Complete biosynthesis of QS-21 in engineered yeast.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Construction of recombinant Lactococcus expressing thymosin and interferon fusion protein and its application as an immune adjuvant.

BACKGROUND: In recent years, biosafety and green food safety standards have increased the demand for immune enhancers and adjuvants. In the present study, recombinant food-grade Lactococcus lactis (r-L. lactis-Tα1-IFN) expressing thymosin Tα1 and chicken interferon fusion protein was constructed. RESULTS: The in vitro interactions with macrophages revealed a mixture of recombinant r-L. lactis-Tα1-IFN could significantly activate both macrophage J774-Dual™ NF-κB and interferon regulator (IRF) signaling pathways. In vitro interactions with chicken peripheral blood mononuclear cells (PBMCs) demonstrated that a mixture of recombinant r-L. lactis-Tα1-IFN significantly enhanced the expression levels of interferon (IFN)-γ, interleukin (IL)-10, CD80, and CD86 proteins in chicken PBMCs. Animal experiments displayed that injecting a lysis mixture of recombinant r-L. lactis-Tα1-IFN could significantly activate the proliferation of T cells and antigen-presenting cells in chicken PBMCs. Moreover, 16S analysis of intestinal microbiota demonstrated that injection of the lysis mixture of recombinant r-L. lactis-Tα1-IFN could significantly improve the structure and composition of chicken intestinal microbiota, with a significant increase in probiotic genera, such as Lactobacillus spp. Results of animal experiments using the lysis mixture of recombinant r-L. lactis-Tα1-IFN as an immune adjuvant for inactivated chicken Newcastle disease vaccine showed that the serum antibody titers of the experimental group were significantly higher than those of the vaccine control group, and the expression levels of cytokines IFN-γ and IL-2 were significantly higher than those of the vaccine control group. CONCLUSION: These results indicate that food-safe recombinant r-L. lactis-Tα1-IFN has potential as a vaccine immune booster and immune adjuvant. This study lays the foundation for the development of natural green novel animal immune booster or immune adjuvant.

Chlorella vulgaris extract conjugated magnetic iron nanoparticles in nile tilapia (Oreochromis niloticus): Growth promoting, immunostimulant and antioxidant role and combating against the synergistic infection with Ichthyophthirius multifiliis and Aeromonashydrophila.

Nile tilapia reared under intensive conditions was more susceptible for Ichthyophthirius multifilii (I. multifiliis) infection eliciting higher mortality, lower productive rate and further bacterial coinfection with Aeromonas hydrophila (A. hydrophila). The higher potency of magnetic field of iron oxide nanoparticles (NPs) can kill pathogens through inhibiting their viability. Herein, coating of Chlorella vulgaris extract (ChVE) with magnetic iron oxide NPs (Mag iron NPs) can create an external magnetic field that facilitates their release inside the targeted tissues. Thus, the current study is focused on application of new functionalized properties of Mag iron NPs in combination with ChVE and their efficacy to alleviate I. multifiliis and subsequent infection with A. hydrophila in Nile tilapia. Four hundred fingerlings were divided into: control group (with no additives), three groups fed control diet supplemented with ChVE, Mag iron NPs and ChVE@Mag iron NPs for 90 days. At the end of feeding trial fish were challenged with I. multifiliis and at 9 days post challenge was coinfected by A. hydrophila. A remarkable higher growth rate and an improved feed conversion ratio were detected in group fed ChVE@Mag iron-NPs. The maximum expression of antioxidant enzymes in skin and gills tissues (GSH-Px, CAT, and SOD) which came in parallel with higher serum activities of these enzymes was identified in groups received ChVE@Mag iron-NPs. Furthermore, group fed a combination of ChVE and Mag iron-NPs showed a boosted immune response (higher lysozyme, IgM, ACH50, and MPO) prior to challenge with I. multifiliis. In contrast, fish fed ChVE@Mag iron-NPs supplemented diet had lower infection (decreased by 62%) and mortality rates (decreased by 84%), as well as less visible white spots (decreased by 92 % at 12 dpi) on the body surfaces and mucous score. Interestingly, post I. multifiliis the excessive inflammatory response in gill and skin tissues was subsided by feeding on ChVE@Mag iron-NPs as proved by down regulation of IL-1ß, TNFα, COX-2 and iNOS and upregulation of IL-10, and IgM, IgT and Muc-2 genes. Notably, group exposed to I. multifiliis-showed higher mortality when exposed to Aeromonas hydrophilia (increased by 43 %) while group fed ChVE@Mag iron-NPs exhibited lower morality (2%). Moreover, the bacterial loads of A. hydrophilia in fish infected by I. multifiliis and fed control diet were higher than those received dietary supplement of ChVE, Mag iron-NPs and the most reduced load was obtained in group fed ChVE@Mag iron-NPs at 7 dpi. In conclusion, ChVE@Mag iron-NPs fed fish had stronger immune barrier and antioxidant functions of skin and gills, and better survival following I. multifiliis and A. hydrophilia infection.

In vivo and ex vivo studies support the immunostimulant and immunoprotective effect of Damiana (Turnera diffusa Willd) in Almaco Jack (Seriola rivoliana).

Damiana (Turnera diffusa Willd) was evaluated in vitro for antioxidant and antibacterial activities against Staphylococcus aureus and Streptococcus pyogenes (as a preliminary screening assessment) by high-performance thin-layer chromatography (HPTLC)-Direct bioautography. A study was performed in vivo to evaluate the effects of Damiana enriched diets at 0.5 % on immune parameters in mucus and serum and gene expression in Almaco Jack (Seriola rivoliana) intestine after two and four weeks; an infection with Aeromonas hydrophila at 1x107 colony forming units (CFU) followed and an ex vivo study was carried out using head-kidney leukocytes. Ferric reducing ability of plasma (FRAP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays showed high antioxidant activities in Damiana leaves; even in the ABTS assay, Damiana at 300 µg/mL showed similar activity to ascorbic acid - the standard control. Damiana exhibited strong in vitro antimicrobial activity against S. aureus and S. pyogenes. In vivo studies showed a strong enhancement of myeloperoxidase, nitric oxide, superoxide dismutase, and catalase activities in mucus and serum of S. rivoliana supplemented with Damiana; their immunological response enhanced after infection with A. hydrophila. IL-1ß, TNF-α, and IL-10 gene expressions upregulated in the fish intestine challenged with the bacterium. Piscidin and macrophage (MARCO) receptor gene expression up-regulated at week 4 and down-regulated after infection. Intestinal histology results confirm that Damiana not cause inflammation or damage. Finally, the ex vivo study confirmed the immunostimulant and protective effects of Damiana through increased phagocytic, respiratory burst, myeloperoxidase activities and nitric oxide generation before and upon the bacterial encounter. These results support the idea that Damiana has the potential as an immunostimulant additive for diets in aquaculture by enhancing immune parameters and protecting Almaco Jack against A. hydrophila infections upon four weeks of supplementation.

Oncolytic Newcastle disease virus expressing the co-stimulator OX40L as immunopotentiator for colorectal cancer therapy.

NDV as an attractive candidate for oncolytic immunotherapy selectively lyses tumor cells but shows limited anti-tumor immunity. Immune co-stimulator OX40 ligand (OX40L) boosts anti-tumor immunity response by delivering a potent costimulatory signal to CD4+ and CD8+ T cells. To improve the anti-tumor immunity of NDV, the recombinant NDV expressing the murine OX40L (rNDV-mOX40L) was engineered. The viral growth kinetics was examined in CT26 cell lines. The ability of rNDV-mOX40L to express mOX40L was detected in the infected tumor cells and tumor tissues. The anti-tumor activity of rNDV-mOX40L was studied in the CT26 animal model. Tumor-specific CD4+, CD8+ and OX40+ T cells were examined by immunohistochemistry staining. The virus growth curve showed that the insertion of the mOX40L gene did not affect the growth kinetics of NDV. rNDV-mOX40L expresses mOX40L and effectively inhibits the growth of CT26 colorectal cancer in vivo. The tumor inhibition rate of the rNDV-mOX40L-treated group was increased by 15.8% compared to that of  NDV-treated group in the CT26 model. Furthermore, immunohistochemistry staining of tumor tissues removed from the CT26 model revealed that intense infiltration of tumor-specific CD4+, CD8+ T cells, especially OX40+ T cells were found in the rNDV-mOX40L-treated group. FACS showed that rNDV-mOX40L significantly enhanced the number of CD4+ and CD8+ T cells in spleen. Moreover, compared to the NDV-treated group, the level of mouse IFN-γ protein in the tumor site increased significantly in the rNDV-mOX40L-treated group. Taken together, rNDV-mOX40L exhibited superior anti-tumor immunity by stimulating tumor-specific T cells and may be a promising agent for cancer immunotherapy.

Asymmetric Assembly and Self-Adjuvanted Antigen Delivery Platform for Improved Antigen Uptake and Antitumor Effect.

The development of effective tumor vaccines is an important direction in the field of cancer prevention/immunotherapy. Efficient antigen delivery is essential for inducing effective antitumor responses for tumor vaccines. Lumazine synthase (BLS) from Brucella spp. is a decameric protein with delivery and adjuvant properties, but its application in tumor vaccines is limited. Here, we developed an antigen delivery platform by combining a BLS asymmetric assembly and the Plug-and-Display system of SpyCatcher/SpyTag. An asymmetric assembly system consisting of BLSke and BLSdr was developed to equally assemble two molecules. Then, the MHC-I-restricted ovalbumin peptide (OVA(257-264) SIINFEKL) was conjugated with BLSke, and a cell-penetrating peptide (CPP) KALA was conjugated with BLSdr using the SpyCatcher/SpyTag system. KALA modification enhanced internalization of OVA peptides by DCs as well as promoted the maturation of DCs and the cross-presentation of SIINFEKL. Moreover, the immunotherapy of a KALA-modified vaccine suppressed tumor growth and enhanced CD8+ T cell responses in E.G7-OVA tumor-bearing mice. In the prophylactic model, KALA-modified vaccination showed the most significant protective effect and significantly prolonged the survival period of tumor challenged mice. In conclusion, the asymmetric assembly platform equally assembles two proteins or peptides, avoiding their spatial or functional interference. This asymmetric assembly and Plug-and-Display technology provide a universal platform for rapid development of personalized tumor vaccines.

Mechanistic insight into immunomodulatory effects of food-functioned plant secondary metabolites.

Medicinally important plant-foods offer a balanced immune function, which is essential for protecting the body against antigenic invasion, mainly by microorganisms. Immunomodulators play pivotal roles in supporting immune function either suppressing or stimulating the immune system's response to invading pathogens. Among different immunomodulators, plant-based secondary metabolites have emerged as high potential not only for immune defense but also for cellular immunoresponsiveness. These natural immunomodulators can be developed into safer alternatives to the clinically used immunosuppressants and immunostimulant cytotoxic drugs which possess serious side effects. Many plants of different species have been reported to possess strong immunomodulating properties. The immunomodulatory effects of plant extracts and their bioactive metabolites have been suggested due to their diverse mechanisms of modulation of the complex immune system and their multifarious molecular targets. Phytochemicals such as alkaloids, flavonoids, terpenoids, carbohydrates and polyphenols have been reported as responsible for the immunomodulatory effects of several medicinal plants. This review illustrates the potent immunomodulatory effects of 65 plant secondary metabolites, including dietary compounds and their underlying mechanisms of action on cellular and humoral immune functions in in vitro and in vivo studies. The clinical potential of some of the compounds to be used for various immune-related disorders is highlighted.

Chimeric Hepatitis B core virus-like particles harboring SARS-CoV2 epitope elicit a humoral immune response in mice.

BACKGROUND: Virus-like particles are an interesting vector platform for vaccine development. Particularly, Hepatitis B virus core antigen has been used as a promising VLP platform. It is highly expressed in different recombinant expression systems, such as E. coli, and self-assembled in vitro. It effectively improves the immunogenicity of foreign antigenic epitopes on its surface. Various foreign antigens from bacteria, viruses, and protozoa can be genetically inserted into such nanoparticles. The effective immunogenicity due to VLP vaccines has been reported. However, no research has been performed on the SARS-CoV2 vaccine within this unique platform through genetic engineering. Considering the high yield of target proteins, low cost of production, and feasibility of scaling up, E. coli is an outstanding expression platform to develop such vaccines. Therefore, in this investigation, we planned to study and develop a unique HBc VLP-based vaccine against SARS-Cov2 utilizing the E. coli expression system due to its importance. RESULTS: Insertion of the selected epitope was done into the major immunodominant region (MIR) of truncated (149 residues) hepatitis B core capsid protein. The chimeric protein was constructed in PET28a+ and expressed through the bacterial E. coli BL21 expression system. However, the protein was expressed in inclusion body forms and extracted following urea denaturation from the insoluble phase. Following the extraction, the vaccine protein was purified using Ni2 + iminodiacetic acid (IDA) affinity chromatography. SDS-PAGE and western blotting were used to confirm the protein expression. Regarding the denaturation step, the unavoidable refolding process was carried out, so that the chimeric VLP reassembled in native conformation. Based on the transmission electron microscopy (TEM) analysis, the HBC VLP was successfully assembled. Confirming the assembled chimeric VLP, we explored the immunogenic effectivity of the vaccine through mice immunization with two-dose vaccination with and without adjuvant. The utilization of adjuvant was suggested to assess the effect of adjuvant on improving the immune elicitation of chimeric VLP-based vaccine. Immunization analysis based on anti-spike specific IgG antibody showed a significant increase in antibody production in harvested serum from immunized mice with HBc-VLP harboring antigenic epitope compared to HBc-VLP- and PBS-injected mice. CONCLUSIONS: The results approved the successful production and the effectiveness of the vaccine in terms of humoral IgG antibody production. Therefore, this platform can be considered a promising strategy for developing safe and reasonable vaccines; however, more complementary immunological evaluations are needed.

Probiotics and Immunostimulant modulate intestinal flora diversity in Reeves' Turtle (Mauremys reevesii) and effects of Clostridium butyricum on its spleen transcriptome.

In this study, we investigated the effects of Clostridium butyricum (group A), Bacillus subtilis (group B), and the immune enhancer algal ß-1,3 glucan (group C) on the intestinal flora of Reeves' turtle Mauremys reevesii and the effects of C. butyricum on the transcriptome of M. reevesii splenic immune tissues. Reeve' turtles were assigned to four groups, each containing three replicates from 18 samples. Juvenile turtles with an initial weight of 106.35 ± 0.03 g were fed a basic diet containing no probiotics (group D), or a basic diet containing C. butyricum TF20201120, B.subtilis, or algal ß-1,3 glucan supplement, respectively. After the turtles had been fed for 60, 90, and 120 d of the experimental period, high-throughput sequencing of the 16S rRNA gene revealed no significant difference in alpha diversity among the four groups at 60 days of feeding (P > 0.05), and at 90 days, the alpha diversity in group A was significantly different (P < 0.05), with an increase of 26.62% in the Shannon index and a decrease of 83.33% in the Simpson index; at 120 d, the alpha diversity (Shannon index) showed a decreasing trend in order for groups A, B, and C, At the phylum level, the abundance of Bacteroidetes, Proteobacteria, and Fusobacteria in group A increased significantly with increasing feeding time (P < 0.05), At the genus level, the abundance of Ruminococcaceae and Anaerotruncus in group A increased significantly compared with that in the other three groups (P < 0.05). Transcriptome analysis showed that 384 genes were differentially expressed in the spleen of M. reevesii, 195 genes were upregulated and 189 genes were downregulated, and C. butyricum TF201120 regulated the hematopoietic cell lineage signaling pathway in the spleen of M. reevesii (P < 0.05). The regulation of several identified immune-related genes was confirmed by qPCR. These results showed that C. butyricum, B. subtilis, and the immune enhancer algal ß-1,3 glucan can improve the intestinal flora of M. reevesii, with C. butyricum TF20201120 being the most effective and significantly enhancing the immunity of M. reevesii.

Identification of Functional Domains of CXCL14 Involved in High-Affinity Binding and Intracellular Transport of CpG DNA.

Some CXC chemokines, including CXCL14, transport CpG oligodeoxynucleotides (ODNs) into dendritic cells (DCs), thereby activating TLR9. The molecular basis of this noncanonical function of CXC chemokines is not well understood. In this study, we investigated the CpG ODN binding and intracellular transport activities of various CXC chemokines and partial peptides of CXCL14 in mouse bone marrow-derived dendritic cells. CXCL14, CXCL4, and CXCL12 specifically bound CpG ODN, but CXCL12 failed to transport it into cells at low dose. CXCL14 N-terminal peptides 1-47, but not 1-40, was capable of transporting CpG ODN into the cell, resulting in an increase in cytokine production. However, both the 1-47 and 1-40 peptides bound CpG ODN. By contrast, CXCL14 peptides 13-50 did not possess CpG ODN binding capacity or transport activity. The chimeric peptides CXCL12 (1-22)-CXCL14 (13-47) bound CpG ODN but failed to transport it. These results suggest that amino acids 1-12 and 41-47 of CXCL14 are required for binding and intracellular transport of CpG ODN, respectively. We found that an anti-CXCL14 Ab blocked cell-surface binding and internalization of the CpG ODN/CXCL14 complex. On the basis of these findings, we propose that CXCL14 has two functional domains, one involved in DNA recognition and the other in internalization of CXCL14-CpG DNA complex via an unidentified CXCL14 receptor, which together are responsible for eliciting the CXCL14/CpG ODN-mediated TLR9 activation. These domains could play roles in CXCL14-related diseases such as arthritis, obesity-induced diabetes, and various types of carcinoma.

Comparative effects of different bacterial lipopolysaccharides on modulation of immune levels to improve survival of the black tiger shrimp.

To prevent loss from disease, immunostimulants have been used as dietary supplements to improve immunity and survival of shrimps. Among the various types of immunostimulants, there is increasing evidence that a diet enriched with bacterial lipopolysaccharide can reduce the mortality rate of shrimp under exposure to pathogens. Here, the immunostimulatory effects of bacterial lipopolysaccharide (LPS) from various bacterial sources were explored. Bacterial LPS was extracted from a shrimp pathogen, Vibrio harveyi and its effects were compared with the commercially available LPS from the non-shrimp pathogen, Escherichia coli. Our results revealed that the LPS from V. harveyi was different in molecular size but contained similar functional groups to that from E. coli. To understand their molecular mechanisms, bacterial LPS from the two sources were applied as a supplementary diet and fed to juvenile shrimp for 4-week feeding period before tissue samples were collected for transcriptomic analysis by next generation sequencing. Gene expression profiling revealed that major immune-related genes such as pattern recognition proteins (PRPs), proteinases and proteinase inhibitors, prophenoloxidase systems (proPO system), antimicrobial peptides (AMPs), signaling transduction pathways, heat shock proteins (HSPs), oxidative stress responses, and other immune-related molecules such as mucins and peritrophins were modulated in the groups of shrimp fed with bacterial LPS from both sources, but at different levels. The results suggest that bacterial LPS could modulate shrimp immune system, and different LPS sources led to different activation of immune pathways. Additionally, metabolic-related genes were affected by LPS, suggesting that energy was required for immune stimulation. In the V. harveyi pathogen challenge trial, all shrimp groups fed with diets containing LPS from both bacterial sources showed better survival than the control group without LPS. When comparing groups fed with LPS supplemented diets, the higher concentration of LPS (8 µg/body weight) from E. coli resulted in a better survival rate than a lower concentration (4 µg/body weight). Conversely, shrimp fed with a diet containing LPS from V. harveyi showed a lower survival rate when a higher dose of LPS (8 µg/body weight) was administered than the group fed with a lower concentration of LPS (4 µg/body weight). This could be due to overstimulation of shrimp immune responses, especially by LPS derived from shrimp pathogens, resulting in a reverse effect. These results confirm that immunity in shrimp upon administration of bacterial LPS depends on the origin and dose of the LPS administered.

Intrinsic Allergenicity Potential of Salt-Soluble Protein Extracts from the Diploid, Tetraploid and Hexaploid Wheats: Validation Using an Adjuvant-Free Mouse Model.

Wheat allergies are potentially life-threatening and, therefore, have become a major health concern at the global level. It is largely unknown at present whether genetic variation in allergenicity potential exists among hexaploid, tetraploid and diploid wheat species. Such information is critical in establishing a baseline allergenicity map to inform breeding efforts to identify hyper-, hypo- and non-allergenic varieties. We recently reported a novel mouse model of intrinsic allergenicity using the salt-soluble protein extract (SSPE) from durum, a tetraploid wheat (Triticum durum). Here, we validated the model for three other wheat species [hexaploid common wheat (Triticum aestivum), diploid einkorn wheat (Triticum monococcum), and the ancient diploid wheat progenitor, Aegilops tauschii], and then tested the hypothesis that the SSPEs from wheat species will exhibit differences in relative allergenicities. Balb/c mice were repeatedly exposed to SSPEs via the skin. Allergic sensitization potential was assessed by specific (s) IgE antibody responses. Oral anaphylaxis was quantified by the hypothermic shock response (HSR). The mucosal mast cell response (MMCR) was determined by measuring mast cell protease in the blood. While T. monococcum elicited the least, but significant, sensitization, others were comparable. Whereas Ae. taushcii elicited the least HSR, the other three elicited much higher HSRs. Similarly, while Ae. tauschii elicited the least MMCR, the other wheats elicited much higher MMCR as well. In conclusion, this pre-clinical comparative mapping strategy may be used to identify potentially hyper-, hypo- and non-allergenic wheat varieties via crossbreeding and genetic engineering methods.

Cancer immunotherapy using artificial adjuvant vector cells to deliver NY-ESO-1 antigen to dendritic cells in situ.

NY-ESO-1 is a cancer/testis antigen expressed in various cancer types. However, the induction of NY-ESO-1-specific CTLs through vaccines is somewhat difficult. Thus, we developed a new type of artificial adjuvant vector cell (aAVC-NY-ESO-1) expressing a CD1d-NKT cell ligand complex and a tumor-associated antigen, NY-ESO-1. First, we determined the activation of invariant natural killer T (iNKT) and natural killer (NK) cell responses by aAVC-NY-ESO-1. We then showed that the NY-ESO-1-specific CTL response was successfully elicited through aAVC-NY-ESO-1 therapy. After injection of aAVC-NY-ESO-1, we found that dendritic cells (DCs) in situ expressed high levels of costimulatory molecules and produced interleukn-12 (IL-12), indicating that DCs undergo maturation in vivo. Furthermore, the NY-ESO-1 antigen from aAVC-NY-ESO-1 was delivered to the DCs in vivo, and it was presented on MHC class I molecules. The cross-presentation of the NY-ESO-1 antigen was absent in conventional DC-deficient mice, suggesting a host DC-mediated CTL response. Thus, this strategy helps generate sufficient CD8+ NY-ESO-1-specific CTLs along with iNKT and NK cell activation, resulting in a strong antitumor effect. Furthermore, we established a human DC-transferred NOD/Shi-scid/IL-2γcnull immunodeficient mouse model and showed that the NY-ESO-1 antigen from aAVC-NY-ESO-1 was cross-presented to antigen-specific CTLs through human DCs. Taken together, these data suggest that aAVC-NY-ESO-1 has potential for harnessing innate and adaptive immunity against NY-ESO-1-expressing malignancies.

Influence of black rice (Oryza sativa L.) bran derived anthocyanin-extract on growth rate, immunological response, and immune-antioxidant gene expression in Nile tilapia (Oreochromis niloticus) cultivated in a biofloc system.

This study investigated the effects of dietary supplementation with anthocyanin extracted from black rice bran (AR) on the growth rate, immunological response, and expression of immune and antioxidant genes in Nile tilapia raised in an indoor biofloc system. A total of 300 Nile tilapia fingerlings (15.14 ± 0.032 g) were maintained in 150 L tanks and acclimatized for two weeks. Five experimental AR diets (0, 1, 2, 4, and 8 g kg-1) with various anthocyanin doses were used to feed the fish. We observed that the growth and feed utilization of fish fed with different dietary AR levels increased significantly after eight weeks (p < 0.05). In addition, the serum immunity of fish fed AR diets was much greater than that of those fed non-AR diets (p < 0.05). However, there were little or no difference in between fish fed AR enriched diets and the control AR-free diet (p > 0.05). After eight weeks, fish fed AR-supplemented diets had significantly higher mRNA transcript levels in immune (interleukin [IL]-1, IL-8, and liposaccharide-binding protein [LBP]) and antioxidant (glutathione transferase-alpha [GST-α] and glutathione reductase [GSR]) genes compared to control fish fed the AR-free diet, with the greatest enhancement of mRNA transcript levels (in the case of IL-8 by up to about 5.8-fold) in the 4 g kg-1 AR diet. These findings suggest that dietary inclusion of AR extract from black rice bran at 4-8 g kg-1 could function as a herbal immunostimulant to enhance growth performance, feed consumption, and immunity in Nile tilapia.

Effects of temperature on the innate immune response on Antarctic and sub-Antarctic fish Harpagifer antarcticus and Harpagifer bispinis challenged with two immunostimulants, LPS and Poly I:C: In vivo and in vitro approach.

Rising ocean temperatures due to climate change combined with the intensification of anthropogenic activity can drive shifts in the geographic distribution of species, with the risks of introducing new diseases. In a changing environment, new host-pathogen interactions or changes to existing dynamics represent a major challenge for native species at high latitudes. Notothenioid fish constitute a unique study system since members of this group are found inside and outside Antarctica, are highly adapted to cold and particularly sensitive to temperature increments. However, data about their immune response remains scarce. Here, we aimed to evaluate the innate immune response under thermal stress in two species of Notothenioid fish, Harpagifer antarcticus and Harpagifer bispinis. Adult individuals from both species were collected on King George Island (Antarctica), and Punta Arenas (Chile), respectively. Specimens were assigned to a control group or injected with one of two agents (LPS and Poly I:C) to simulate either a bacterial or viral infection, and subjected to three different temperatures 2, 5 and 8 °C for 1 week. In parallel, we established leukocytes primary cell cultures from head kidney, which were also subjected to the immunostimulants at the same three temperatures, and incubated for 0.5, 1, 3, 6, 12, 24, and 48 h. We evaluated the relative gene expression of genes involved in the innate immune response (TLR1, TLR3, NF-kB, MYD88, IFNGR e IL-8) through real time qPCR. We found differences between species mainly in vivo, where H. antarcticus exhibited upregulation at high temperatures and H. bispinis seemed to have reached their physiological minimum at 2 °C. Although temperature had a strong effect during the in vivo assay for both species, it was negligible for primary cell cultures, which responded primarily to condition and time. Moreover, while leukocytes responded with fluctuations across time points, in vivo both species manifested strong and clear patterns of gene expression. These results highlight the importance of evaluating the effect of multiple stressors and set a precedent for future research.

Characterization of aquaporin-1ab (Aqp1ab) mRNA in mud loach (Misgurnus mizolepis) exposed to heavy metal and immunostimulant stimuli.

Aquaporins (AQPs) are key proteins that regulate fluid homeostasis in cells via modulating osmotic water transport. In the present study, we identified three variants of Aqp1ab transcript (mmAQP1ab x1, mmAQP1ab x2, and mmAQP1ab x3) in mud loaches (Misgurnus mizolepis), and their expression patterns were examined in response to heavy metal and immunostimulant exposure. Mud loach Aqp1ab gene has a somewhat different organizational structure (i.e. five exons interrupted by four introns) compared to most other teleostean Aqp1ab orthologues, which have four exons. The 5'-flanking regulatory region of Aqp gene showed diverse transcription factor binding motifs, particularly those associated with stress/immune responses. Developmental expression patterns indicated that Aqp1ab mRNA was maternally inherited, presumably important for fine-tuning gene expression during embryonic and early larval developments. Expression of mud loach Aqp1ab mRNA was significantly and differentially modulated in several tissues (intestine, kidneys, spleen, and liver) in response to various heavy metal treatments. In addition, Aqp1ab gene expression was highly induced in response to immune challenge (LPS and polyI:C injections). Collectively, our results suggested that AQPs are multifunctional effectors playing diverse roles in cellular pathways relevant to immune and/or stress adaptation responses, in addition to their involvement in osmoregulation.

Glucan particles as a novel adjuvant for the induction of experimental autoimmune encephalomyelitis.

For over 70 years experimental autoimmune encephalomyelitis (EAE) has been induced with myelin autoantigens emulsified in complete Freund's adjuvant (CFA) which has significant side effects such as pain, inflammation, and tissue necrosis at the injection site. ß-1,3-d-glucan particles (GPs) are hollow microcapsules prepared from Saccharomyces cerevisiae cell walls that induce potent Th17 cell responses without causing strong injection site tissue reactions. We evaluated the potential of GPs complexed with neuroantigens to induce EAE while avoiding undesirable side effects. GPs loaded with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) or proteolipid protein 139-151 (PLP139-151) peptides effectively induced EAE in C57BL/6 mice and SJL mice. Disease severity, CNS pathology and immune responses were comparable between GP- and CFA-immunized mice. Importantly, injection with GPs resulted in significantly decreased inflammation compared with CFA. We posit that use of GPs provides an alternative means for inducing EAE that results in comparable disease, but less discomfort to animals.

Bioresorbable Depot for Sustained Release of Immunostimulatory Resiquimod in Suppressing Both Primary Triple-Negative Breast Tumors and Metastatic Occurrence.

In light of immune facilities trafficking toward the pathological sites along upward gradient of immunostimulatory cytokines, a localized resiquimod (Toll-like receptor 7/8 agonist) release depot was manufactured for pursuit of precision immunostimulation toward intractable triple-negative breast carcinoma. In principle, resiquimod/poly(lactic-co-glycolic acid) microspheres were fabricated and embedded into injectable and biodegradable poly(ethylene glycol) (PEG)-based hydrogel. The subsequent investigations approved persistent retention of immunostimulatory resiquimod in tumors upon peritumoral administration, which consequently led to localized and consistent secretion of immunostimulatory cytokines. Initially, not only innate tumor phagocytosis but also adaptive antitumor immunities were successfully cultivated for in situ suppression of the growth of primary solid tumors, more importantly, capable of inhibiting distant pulmonary metastasis, as evidenced by observation of enormous lymphocytes selectively gathering in the pulmonary artery. Hence, our presented study provided an important clinical indication of using immunostimulatory drugs to activate potent innate and adaptive antitumor immunities for precision antitumor therapy. Further immunomodulatory strategies, such as checkpoint blockage and tumor immunogenicity, could also be complementary for development of advanced antitumor immunotherapeutics in treatment of a number of intractable tumors.

Nano-Immune-Engineering Approaches to Advance Cancer Immunotherapy: Lessons from Ultra-pH-Sensitive Nanoparticles.

Immunotherapy has transformed the field of oncology and patient care. By leveraging the immune system of the host, immunostimulatory compounds exert a durable, personalized response against the patient's own tumor. Despite the clinical success, the overall response rate from current therapies (e.g., immune checkpoint inhibitors) remains low (∼20%) because tumors develop multiple resistance pathways at molecular, cellular, and microenvironmental levels. Unlike other oncologic therapies, harnessing antitumor immunity requires precise activation of a complex immunological system with multiple levels of regulation over its function. This requires the ability to exert control over immune cells in both intracellular compartments and various extracellular sites, such as the tumor microenvironment, in a spatiotemporally coordinated fashion.The immune system has evolved to sense and respond to nano- and microparticulates (e.g., viruses, bacteria) as foreign pathogens. Through the versatile control of composition, size, shape, and surface properties of nanoparticles, nano-immune-engineering approaches are uniquely positioned to mount appropriate immune responses against cancer. This Account highlights the development and implementation of ultra-pH-sensitive (UPS) nanoparticles in cancer immunotherapy with an emphasis on nanoscale cooperativity. Nanocooperativity has been manifested in many biological systems and processes (e.g., protein allostery, biomolecular condensation), where the system can acquire emergent properties distinct from the sum of individual parts acting in isolation.Using UPS nanoparticles as an example, we illustrate how all-or-nothing protonation cooperativity during micelle assembly/disassembly can be leveraged to augment the cancer-immunity cycle toward antitumor immunity. The cooperativity behavior enables instant and pH-triggered payload release and dose accumulation in acidic sites (e.g., endocytic organelles of antigen presenting cells, tumor microenvironment), intercepting specific immunological and tumor pathophysiological processes for therapy. These efforts include T cell activation in lymph nodes by coordinating cytosolic delivery of tumor antigens to dendritic cells with simultaneous activation of stimulator of interferon genes (STING), or tumor-targeted delivery of acidotic inhibitors to reprogram the tumor microenvironment and overcome T cell retardation. Each treatment strategy represents a nodal intervention in the cancer-immunity cycle, featuring the versatility of UPS nanoparticles. Overall, this Account aims to highlight nanoimmunology, an emerging cross field that exploits nanotechnology's unique synergy with immunology through nano-immune-engineering, for advancing cancer immunotherapy.

Cytokine responses to various larval stages of equine strongyles and modulatory effects of the adjuvant G3 in vitro.

AIMS: To generate different larval stages of Strongylus vulgaris and to study cytokine responses in cultures of eqPBMC exposed to defined larval stages of S. vulgaris and cyathostomins with the aim to understand the early immune reaction to these parasites. METHODS AND RESULTS: EqPBMC were exposed to S. vulgaris larvae (L3, exsheated L3 and L4) and cyathostomin L3 and analysed for cytokine gene expression. Procedures for decontamination, culturing and attenuation of larvae were established. Transcription of IL-4, IL-5 and IL-13 was induced by both S. vulgaris and cyathostomin L3. Moulting of S. vulgaris from L3 to L4 stage was accompanied by a shift to high expression of IL-5 and IL-9 (exsheated L3 and L4) and IFN-γ (L4 only). In parallel, the adjuvant G3 modified the cytokine profile induced by both parasites by reducing the expression of IL-4, IL-5 and IL-10 while concomitantly enhancing the expression of IFN-γ. CONCLUSION: The L4 stage of S. vulgaris generated a cytokine profile different from that induced by the earlier L3 stage of S. vulgaris and cyathostomins. This diversity depending on the life cycle stage will have implications for the choice of antigen and adjuvant in future vaccine design.