N-dihydrogalactochitosan serves as an effective mucosal adjuvant for intranasal vaccine in combination with recombinant viral proteins against respiratory infection.

Mucosal vaccinations for respiratory pathogens provide effective protection as they stimulate localized cellular and humoral immunities at the site of infection. Currently, the major limitation of intranasal vaccination is using effective adjuvants capable of withstanding the harsh environment imposed by the mucosa. Herein, we describe the efficacy of using a unique biopolymer, N-dihydrogalactochitosan (GC), as a nasal mucosal vaccine adjuvant against respiratory infections. Specifically, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV), an MF-59 equivalent. In contrast to AV, intranasal application of GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. Moreover, GC+S+NC-vaccinated animals were largely resistant to the lethal SARS-CoV-2 challenge and experienced drastically reduced morbidity and mortality, with animal weights and behavior returning to normal 22 days post-infection. In contrast, animals intranasally vaccinated with AV+S+NC experienced severe weight loss, mortality, and respiratory distress, with none surviving beyond 6 days post-infection. Our findings demonstrate that GC can serve as a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses. STATEMENT OF SIGNIFICANCE: We demonstrated that a unique biopolymer, N-dihydrogalactochitosan (GC), was an effective nasal mucosal vaccine adjuvant against respiratory infections. Specifically, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV). In contrast to AV, GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. About 90 % of the GC+S+NC-vaccinated animals survived the lethal SARS-CoV-2 challenge and remained healthy 22 days post-infection, while the AV+S+NC-vaccinated animals experienced severe weight loss and respiratory distress, and all died within 6 days post-infection. Our findings demonstrate that GC is a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses.

Grass carp (Ctenopharyngodon idellus) TRAF6 up-regulates IFN1 expression by activating IRF5.

In mammals, interferon regulatory factor 5 (IRF5) can be activated by tumor necrosis factor receptor-associated factor 6 (TRAF6). Upon activation, IRF5 translocates into the nucleus, where it binds to IFN promoter and up-regulates IFN expression. However, there are few reports on the molecular mechanism by which TRAF6 up-regulates IFN expression in fish. In this study, we explored how Grass carp (Ctenopharyngodon idellus) TRAF6 initiated innate immunity by activating IRF5. We found that CiTRAF6, CiIRF5 and CiIFN1 were all significantly up-regulated in LPS-stimulated CIK cells and the expression of CiTRAF6 was earlier than the expressions of CiIRF5 and CiIFN1. These findings suggested that CiIFN1 expression might be induced by CiTRAF6 in fish. CiIFN1 expression, CiIFN1 promoter activity and CO cells viability were all significantly up-regulated in the overexpression experiments, but they were significantly down-regulated in the gene silencing experiments. This indicated that CiTRAF6, along with CiIRF5, regulated CiIFN1 expression. The localization analysis found that both CiTRAF6 and CiIRF5 located in the cytoplasm. Following LPS stimulation, CiIRF5 was observed to translocate to the nucleus. GST-pull down and co-IP experiments revealed that CiTRAF6 interacted with CiIRF5. The colocalization analysis also showed that CiTRAF6 bound with CiIRF5 in the cytoplasm. Overexpression of CiTRAF6 increased the endogenous CiIRF5, promoted its ubiquitination and nuclear translocation. In conclusion, CiTRAF6 bound to CiIRF5 in the cytoplasm, and then activated CiIRF5, resulting in up-regulating the expression of CiIFN1.

Grass carp (Ctenopharyngodon idella) Bcl-xl: transcriptional regulation and anti-apoptosis analysis.

Bcl-xl, Bax2, and NF-κB are well-known to be involved in anti-apoptosis response. Although Bcl-xl has been reported in fish, the NF-κB-mediated regulatory mechanism and anti-apoptotic function are still unclear. Here, we cloned and characterized the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) Bcl-xl (CiBcl-xl) and its promoter region sequence. The full-length cDNA of CiBcl-xl is 2836 bp with an ORF of 627 bp encoding a polypeptide of 208 amino acids. Phylogenetic tree analysis revealed that CiBcl-xl shared high homology with Dario rerio Bcl-xl (DrBcl-xl). After stimulation with Poly I:C, the expression of CiBcl-xl in CIK cells and various tested tissues of grass carp were significantly upregulated. To further understand the transcriptional control of fish Bcl-xl induced by NF-κB, CiC-rel and Cip65 were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. In vitro, gel mobility shift assays demonstrated the high affinity of CiC-rel and Cip65 with CiBcl-xl promoter. Dual-luciferase reporter assays showed that CiC-rel and Cip65 activated CiBcl-xl promoter. Also, knockdown of CiC-rel and Cip65 reduced the expression of Bcl-xl. Therefore, similar to those of mammals, fish C-rel and p65 can upregulate the transcription of Bcl-xl. In addition, we found that overexpression of CiBcl-xl in CIK cells increased the cell activity and inhibited cell apoptosis, while overexpression of Bax2 promoted cell apoptosis. Meanwhile, co-transfection of CiBcl-xl and CiBax2 into cells can ease up apoptotic rate. To further investigate the molecular basis of synergistic effect of Bcl-xl and Bax2, we showed that Bcl-xl and Bax2 interacted with each other. The results suggested that Bcl-xl executed its anti-apoptotic function by binding to and inhibiting the pro-apoptotic activity of Bax2.

Ctenopharyngodon idella TBK1 activates innate immune response via IRF7.

In mammals, IFN regulatory factor (IRF) 7 is a central regulator of IFN-α expression in response to variable pathogenic infections. There are several pathogenic sensors involved in monitoring pathogen intrusion in mammals. These sensors trigger IRF7-mediated responses through different pathways. TANK-binding kinase 1 (TBK1) is a critical mediator of IRF7 activation upon pathogen infection. In fish, there are many reports on TBK1, IRF3 and IRF7, especially on TBK1-IRF3 signaling pathway. However, it is not very clear how TBK1-IRF7 works in innate immune signaling pathway. In this study, we explored how TBK1 up-regulates IFN, ISG expression, and how TBK1 initiates innate immune response through IRF7 in fish under lipopolysaccharides (LPS) stimulation. After stimulation with LPS, grass carp IRF3 and IRF7 transcriptions were up-regulated, indicating they participate in TLR-mediated antiviral signaling pathway. It is interesting that the response time of grass carp IRF3 to LPS was earlier than that of IRF7. In addition, IRF7 rather than IRF3 acted as a stronger positive regulator of IFN and ISG transcription in Ctenopharyngodon idella kidney cells (CIKs). It is suggested the potential function differentiation between IRF3 and IRF7 upon LPS infection in fish. Dual luciferase assays also showed that overexpression of grass carp IRF7 and TBK1 up-regulated the transcription level of IFN and PKR. However, knockdown of IRF7 inhibits ISG expression, suggesting that grass carp TBK1 regulates the transcription via IRF7. Co-immunoprecipitation and GST pull-down assays proved the binding of grass carp IRF7 to TBK1. Furthermore, grass carp TBK1 can promote the nuclear translocation of IRF7. The results indicated that grass carp TBK1 can bind directly to and activate IRF7.

Activated grass carp STAT6 up-regulates the transcriptional level and expression of CCL20 and Bcl-xl.

In mammals, signal transducer and activator of transcription 6 (STAT6) is a broad-spectrum transcriptional regulator involved in cellular immune responses and apoptosis by regulating the immune-related genes and various functional genes. The structure, expression and tyrosine-based phosphorylation of STAT6 are conserved from fish to mammal. However, except the sporadic reports from zebra fish, the function of fish STAT6 has not been well reported. Here, we cloned and characterized the full length cDNA sequence of grass carp (Ctenopharyngodon idella) STAT6 (CiSTAT6). Meanwhile, the activation mechanism and the potential function of CiSTAT6 were studied. The full length cDNA of CiSTAT6 is 2747 bp with an ORF of 2313 bp encoding a polypeptide of 770 amino acids. Phylogenetic tree analysis revealed that CiSTAT6 shares the maximum homology with Cyprinus carpio STAT6. CiSTAT6 was significantly up-regulated and interacted with each other to form the homodimer after treatment with poly I:C. The transfected CiSTAT6 in fish cell lines can activate the promoter activities of CCL20 and Bcl-xl and increase their mRNA levels. In addition, we also found that CiSTAT6 can increase cell viability and inhibit cell apoptosis. Taken together, grass carp STAT6 plays an important part in innate immunity and anti-apoptosis.

Purification and Characterization of the Trypsin Inhibitor from Sophora alopecuroide L.

Sophora alopecuroide trypsin inhibitor (SATI) was purified from the seeds of Sophora alopecuroide L. by affinity chromatography on trypsin-sepharose 4B. The inhibitor is homogenous on SDS-PAGE (12%), which is a single polypeptide chain with alanine as its N-terminal. It has the molecular weight of 18 kD and pI at 9.3. Results also show that the inhibitor has a 1:2 molar inhibitory ratio of inhibitor to trypsin. In further study with the method of modification, the residues of argenine and lysine are found to be the only 2 reactive centers on the inhibitor. Feeding trials on cotton aphid indicated that the inhibitor has relatively strong anti-cotton aphid activity with LC(50) (half lethal concentration) of 87.2 mg/L and LD(50) (half lethal dosage) of 6.796 &mgr;g (feeding for 120 h).