The Papain-like Protease Domain of Severe Acute Respiratory Syndrome Coronavirus 2 Conjugated with Human Beta-Defensin 2 and Co1 Induces Mucosal and Systemic Immune Responses against the Virus.

Most of the licensed vaccines against SARS-CoV-2 target spike proteins to induce viral neutralizing antibodies. However, currently prevalent SARS-CoV-2 variants contain many mutations, especially in their spike proteins. The development of vaccine antigens with conserved sequences that cross-react with variants of SARS-CoV-2 is needed to effectively defend against SARS-CoV-2 infection. Given that viral infection is initiated in the respiratory mucosa, strengthening the mucosal immune response would provide effective protection. We constructed a mucosal vaccine antigen using the papain-like protease (PLpro) domain of non-structural protein 3 of SARS-CoV-2. To potentiate the mucosal immune response, PLpro was combined with human beta-defensin 2, an antimicrobial peptide with mucosal immune adjuvant activity, and Co1, an M-cell-targeting ligand. Intranasal administration of the recombinant PLpro antigen conjugate into C57BL/6 and hACE2 knock-in (KI) mice induced antigen-specific T-cell and antibody responses with complement-dependent cytotoxic activity. Viral challenge experiments using the Wuhan and Delta strains of SARS-CoV-2 provided further evidence that immunized hACE2 KI mice were protected against viral challenge infections. Our study shows that PLpro is a useful candidate vaccine antigen against SARS-CoV-2 infection and that the inclusion of human beta-defensin 2 and Co1 in the recombinant construct may enhance the efficacy of the vaccine.

Marked enhancement of the immunogenicity of plant-expressed IgG-Fc fusion proteins by inclusion of cholera toxin non-toxic B subunit within the single polypeptide.

Immunoglobulin G (IgG)-based fusion proteins have been widely exploited as a potential vaccine delivery platform but in the absence of exogenous adjuvants, the lack of robust immunity remains an obstacle. Here, we report on a key modification that overcomes that obstacle. Thus, we constructed an IgG-Fc vaccine platform for dengue, termed D-PCF, which in addition to a dengue antigen incorporates the cholera toxin non-toxic B subunit (CTB) as a molecular adjuvant, with all three proteins expressed as a single polypeptide. Following expression in Nicotiana benthamiana plants, the D-PCF assembled as polymeric structures of similar size to human IgM, a process driven by the pentamerization of CTB. A marked improvement of functional properties in vitro and immunogenicity in vivo over a previous iteration of the Fc-fusion protein without CTB [1] was demonstrated. These include enhanced antigen presenting cell binding, internalization and activation, complement activation, epithelial cell interactions and ganglioside binding, as well as more efficient polymerization within the expression host. Following immunization of mice with D-PCF by a combination of systemic and mucosal (intranasal) routes, we observed robust systemic and mucosal immune responses, as well as systemic T cell responses, significantly higher than those induced by a related Fc-fusion protein but without CTB. The induced antibodies could bind to the domain III of the dengue virus envelope protein from all four dengue serotypes. Finally, we also demonstrated feasibility of aerosolization of D-PCF as a prerequisite for vaccine delivery by the respiratory route.

Lipid mediators obtained from docosahexaenoic acid by soybean lipoxygenase attenuate RANKL-induced osteoclast differentiation and rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by persistent inflammation and joint destruction. A lipid mediator (LM, namely, 17S-monohydroxy docosahexaenoic acid, resolvin D5, and protectin DX in a ratio of 3:47:50) produced by soybean lipoxygenase from DHA, exhibits anti-inflammatory activity. In this study, we determined the effect of LM on collagen antibody-induced arthritis (CAIA) in mice and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation in RAW264.7 cells. LM effectively downregulated the expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K, inhibited osteoclast formation, and suppressed the NF-κB signaling pathway in vitro. In vivo, LM at 10 µg/kg/day significantly decreased paw swelling and inhibited progression of arthritis in CAIA mice. Moreover, proinflammatory cytokine (tumor necrosis factor-α, interleukin (IL)-6, IL-1ß, IL-17, and interferon-γ) levels in serum were decreased, whereas IL-10 levels were increased following LM treatment. Furthermore, LM alleviated joint inflammation, cartilage erosion, and bone destruction in the ankles, which may be related to matrix metalloproteinase and Janus kinase (JAK)-signal transducer and activators of transcription (STAT) signaling pathway. Our findings suggest that LM attenuates arthritis severity, restores serum imbalances, and modifies joint damage. Thus, LM represents a promising therapy for relieving RA symptoms.

Lipid mediators derived from DHA alleviate DNCB-induced atopic dermatitis and improve the gut microbiome in BALB/c mice.

Atopic dermatitis (AD) is a chronic inflammatory skin condition that primarily results from immune dysregulation. We determined the potential therapeutic benefits of lipid mediators (LM, 17S-monohydroxy DHA, resolvin D5, and protectin DX in a ratio of 3:47:50) produced by soybean lipoxygenase from DHA. The underlying molecular mechanisms involved in TNF-α/IFN-γ-stimulated HaCaT cells as well as its effect in an AD mouse model induced by DNCB in BALB/c mice were examined. The results indicated that LM effectively attenuates the production of inflammatory cytokines (IL-6 and IL-1ß) and chemokines (IL-8 and MCP-1) by inhibiting the NF-κB signaling pathway in TNF-α/IFN-γ-stimulated HaCaT cells. The oral administration of LM at 5 or 10 µg/kg/day significantly reduced skin lesions, epidermal thickness, and mast cell infiltration in AD mice. Furthermore, LM reduced the production of IgE and inflammatory cytokines (TNF-α, IL-6, and IL-1ß) in the serum, modulated gut microbiota diversity, and restored the microbial composition. Overall, our findings suggest that LM represents a potential therapeutic agent for improving AD symptoms through its ability to suppress inflammatory cytokines and alter the composition of gut microbiota.

C5aR+ dendritic cells fine-tune the Peyer's patch microenvironment to induce antigen-specific CD8+ T cells.

The mucosal delivery route is considered ideal for immunization. However, induction of antigen-specific mucosal immunity is difficult due to the tolerogenic environment. Therefore, developing an immunogenic mucosal dendritic cell (DC)-targeting strategy is required. Herein, we investigated the characteristics and immunogenic potential of Peyer's patch (PP) DCs as an oral vaccination-targeting strategy. Single-cell RNA sequencing analysis of the PP DCs showed that complement C5a receptor- and lysozyme-expressing DCs exhibit increased expression of genes related to chemotaxis. Administration of the Co1 peptide, a C5aR ligand, increased CD8+ T cell infiltration and response to the co-delivered model antigen in mice. Furthermore, in the SARS-CoV-2 vaccine model, vaccination with Co1 elicited both systemic and mucosal immunity. Collectively, these findings demonstrate that C5aR signaling in mucosal DCs plays a role in regulating adjuvant activity by modulating the tissue microenvironment.

Understanding the Roles of Host Defense Peptides in Immune Modulation: From Antimicrobial Action to Potential as Adjuvants.

Host defense peptides are expressed in various immune cells, including phagocytic cells and epithelial cells. These peptides selectively alter innate immune pathways in response to infections by pathogens, such as bacteria, fungi, and viruses, and modify the subsequent adaptive immune environment. Consequently, they play a wide range of roles in both innate and adaptive immune responses. These peptides are of increasing importance due to their broad-spectrum antimicrobial activity and their functions as mediators linking innate and adaptive immune responses. This review focuses on the pleiotropic biological functions and related mechanisms of action of human host defense peptides and discusses their potential clinical applications.

7S,15R-Dihydroxy-16S,17S-epoxy-docosapentaenoic Acid Overcomes Chemoresistance of 5-Fluorouracil by Suppressing the Infiltration of Tumor-Associated Macrophages and Inhibiting the Activation of Cancer Stem Cells in a Colorectal Cancer Xenograft Model.

Although the tumor bulk is initially reduced by 5-fluorouracil (5-FU), chemoresistance developed due to prolonged chemotherapy in colorectal cancer (CRC). The enrichment of cancer stem cells (CSCs) and the infiltration of tumor-associated macrophages (TAMs) contribute to chemoresistance and poor outcomes. A docosahexaenoic acid derivative developed by our group, 7S,15R-dihydroxy-16S,17S-epoxy-docosapentaenoic acid (diHEP-DPA), exerts antitumor effects against TAMs infiltration and CSCs enrichment in our previous study. The current study aimed to investigate whether diHEP-DPA was able to overcome chemoresistance to 5-FU in CRCs, together with the potential synergistic mechanisms in a CT26-BALB/c mouse model. Our results suggested that although 5-FU inhibited tumor growth, 5-FU enriched CSCs via the WNT/ß-catenin signaling pathway, resulting in chemoresistance in CRCs. However, we revealed that 5-FU promoted the infiltration of TAMs via the NF-kB signaling pathway and improved epithelial-mesenchymal transition (EMT) via the signal transducer and activator of the transcription 3 (STAT3) signaling pathway; these traits were believed to contribute to CSC activation. Furthermore, supplementation with diHEP-DPA could overcome drug resistance by decreasing the CSCs, suppressing the infiltration of TAMs, and inhibiting EMT progression. Additionally, the combinatorial treatment of diHEP-DPA and 5-FU effectively enhanced phagocytosis by blocking the CD47/signal regulatory protein alpha (SIRPα) axis. These findings present that diHEP-DPA is a potential therapeutic supplement to improve drug outcomes and suppress chemoresistance associated with the current 5-FU-based therapies for colorectal cancer.

Recombinant Protein Mimicking the Antigenic Structure of the Viral Surface Envelope Protein Reinforces Induction of an Antigen-Specific and Virus-Neutralizing Immune Response Against Dengue Virus.

Dengue virus (DENV), belonging to the family Flaviviridae, is the causative agent of dengue and comprises four serotypes. A second heterologous DENV infection is a critical risk factor for severe dengue, and no effective vaccine is available to prevent infection by all four DENV serotypes. Recombinant DENV vaccines are primarily based on the envelope proteins, prM and E. The E protein and its envelope domain III (EDIII) have been investigated as candidate antigens (Ags) for recombinant subunit vaccines. However, most EDIII-based Ags are monomers that do not display the cognate antigenic structure of E protein, which is essential for induction of virus-neutralizing immunity. Here, we developed recombinant DENV-2 envelope domain (r2ED) protein as an Ag that mimics the quaternary structure of E protein on the DENV surface. We confirmed that r2ED retained the conformational epitope displayed at the E-dimer interface, which reportedly exhibits broad virus-neutralizing capacity, without displaying the fusion loop epitope that causes antibody (Ab)-dependent enhancement. Furthermore, compared with EDIII alone, r2ED elicited stronger Ag-specific and cross-reactive neutralizing Ab and T cell-mediated immune responses in mice. This Ag-specific immunity was maintained at an elevated level 6 months after the last immunization, suggesting sustained Ag-specific immune memory. Taken together, these observations suggest that r2ED could be used to develop an improved subunit vaccine capable of inducing a broadly cross-reactive and long-lasting immune response against DENV infection.

Effects of Fish Oil, Lipid Mediators, Derived from Docosahexaenoic Acid, and Their Co-Treatment against Lipid Metabolism Dysfunction and Inflammation in HFD Mice and HepG2 Cells.

Although fish oil (FO) and lipid mediators (LM) derived from polyunsaturated fatty acids can prevent obesity, their combined effects and cellular metabolism remain unclear. Therefore, this study aimed to examine the potential protective and metabolic effects of FO in combination with LM (a mixture of 17S-monohydroxy docosahexaenoic acid, resolvin D5, and protectin DX [3:47:50], derived from docosahexaenoic acid (DHA)) on palmitic acid (PA)-induced HepG2 cells and high-fat- diet (HFD)-induced C57BL/6J mice after 9-week treatment. Lipid metabolism disorders and inflammation induced by HFD and PA were substantially reduced after FO and LM treatment. Further, FO and LM treatments reduced lipid accumulation by increasing fatty acid oxidation via peroxisome proliferator-activated receptor α and carnitine-palmitoyl transferase 1 as well as by decreasing fatty acid synthesis via sterol regulatory element-binding protein-1c and fatty acid synthase. Finally, FO and LM treatment reduced inflammation by blocking the NF-κB signaling pathway. Importantly, the combination of FO and LM exhibited more robust efficacy against nonalcoholic fatty liver disease, suggesting that FO supplemented with LM is a beneficial dietary strategy for treating this disease.

Application of Antimicrobial Peptide LL-37 as an Adjuvant for Middle East Respiratory Syndrome-Coronavirus Antigen Induces an Efficient Protective Immune Response Against Viral Infection After Intranasal Immunization.

The human antimicrobial peptide LL-37 has chemotactic and modulatory activities in various immune cells, including dendritic cells. Because of its characteristics, LL-37 can be considered an adjuvant for vaccine development. In this study, we confirmed the possible adjuvant activity of LL-37 in mucosal vaccine development against Middle East respiratory syndrome-coronavirus (MERS-CoV) by means of intranasal immunization in C57BL/6 and human dipeptidyl peptidase 4 (hDPP4)-transgenic (hDPP4-Tg) mice. Intranasal immunization using the receptor-binding domain (RBD) of MERS-CoV spike protein (S-RBD) recombined with LL-37 (S-RBD-LL-37) induced an efficient mucosal IgA and systemic IgG response with virus-neutralizing activity, compared with S-RBD. Ag-specific CTL stimulation was also efficiently induced in the lungs of mice that had been intranasally immunized with S-RBD-LL-37, compared with S-RBD. Importantly, intranasal immunization of hDPP4-Tg mice with S-RBD-LL-37 led to reduced immune cell infiltration into the lungs after infection with MERS-CoV. Finally, intranasal immunization of hDPP4-Tg mice with S-RBD-LL-37 led to enhanced protective efficacy, with increased survival and reduced body weight loss after challenge infection with MERS-CoV. Collectively, these results suggest that S-RBD-LL-37 is an effective intranasal vaccine candidate molecule against MERS-CoV infection.

Protective effect of 17S­epoxy­docosapentaenoic acid against dextran sulfate sodium induced ulcerative colitis in BALB/c mice.

Ulcerative colitis (UC) is difficult to eradicate as it leads to chronic inflammation in the digestive tract due to immune system malfunction. The present study demonstrated the protective effect of 7S,15R­dihydroxy­16S,17S­epoxy­docosapentaenoic acid (diHEP­DPA), which had been previously synthesized, on a dextran sulfate sodium (DSS)­induced BALB/c mouse model of UC. UC was induced with 4% DSS drinking water for 7 days. Initially, the anti­inflammatory effect of diHEP­DPA was confirmed by demonstrating that lipopolysaccharide­stimulated THP1 cells treated with diHEP­DPA decreased IL­6, TNF­α and nitrite levels by fluorescence­activated cell sorting (FACS) and Griess reagent kit. The results indicated that the administration of diHEP­DPA at 20 µg/kg significantly reduced the severity of colitis, as determined by hematoxylin and eosin staining. The levels of TNF­α, IL­6 and IL­1ß in the colon tissue and serum were significantly reduced in the diHEP­DPA + DSS­treated group compared with in the control group, as determined by FACS and ELISA kit. It was also observed that diHEP­DPA decreased myeloperoxidase (MPO) and nitrite levels in the colon tissues of diHEP­DPA + DSS­treated mice, as indicated using commercial MPO and nitric oxide kits. The diHEP­DPA+DSS­treated mice also exhibited decreased expression levels of phosporylated (p)­inhibitor κB protein, p­p65 and inducible nitric oxide synthase in the colon tissue by inhibiting inflammation, which were measured by reverse transcription­quantitative PCR and weatern blot analysis. Overall, the present study demonstrated the protective effect of diHEP­DPA against a severe colitis condition in vivo.

Enzymatic bioconversion of ginseng powder increases the content of minor ginsenosides and potentiates immunostimulatory activity.

Background: Ginsenosides are biologically active components of ginseng and have various functions. In this study, we investigated the immunomodulatory activity of a ginseng product generated from ginseng powder (GP) via enzymatic bioconversion. This product, General Bio compound K-10 mg solution (GBCK10S), exhibited increased levels of minor ginsenosides, including ginsenoside-F1, compound K, and compound Y. Methods: The immunomodulatory properties of GBCK10S were confirmed using mice and a human natural killer (NK) cell line. We monitored the expression of molecules involved in immune responses via enzyme-linked immunosorbent assay, flow cytometry, NK cell-targeted cell destruction, quantitative reverse-transcription real-time polymerase chain reaction, and Western blot analyses. Results: Oral administration of GBCK10S significantly increased serum immunoglobulin M levels and primed splenocytes to express pro-inflammatory cytokines such as interleukin-6, tumor necrosis factor-α, and interferon-γ. Oral administration of GBCK10S also activated NK cells in mice. Furthermore, GBCK10S treatment stimulated a human NK cell line in vitro, thereby increasing granzyme B gene expression and activating STAT5. Conclusion: GBCK10S may have potent immunostimulatory properties and can activate immune responses mediated by B cells, Th1-type T cells, and NK cells.

Current status and perspectives on vaccine development against dengue virus infection.

Dengue virus (DENV) consists of four serotypes in the family Flaviviridae and is a causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. DENV is transmitted by mosquitoes, Aedes aegypti and A. albopictus, and is mainly observed in areas where vector mosquitoes live. The number of dengue cases reported by the World Health Organization increased more than 8-fold over the last two decades from 505,430 in 2000 to over 2.4 million in 2010 to 5.2 million in 2019. Although vaccine is the most effective method against DENV, only one commercialized vaccine exists, and it cannot be administered to children under 9 years of age. Currently, many researchers are working to resolve the various problems hindering the development of effective dengue vaccines; understanding of the viral antigen configuration would provide insight into the development of effective vaccines against DENV infection. In this review, the current status and perspectives on effective vaccine development for DENV are examined. In addition, a plausible direction for effective vaccine development against DENV is suggested.

The docosahexaenoic acid derivatives, diHEP-DPA and TH-DPA, synthesized via recombinant lipoxygenase, ameliorate disturbances in lipid metabolism and liver inflammation in high fat diet-fed mice.

7S,15R-Dihydroxy-16S,17S-epoxy-docosapentaenoic acid (diHEP-DPA) and 7S,15R,16S,17S-tetrahydroxy-docosapentaenoic acid (TH-DPA) are two novel lipid mediators derived from docosahexaenoic acid (DHA) that we previously synthesized via combined enzymatic and chemical reactions. In the present study, we investigated the effects of these compounds on disturbances in lipid metabolism and liver inflammation induced by a high fat diet (HFD) in mice. Male BALB/c mice were randomly divided into four groups (n = 10/group): controls, HFD only, HFD + diHEP-DPA, and HFD + TH-DPA. Mice in HFD + diHEP-DPA and HFD + TH-DPA groups were orally administered 20 µg/kg of diHEP-DPA or TH-DPA, respectively. Measurements of adipose accumulation and liver inflammation showed that both diHEP-DPA and TH-DPA decreased adipose tissue mass and liver color depth, as well as total cholesterol, triglycerides, and low-density lipoprotein-cholesterol in the serum of HFD-fed mice compared with mice in the HFD-only group, while elevating high-density lipoprotein-cholesterol. Both of them also decreased hepatic expression of genes encoding lipid synthesis-related proteins (PPARγ, SIRT1, SREBP-1c and FASN) and increased the expression of genes encoding proteins involved in lipid degradation (PPARα and CPT-1) in the liver. Western blotting and quantitative RT-PCR confirmed that diHEP-DPA or TH-DPA administration modulated the expression of inflammation-related genes (TNF-α and IL-6) and inhibited activation of the NF-κB signaling pathway in livers of HFD-fed mice. Taken together, our data indicate that diHEP-DPA and TH-DPA ameliorate liver inflammation and inhibit HFD-induced obesity in mice.

Intranasal immunization with a Middle East respiratory syndrome-coronavirus antigen conjugated to the M-cell targeting ligand Co4B enhances antigen-specific mucosal and systemic immunity and protects against infection.

Middle East respiratory syndrome (MERS) is a threat to public health worldwide. A vaccine against the causative agent of MERS, MERS-coronavirus (MERS-CoV), is urgently needed. We previously identified a peptide ligand, Co4B, which can enhance antigen (Ag) delivery to the nasal mucosa and promote Ag-specific mucosal and systemic immune responses following intranasal immunization. MERS-CoV infects via the respiratory route; thus, we conjugated the Co4B ligand to the MERS-CoV spike protein receptor-binding domain (S-RBD), and used this to intranasally immunize C57BL/6 and human dipeptidyl peptidase 4-transgenic (hDPP4-Tg) mice. Ag-specific mucosal immunoglobulin (Ig) A and systemic IgG, together with virus-neutralizing activities, were highly induced in mice immunized with Co4B-conjugated S-RBD (S-RBD-Co4B) compared to those immunized with unconjugated S-RBD. Ag-specific T cell-mediated immunity was also induced in the spleen and lungs of mice intranasally immunized with S-RBD-Co4B. Intranasal immunization of hDPP4-Tg mice with S-RBD-Co4B reduced immune cell infiltration into the tissues of virus-challenged mice. Finally, S-RBD-Co4B-immunized mice exhibited were better protected against infection, more likely to survive, and exhibited less body weight loss. Collectively, our results suggest that S-RBD-Co4B could be used as an intranasal vaccine candidate against MERS-CoV infection.

7S,15R-Dihydroxy-16S,17S-Epoxy-Docosapentaenoic Acid, a Novel DHA Epoxy Derivative, Inhibits Colorectal Cancer Stemness through Repolarization of Tumor-Associated Macrophage Functions and the ROS/STAT3 Signaling Pathway.

Colorectal cancer is a highly malignant cancer that is inherently resistant to many chemotherapeutic drugs owing to the complicated tumor-supportive microenvironment (TME). Tumor-associated macrophages (TAM) are known to mediate colorectal cancer metastasis and relapse and are therefore a promising therapeutic target. In the current study, we first confirmed the anti-inflammatory effect of 7S,15R-dihydroxy-16S,17S-epoxy-docosapentaenoic acid (diHEP-DPA), a novel DHA dihydroxy derivative synthesized in our previous work. We found that diHEP-DPA significantly reduced lipopolysaccharide (LPS)-induced inflammatory cytokines secretion of THP1 macrophages, IL-6, and TNF-α. As expected, diHEP-DPA also modulated TAM polarization, as evidenced by decreased gene and protein expression of the TAM markers, CD206, CD163, VEGF, and TGF-ß1. During the polarization process, diHEP-DPA treatment decreased the concentration of TGF-ß1, IL-1ß, IL-6, and TNF-α in culture supernatants via inhibiting the NF-κB pathway. Moreover, diHEP-DPA blocked immunosuppression by reducing the expression of SIRPα in TAMs and CD47 in colorectal cancer cells. Knowing that an inflammatory TME largely serves to support epithelial-mesenchymal transition (EMT) and cancer stemness, we tested whether diHEP-DPA acted through polarization of TAMs to regulate these processes. The intraperitoneally injected diHEP-DPA inhibited tumor growth when administered alone or in combination with 5-fluorouracil (5-FU) chemotherapy in vivo. We further found that diHEP-DPA effectively reversed TAM-conditioned medium (TCCM)-induced EMT and enhanced colorectal cancer stemness, as evidenced by its inhibition of colorectal cancer cell migration, invasion and expression of EMT markers, as well as cancer cell tumorspheres formation, without damaging colorectal cancer cells. DiHEP-DPA reduced the population of aldehyde dehydrogenase (ALDH)-positive cells and expression of colorectal stemness marker proteins (CD133, CD44, and Sox2) by modulating TAM polarization. Additionally, diHEP-DPA directly inhibited cancer stemness by inducing the production of reactive oxygen species (ROS), which, in turn, reduced the phosphorylation of nuclear signal transducer and activator of transcription 3 (STAT3). These data collectively suggest that diHEP-DPA has the potential for development as an anticancer agent against colorectal cancer.

Systemic and Oral Immunogenicity of Porcine Epidemic Diarrhea Virus Antigen Fused to Poly-Fc of Immunoglobulin G and Expressed in ΔXT/FT Nicotiana benthamiana Plants.

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family has become increasingly probelmatic in the pig farming industry. Currently, there are no effective, globally applicable vaccines against PEDV. Here, we tested a recombinant PEDV vaccine candidate based on the expression of the core neutralising epitope (COE) of PEDV conjugated to polymeric immunoglobulin G scaffold (PIGS) in glycoengineered Nicotiana be nthamiana plants. The biological activity of COE-PIGS was demonstrated by binding to C1q component of the complement system, as well as the surface of antigen-presenting cells (APCs) in vitro. The recombinant COE-PIGS induced humoral and cellular immune responses specific for PEDV after both systemic and mucosal vaccination. Altogether, the data indicated that PEDV antigen fusion to poly-Fc could be a promising vaccine platform against respiratory PEDV infection.

Identification of a Direct-Acting Antiviral Agent Targeting RNA Helicase via a Graphene Oxide Nanobiosensor.

Dengue virus (DENV), an arbovirus transmitted by mosquitoes, causes infectious diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Despite the dangers posed by DENV, there are no approved antiviral drugs for treatment of DENV infection. Considering the potential for a global dengue outbreak, rapid development of antiviral agents against DENV infections is crucial as a preemptive measure; thus, the selection of apparent drug targets, such as the viral enzymes involved in the viral life cycle, is recommended. Helicase, a potential drug target in DENV, is a crucial viral enzyme that unwinds double-stranded viral RNA, releasing single-stranded RNA genomes during viral replication. Therefore, an inhibitor of helicase activity could serve as a direct-acting antiviral agent. Here, we introduce an RNA helicase assay based on graphene oxide, which enables fluorescence-based analysis of RNA substrate-specific helicase enzyme activity. This assay demonstrated high reliability and ability for high-throughput screening, identifying a new helicase inhibitor candidate, micafungin (MCFG), from an FDA-approved drug library. As a direct-acting antiviral agent targeting RNA helicase, MCFG inhibits DENV proliferation in cells and an animal model. Notably, in vivo, MCFG treatment reduced viremia, inflammatory cytokine levels, and viral loads in several tissues and improved survival rates by up to 40% in a lethal mouse model. Therefore, we suggest MCFG as a potential direct-acting antiviral drug candidate.

13R,20-Dihydroxydocosahexaenoic Acid, a Novel Dihydroxy- DHA Derivative, Inhibits Breast Cancer Stemness through Regulation of the Stat3/IL-6 Signaling Pathway by Inducing ROS Production.

Breast cancer is a major health problem worldwide. Cancer stem cells (CSCs) are known to mediate breast cancer metastasis and recurrence and are therefore a promising therapeutic target. In this study, we investigated the anti-inflammatory effect of 13R,20-dihydroxydocosahexaenoic acid (13R,20-diHDHA), a novel dihydroxy-DHA derivative, which was synthesized through an enzymatic reaction using cyanobacterial lipoxygenase. We found that 13R,20-diHDHA reduced the macrophage secretion of the inflammatory cytokines, IL-6 and TNF-α, and thus appeared to have anti-inflammatory effects. As the inflammatory tumor microenvironment is largely devoted to supporting the cancer stemness of breast cancer cells, we investigated the effect of 13R,20-diHDHA on breast cancer stemness. Indeed, 13R,20-diHDHA effectively inhibited breast cancer stemness, as evidenced by its ability to dose-dependently inhibit the mammospheres formation, colony formation, migration, and invasion of breast CSCs. 13R,20-diHDHA reduced the populations of CD44high/CD24low and aldehyde dehydrogenase (ALDH)-positive cells and the expression levels of the cancer stemness-related self-renewal genes, Nanog, Sox2, Oct4, c-Myc, and CD44. 13R,20-diHDHA increased reactive oxygen species (ROS) production, and the generated ROS reduced the phosphorylation of nuclear signal transducer and activator of transcription 3 (Stat3) and the secretion of IL-6 by mammospheres. These data collectively suggest that 13R,20-diHDHA inhibits breast cancer stemness through ROS production and downstream regulation of Stat3/IL-6 signaling, and thus might be developed as an anti-cancer agent acting against CSCs.

Complement C5a promotes antigen cross-presentation by Peyer's patch monocyte-derived dendritic cells and drives a protective CD8+ T cell response.

The complement fragment C5a is closely associated with adaptive immune induction in the mucosa. However, the mechanisms that control CD8+ T cell responses by C5a have not been extensively explored. This study reveals that C5/C5a in the Peyer's patch (PP) subepithelial dome increases upon oral Listeria infection. We hypothesize that C5aR+ PP cells play an important role in the induction of antigen-specific T cell immunity. Using single-cell RNA sequencing, we identify C5aR- and lysozyme-expressing dendritic cells (C5aR+ LysoDCs) in PP and examine their role in CD8+ T cell immune induction. Stimulation of C5aR+ LysoDCs by C5a increases reactive oxygen species levels, leading to efficient antigen cross-presentation, which elicits an antigen-specific CD8+ T cell response. In C5-deficient mice, oral co-administration of C5a and Listeria enhances Listeria-specific cytotoxic T cell levels. Collectively, these findings suggest a role of the complement system in intestinal T cell immunity.