, such as liposomal hesperetin, are considered novel substitutes for antibiotics in the broiler industry owing to their improved nutritional and therapeutic properties. Therefore, our key goal was to investigate liposomal hesperetin impact on broiler growth performance, health, antioxidant status, tight junction proteins (TJP), and resistance against Listeria monocytogenes. Four broiler groups were fed 0, 150, 250, or 400 mg/kg of liposomal hesperetin-supplemented diets and experimentally infected with L. monocytogenes strain. Herein, liposomal hesperetin, especially at higher concentrations, augmented broilers FCR with upregulation of genes encoding TJP (occludin, JAM-2, MUC-2), and antioxidant attributes (GPX-1, SOD-1, CAT, HO-1, NQO1, COX2), which reflect enhancing health and welfare of broilers. Muscle antioxidant biomarkers were enhanced; meanwhile, muscle MDA, ROS, and H2O2 levels were reduced in response to 400 mg/kg of liposomal hesperetin. Liposomal hesperetin fortification reduced L. monocytogenes loads and expression levels of its virulence-related genes (flaA, hlyA, and ami). Remarkably, histopathological alterations in intestinal and brain tissues of L. monocytogenes-infected broilers were restored post-inclusion at higher levels of liposomal hesperetin, which reflects increase of the birds' resistance to L. monocytogenes infection. Transcription levels of genes encoding cytokines/chemokines (MyD88, AVBD6, CCL20, IL-1ß, IL-18), and autophagy (Bcl-2, LC3, AMPK, AKT, CHOP, Bip, p62, XBP1) were ameliorated following dietary liposomal hesperetin fortification, which suggests enhancement of the birds' immunity and health. Collectively, our research recommends liposomal hesperetin application in broiler diets owing to its promoting impact on growth performance, antioxidant status, immunity, health, and welfare besides its antibacterial, and antivirulence characteristics to fight against L. monocytogenes.

PACAP binds conserved receptors and modulates cytokine gene expression and protein secretion in trout cell lines.

Antimicrobial peptides (AMPs) are an alternative to antibiotics for treatment and prevention of infections with a lower risk of bacterial resistance. Pituitary adenylate cyclase activating polypeptide (PACAP) is an outstanding AMP with versatile effects including antimicrobial activity and modulation of immune responses. The objective of this research was to study PACAP immunomodulatory effect on rainbow trout cell lines infected with Aeromonas salmonicida. PACAP from Clarias gariepinus (PACAP1) and a modified PACAP (PACAP5) were tested. RT-qPCR results showed that il1b and il8 expression in RTgutGC was significantly downregulated while tgfb expression was upregulated after PACAP treatment. Importantly, the concentration of IL-1ß and IFN-γ increased in the conditioned media of RTS11 cells incubated with PACAP1 and exposed to A. salmonicida. There was a poor correlation between gene expression and protein concentration, suggesting a stimulation of the translation of IL-1ß protein from previously accumulated transcripts or the cleavage of accumulated IL-1ß precursor. In-silico studies of PACAP-receptor interactions showed a turn of the peptide characteristic of PACAP-PAC1 interaction, correlated with the higher number of interactions observed with this specific receptor, which is also in agreement with the higher PACAP specificity described for PAC1 compared to VPAC1 and VPACA2. Finally, the in silico analysis revealed nine amino acids related to the PACAP receptor-associated functionality.

Immunostimulatory Effects of Korean Mineral-Rich Seawaters on Cyclophosphamide-Induced Immunosuppression in Mice.

Deep seawater (DS), obtained from a depth over 200 m, has health benefits due to its rich nutrients and minerals, and intake of DS has shown diverse immunomodulatory effects in allergies and cancer. Therefore, the immunostimulatory effects of Korean mineral-rich seawaters were examined in a cyclophosphamide (CPA)-induced immunosuppression model. Three samples of Korean seawater, namely DS from the East Sea off the coasts of Pohang (PDS) and Uljin (UDS), and seawater from the West Sea off the coast of Boryeong (BS), were collected. The seawaters were abundant in several minerals (calcium, iron, zinc, selenium, etc.). Mice were orally administered the seawaters for 42 days, followed by CPA-induced immunosuppression. The CPA induction reduced the weight of the spleen and lymph nodes; however, the administration of seawaters increased the weight of the lymphoid organs, accompanied by stimulation of natural killer cells' activity and NF-kB-mediated cytokine production (IFNγ, TNFα, IL1ß, IL6, and IL12). The mouse-derived splenocytes showed lymphoproliferation without cytotoxicity in the seawater groups. Histopathological analysis revealed that the seawaters improved the CPA-induced atrophic changes by promoting lymphoproliferation in the spleen and lymph nodes. These results provide useful information for the use of Korean mineral-rich seawaters, particularly PDS and UDS, as alternative immunostimulants under immunosuppressive conditions.

Pharmacologic Interventions to Immunologic and Immune-Mediated Conditions in Horses.

Immunomodulators can stimulate, suppress, or regulate one or many aspects of the immune response. Use of a variety of immunostimulants, immunosuppressors, and anti-inflammatory drugs are described in horses, but the evidence supporting their efficacy is variable. Corticosteroids and nonsteroidal anti-inflammatory drugs are the best characterized immunomodulators in horses, but further study is needed to fully define their ideal dosing protocols and indications and to characterize the efficacy of other immunomodulators in equine medicine.

Polyvalent Bacterial Lysate with Potential Use to Treatment and Control of Recurrent Urinary Tract Infections.

Overuse of antimicrobials has greatly contributed to the increase in the emergence of multidrug-resistant bacteria, a situation that hinders the control and treatment of infectious diseases. This is the case with urinary tract infections (UTIs), which represent a substantial percentage of worldwide public health problems, thus the need to look for alternatives for their control and treatment. Previous studies have shown the usefulness of autologous bacterial lysates as an alternative for the treatment and control of UTIs. However, a limitation is the high cost of producing individual immunogens. At the same time, an important aspect of vaccines is their immunogenic amplitude, which is the reason why they must be constituted of diverse antigenic components. In the case of UTIs, the etiology of the disease is associated with different bacteria, and even Escherichia coli, the main causal agent of the disease, is made up of several antigenic variants. In this work, we present results on the study of a bacterial lysate composed of 10 serotypes of Escherichia coli and by Klebsiella pneumoniae, Klebsiella aerogenes, Enterococcus faecalis, Proteus mirabilis, Citrobacter freundii, and Staphylococcus haemolyticus. The safety of the compound was tested on cells in culture and in an animal model, and its immunogenic capacity by analysing in vitro human and murine macrophages (cell line J774 A1). The results show that the polyvalent lysate did not cause damage to the cells in culture or alterations in the animal model used. The immunostimulatory activity assay showed that it activates the secretion of TNF-α and IL-6 in human macrophages and TNF-α in murine cells. The obtained results suggest that the polyvalent lysate evaluated can be an alternative for the treatment and control of chronic urinary tract infections, which will reduce the use of antimicrobials.

The efficacy of intravenous application of the Mycobacterium Cell Wall Fraction (AMPLIMUNE) in the treatment of subclinical mastitis caused by S. Aureus.

The aim of the study was to investigate the effect of the immunostimulant Mycobacterium Cell Wall Fraction (MCWF) on the treatment of S. aureus SCM by intravenous application. The study included 45 HF dairy cows in 2nd and 3rd month after parturition divided into three groups (n = 15 per group): the MC + group - cows with S. aureus SCM treated with MCWF; the MC- group - cows with S. aureus SCM, with no treatment; and the C group - the control group of healthy cow with no treatment. Samples were collected 0th (I sample), 7th (II), and 14th day (III) from the day of SCM diagnosis and on day 21st (IV). A greater influx of leukocytes was confirmed into milk after 7 days after MCWF treatment in MC + group, which was followed by increase of WBC and LYM in blood. These results support the hypothesis of effective action of MCWF, and in quarters with lower-grade infection, bacteriological cure was achieved. The MC- group had a statistically higher concentration of TBARS and CAT activity in milk, while MC + group had lower blood serum LDH activity, which indicates a positive effect of the MCWF application and a lower exposure of the tissue to lipide peroxidation and inflammation caused by S. aureus. The application of MCWF would give new possibilities in the prevention and therapy of mammary gland diseases without fear of the presence of residues and the emergence of bacterial resistance. In future studies, the effects of local and systemic application of MCWF in the treatment of S. aureus SCM should be compared.

Dietary Black Soldier Fly (Hermetia illucens)-Dipterose-BSF-Enhanced Zebrafish Innate Immunity Gene Expression and Resistance to Edwardsiella tarda Infection.

Dietary management using immunostimulants to protect fish health and prevent bacterial infection is widely practiced. Many insect species possess various bioactive substances that can improve animal health. We previously identified several bioactive polysaccharides derived from insects, including dipterose-BSF from black soldier fly (Hermetia illucens) larvae; this can stimulate innate immunity in mammalian macrophage RAW264.7 cells. However, the effect of dietary dipterose-BSF on the immune system of teleosts remains unclear. Here, we analyzed the immune status of zebrafish (Danio rerio) after 14 days of dietary inclusion of dipterose-BSF (0.01, 0.1, and 1 µg/g), followed by an immersion challenge using Edwardsiella tarda. To identify changes in the transcriptional profile induced by dipterose-BSF, we performed RNA-sequencing analyses of the liver and intestine. Differentially expressed genes were investigated, with both organs showing several upregulated genes, dominated by nuclear factor and tumor necrosis factor family genes. Gene Ontology analysis revealed several terms were significantly higher in the experimental group compared with the control group. Challenge tests suggested that dietary dipterose-BSF had some positive effects on disease resistance in fish, but these effects were not pronounced.

In vitro Immunostimulant Activity of the Polyphenolic Extract from the Arctic Brown Algae Fucus vesiculosus.

Polyphenols (PP) found in brown algae are known for their wide range of biological activities including noteworthy antitumor properties. This article presents a method for obtaining an active polyphenolic extract from the Arctic alga Fucus vesiculosus with 98% purity and radical scavenging activity equivalent to 862 mg of ascorbic acid per gram of extract. Immunostimulant effects of polyphenols were assessed in vitro using venous blood from two groups of people: healthy people (HP) and people with chronic undifferentiated lymphocytic leukemia (LP). Polyphenols activated the surface properties of immunocompetent cells. Specifically, polyphenols dose-dependently increased the percentage of cells' spreading and adhesion by 2-3 times. Additionally, polyphenols increased the number of activated lymphocytes in the LP blood to levels characteristic of HP. Given their natural origin, high activity, non-toxicity, and straightforward production process, these studied polyphenols exhibit immense potential for use as new pharmaceuticals or as active components with immunostimulatory effects.

ß-Glucan fragmentation by microfluidization and TNF-α-immunostimulating activity of fragmented ß-glucans.

Fragmentation of ß-glucans secreted by the fungus Ophiocordyceps dipterigena BCC 2073 achieved by microfluidization was investigated. The degree of ß-glucan fragmentation was evaluated based on the average number of chain scissions (α). The effects on the α value of experimental variables like solid concentration of the ß-glucan suspension, interaction chamber pressure, and number of passes through the microfluidizer were examined. Kinetic studies were conducted using the relationships of the α and suspension viscosity values with the number of passes. Evidence indicated that α increases with the interaction chamber pressure and the number of passes, whereas the solid concentration shows the inverted effect. Kinetic data indicated that the fragmentation rate increases with ß-glucan solid concentration and interaction chamber pressure. Furthermore, since ß-glucan molecular weight is a key factor determining its biological activity, the effect of ß-glucans of different molecular weights produced by fragmentation on tumor necrosis factor (TNF)-α-stimulating activity in THP-1 human macrophage cells was investigated. Evidence suggested that ß-glucans have an immunostimulating effect on macrophage function, in the absence of cytotoxic effects. Indeed, ß-glucans characterized by a range of molecular weights produced via microfluidization exhibited promise as immunostimulatory agents.

A Comparative Analysis of Polysaccharides and Ethanolic Extracts from Two Egyptian Sweet Potato Cultivars, Abees and A 195: Chemical Characterization and Immunostimulant Activities.

Sweet potato (Ipomoea batatas (L.) Lam.) belongs to family Convolvulaceae. The plant is distributed worldwide and consumed, especially for its edible tubers. Many studies have proved that the plant has variable biological activities such as antidiabetic, anti-cancer, antihypertensive, antimicrobial, and immunostimulant activities. The roots of sweet potatoes are rich in valuable phytochemical constituents that vary according to the flesh color. Our investigation focused on the chemical profiling of two Egyptian sweet potato cultivars, Abees and A 195, using UPLC-QTOF and the analysis of their polysaccharide fractions by GC-MS. Furthermore, we assessed the immunostimulant properties of these extracts in immunosuppressed mice. The study revealed that sweet potato roots contain significant concentrations of phenolic acids, including caffeoylquinic, caffeic, caffeoyl-feruloyl quinic, and p-coumaric acids, as well as certain flavonoids, such as diosmin, diosmetin, and jaceosidin, and coumarins, such as scopoletin and umbelliferone. Moreover, polysaccharides prepared from both studied cultivars were analyzed using GC-MS. Further biological analysis demonstrated that all the tested extracts possessed immunostimulant properties by elevating the level of WBCs, IL-2, TNF, and IFN-γ in the immunosuppressed mice relative to the control group with the highest values in polysaccharide fractions of A195 (the ethanolic extract showed a higher effect on TNF and IFN-γ, while its polysaccharide fraction exhibited a promising effect on IL-2 and WBCs). In conclusion, the roots of the Egyptian sweet potato cultivars Abees and A 195 demonstrated significant immunostimulant activities, which warrants further investigation through clinical studies.

Corrigendum: Future impact of thymoquinone-loaded nanoemulsion in rabbits: prospects for enhancing growth, immunity, antioxidant potential and resistance against Pasteurella multocida.

[This corrects the article DOI: 10.3389/fvets.2023.1340964.].

Effects of Dietary Supplementation with Caesalpinia sappan Linn. Extract for Promoting Flock Health and Performance in Late-Phase Laying Hens.

The present study investigated the effects of dietary supplementation of Caesalpinia sappan Linn Extract (CSE) on the health and productive performance of late-phase laying hens on farms. Proximate composition and antioxidant markers of CSE powder revealed favorable characteristics with high total dry matter; phenolic content, and antioxidant potency. Three hundred and sixty (64-week-old) Hy-line Brown hens were divided into five groups with 0 (control diet), 250, 500, 1000, and 2000 mg/kg CSE, respectively. The laying performance and egg quality of the CSE supplementation groups demonstrated significant improvements in egg weight and albumin weight (p < 0.05), and a tendency for enhanced egg mass and feed conversion ratio. Additionally, the intestinal morphostructural indices in the 2000 mg CSE/kg diet group showed the greatest statistical significance (p < 0.05), with a detectable trend suggesting an increase in the villus height to crypt depth ratio. In addition, significant downregulation of proinflammatory genes occurred in their liver tissues, coupled with a greater expression of genes linked to antioxidants and anti-inflammatory processes. Furthermore, the blood biochemical parameters and the organ weights may suggest a favorable safety profile of CSE supplementation. These findings highlight the potential of CSE as a dietary supplement to enhance the productive performance and flock health of late-phase laying hens. Further research is warranted to explore the long-term effects and optimal dosage of CSE supplementation for laying hens in farming practices.

Liposomal co-encapsulation of a novel gemini lipopeptide and a CpG-ODN induces a strong Th1 response with the co-activation of a Th2/Th17 profile and high antibody levels.

A successful vaccine depends on its capacity to elicit a protective immune response against the target pathogen. The adjuvant used plays an important role in enhancing and directing the immune response. Liposomes are vaccine adjuvants that allow the co-encapsulation of antigens and immunostimulants. Our aim was to evaluate the adjuvanticity of a cationic liposome (Lip) formulated with a novel gemini lipopeptide (AG2-C16) alone or in combination with CpG-ODN as immunostimulants. To achieve this, we used the recombinant clumping factor of Staphylococcus aureus (rClfA) as a model antigen, in a murine model. We characterized the formulations by DLS, Cryo-SEM, and TEM, and analyzed the humoral and cellular immune responses induced in BALB/c and C57BL/6J mice injected with free rClfA and three formulations: Lip + CpG-ODN + rClfA, Lip + AG2-C16 + rClfA and Lip + AG2-C16 + CpG-ODN + rClfA. The addition of immunostimulants to the liposomes did not change the membrane diameter but affected their hydrodynamic diameter, z-potential, and homogeneity. All liposomal formulations were able to stimulate a specific humoral response, with high serum IgG, IgG1 and IgG2a or IgG2c titers in BALB/c or C57BL/6J mice, respectively. In addition, increased vaginal IgG levels were detected after injection, with no specific IgA. The cellular immunity induced by Lip + AG2-C16 + CpG-ODN + rClfA was characterized by a predominant Th1 profile, with the co-induction of Th2 and Th17 cells, and IFN-γ+ cytotoxic T cells. Furthermore, we studied the capacity of the different formulations to stimulate murine keratinocytes and fibroblasts in vitro. While no formulation activated keratinocytes, Lip + AG2-C16 + CpG-ODN increased the expression of CXCL9 in fibroblasts. These results suggest Lip + AG2-C16 + CpG-ODN as a promising adjuvant candidate to be used in vaccines against pathogens that require Th1/Th2/Th17 combined profiles, like S. aureus. Additionally, based on the IFN-γ+ cytotoxic T cells stimulation and the CXCL9 production by fibroblasts, we propose the use of this adjuvant formulation for the stimulation of a Th1 profile.

Design and functional characterization of Salmo salar TLR5 agonist peptides derived from high mobility group B1 acidic tail.

Toll-like receptor 5 (TLR5) responds to the monomeric form of flagellin and induces the MyD88-depending signaling pathway, activating proinflammatory transcription factors such as NF-κB and the consequent induction of cytokines. On the other hand, HMGB1 is a highly conserved non-histone chromosomal protein shown to interact with and activate TLR5. The present work aimed to design and characterize TLR5 agonist peptides derived from the acidic tail of Salmo salar HMGB1 based on the structural knowledge of the TLR5 surface using global molecular docking platforms. Peptide binding poses complexed on TLR5 ectodomain model from each algorithm were filtrated based on docking scoring functions and predicted theoretical binding affinity of the complex. Circular dichroism spectra were recorded for each peptide selected for synthesis. Only intrinsically disordered peptides (6W, 11W, and SsOri) were selected for experimental functional assay. The functional characterization of the peptides was performed by NF-κB activation assays, RT-qPCR gene expression assays, and Piscirickettsia salmonis challenge in SHK-1 cells. The 6W and 11W peptides increased the nuclear translation of p65 and phosphorylation. In addition, the peptides induced the expression of genes related to the TLR5 pathway activation, pro- and anti-inflammatory response, and differentiation and activation of T lymphocytes towards phenotypes such as TH1, TH17, and TH2. Finally, it was shown that the 11W peptide protects immune cells against infection with P. salmonis bacteria. Overall, the results indicate the usefulness of novel peptides as potential immunostimulants in salmonids.