Background: Nuclear receptor NR1H3 is a key regulator of macrophage function and lipid homeostasis. Here, we aimed to visualize the prognostic value and immunological characterization of NR1H3 in breast cancer.Methods: The expression pattern and prognostic value of NR1H3 were analyzed via multiple databases, including TIMER2, GEPIA2 and Kaplan-Meier Plotter. TISIDB, TIMER2 and immunohistochemical analysis were used to investigate the correlation between NR1H3 expression and immune infiltration. GO enrichment analysis, KEGG analysis, Reactome analysis, ConsensusPathDB and GeneMANIA were used to visualize the functional enrichment of NR1H3 and signaling pathways related to NR1H3.Results: We demonstrated that the expression of NR1H3 was significantly lower in breast cancer compared with adjacent normal tissues. Kaplan-Meier survival curves showed shorter overall survival in basal breast cancer patients with low NR1H3 expression, and poorer prognosis of relapse-free survival in breast cancer patients with low NR1H3 expression. NR1H3 was mainly expressed in immune cells, and its expression was closely related with infiltrating levels of tumor-infiltrating immune cells in breast cancer. Additionally, univariate and multivariate analysis indicated that the expression of NR1H3 and the level of macrophage infiltration were independent prognostic factors for breast cancer. Gene interaction network analysis showed the function of NR1H3 involved in regulating of innate immune response and macrophage activation. Moreover, NR1H3 may function as a predictor of chemoresponsiveness in breast cancer.Conclusion: These findings suggest that NR1H3 serves as a prognostic biomarker and contributes to the regulation of macrophage activation in breast cancer. Breast cancer is the most common malignancy and the second leading cause of cancer related-deaths among women worldwide . DespiteMacrophages are an important part of infiltrating immune cells in tumor microenvironment (TME).They are always abundantly present in breast cancer . In the via its interaction with infiltrating macrophages. Immune-related gene NR1H3 is likely to be one of potential immune markers for breast cancer immunotherapy.In this study, we visualized the prognostic landscape of NR1H3 in breast cancer using databases, including TIMER2, GEPIA2, and Kaplan-Meier Plotter. We also explored the potential relationship between NR1H3 expression and macrophage infiltration level using the TIMER2 and TISIDB databases. Our results indicate that NR1H3 influences the prognosis of patients with breast cancer, probably http://timer.cistrome.org/) was used to show the expression difference of NR1H3 between tumor and adjacent normal tissues in different cancer types of the TCGA project  was used to obtain box plot of the expression difference between the breast tumor tissues and the corresponding normal tissues of the GTEx database  (http://www.oncomine.org) was used to validate the expression of NR1H3 in breast cancer (TIMER2 database ( project . The “Exdatabase . Additioot.html) . The Onct cancer .Paired human breast cancer and adjacent non-tumor paraffin tissue microarrays were purchased from Shanghai Zuocheng Biotech . The sections were subjected to antigen retrieval and incubated with primary antibodies against NR1H3  and CD68  at 4°C in a humid chamber overnight. The next day, the sections were incubated with biotinylated secondary antibody for 60 min. Protein levels of NR1H3 and CD68 were evaluated as follows: the slides were appraised for the intensity of the staining (0–3) and the percentage of positively stained cells (0–4). Index of protein levels was calculated as the intensity of the staining × the percentage of positively stained cells. Therefore, slices were divided into 4 groups: negative (score 0), low expression (score 1–4), medium expression (score 5–8) and high expression group (score 9–12).The subtypes of breast cancer for sub-group analysis are divided based on the 2013 StGallen criteria using the expression of HER2, ESR1 and MKI67, including basal (ESR1-/HER2-), luminal A (ESR1+/HER2-/MKI67 low), luminal B (ESR1+/HER2+/MKI67 high) and HER2(HER2+/ESR1-).http://bioinfo.life.hust.edu.cn/GSCA/#/) databases were used to reveal the correlation between NR1H3 expression and overall survival (OS), disease-free survival (DFS) or progress free survival (PFS) of breast cancer patients  was used to assess the effect of NR1H3 on OS, relapse-free survival (RFS), distant metastasis-free survival (DMFS) and post-progression survival (PPS) in breast cancer  was used to show the relations between NR1H3 expression and abundance of 28 tumor-infiltrating lymphocytes (TILs) types, immunoinhibitors, immunostimulators, MHC molecules, chemokines and chemokine receptors (http://timer.cistrome.org/) was used to analyze the correlation of NR1H3 with the infiltration level and prognostic value of immune cells, including macrophages, CD4+ T Cells, CD8+ T Cells, monocytes, B Cells, dendritic cells (DCs), neutrophils and natural killer (NK) cells  cells . We alsohttp://tisch.comp-genomics.org) was used to show the detailed cell-type annotation at the single-cell level in breast cancer (http://gepia2021.cancer-pku.cn/) visualized the NR1H3 expression in each immune cell type  available in TCGA/GTEx sub-datasets.The scRNA-seq database TISCH (t cancer . Sub-exphttp://www.mutarget.com) is a cancer biomarker/target discovery tool that can identify mutations resulting in expression change. We used the database to predict the mutant genes that affect the expression of NR1H3 gene.The muTarget database (http://www.linkedomics.org/) pathway analysis. Gene Set Enrichment Analysis (GSEA) was used to search for Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathways enrichment analysis. The network neighborhoods of NR1H3 were visualized by ConsensusPathDB-human (http://consensuspathdb.org). These data originate from currently 32 public resources for interactions (http://genemania.org/), an online tool for investigation into associated or similar genes for target genes, was used to validate the gene interaction network results and conduct functional enrichment analysis (The gene ontology (GO) term enrichment analysis was performed by the LinkedOmics database  was used to identify NR1H3 whether predicts benefit from endocrine therapy and chemotherapy . In addition, according to the clinical data of these cancer cases, we found that the lower expression of NR1H3, the more lymph node metastasis (p > 0.05). Then we analyzed the expression of NR1H3 in BRCA based on tumor subclasses using the UALCAN database. Luminal (p < 1.0e-12), HER2Pos (p = 4.1e-10), TNBC Basal-like 1 (TNBC-BL1) (p = 5.2e-03), TNBC Basal-like 2 (TNBC-BL2) (p = 1.54e-5), TNBC mesenchymal stem-like (TNBC-MSL) (p = 6.26e-5), TNBC Mesenchymal (TNBC-M) (p = 4.4e-16), TNBC unspecified (TNBC-UNS) (p = 1.0e-4) showed lower NR1H3 expression compared with normal tissues (p > 0.05)  . Moreove > 0.05) .p = 0.007; RFS p = 1.9e-8; DMFS p = 0.004; PPS p = 0.011) was shown to correlate with lower NR1H3 expression (p = 0.015), DMFS (p = 0.004) and PPS (p = 0.023) were shorter in basal breast cancer patients with low NR1H3 expression, but not in luminal A, luminal B and HER2+ breast cancer patients (p > 0.05). Moreover, low NR1H3 expression was correlated with poorer prognosis of RFS in basal (p = 5.3e-6), luminal A (p = 3.0e-4), luminal B (p = 0.002) and HER2+ breast cancer patients (p = 0.0003)  .p = 0.023) in breast cancer with low NR1H3 expression (p = 0.03) (p = 0.006) and PR-negative  subtypes were also statistically associated with clinical prognosis of RFS and DMFS, but only a trend towards poor survival without statistical significance of OS and PPS in NR1H3-low breast cancer (p = 0.047), RFS (p = 3e-04) and PPS (p = 0.033) in HER2-positive breast cancer (p = 5.8e-06) and DMFS (p = 0.023) showed statistical survival differences (p = 0.015), but not in lymph node negative breast cancer patients (p = 0.058). For grade 3 breast cancer patients, low expression of NR1H3 indicated a worse survival prognosis of OS (p = 0.01), RFS (p = 0.025) and DMFS (p = 0.035). These results suggest that low NR1H3 expression may be a risk factor for a poor prognosis in breast cancer patients.We further explored the prognostic characteristics of NR1H3 under different ER, PR, and HER-2 status. ER-positive subtype had shorter RFS (pression . Low NR1 = 0.03) . ER-negat cancer . Comparet cancer . Among Hferences . In addi+ T Cells, CD8+ T Cells, neutrophils, macrophages and DCs) in BRCA using TIMER database. Consistently, p = 2.18e-8), CD8+ T Cells , CD4+ T Cells , macrophages , neutrophils , and DCs  in BRCA and with negative correlation with tumor purity . Moreover, the same trend results were found in each subtype  in diverse cancer types of TCGA. As shown in + T Cells and macrophages were two immune cell types most strongly correlated with NR1H3 expression in BRCA.Next, TIMER, CIBERSORT, CIBERSORT-ABS, QUANTISEQ, XCELL, MCPCOUNTER and EPIC algorithms were further used to validate the potential relation between the expression of NR1H3 and the infiltration level of 8 types of immune cells  and high amplification (p = 0.021) of NR1H3 had significant correlation with CD4+ Th2 cell infiltration level using XCELL algorithms. High amplification of NR1H3 was associated with low M2 macrophage infiltration level, compare with the “diploid/normal” status (p = 0.039). By CIBERSORT and CIBERSORT-ABS algorithms, high amplification of NR1H3 had high B Cell and low DC cell infiltration . These findings indicate the potential mechanism by which NR1H3 alterations affect immune infiltration distribution.The association between NR1H3 copy number variations  and immune infiltrates in BRCA was investigated using different algorithms of TIMER2. The immune infiltration distribution by the somatic copy number alterations (sCNA) status of NR1H3 across TCGA cancer types was demonstrated in ltration . However+ T Cells were the most abundant immune cells , whereas NR1H3 was highly expressed in monocyte/macrophage  in the TISCH platform to analyze the expression of NR1H3 at the single-cell level. The results showed higher NR1H3 expressions in immune cells, mainly in monocyte/macrophage, compared with malignant cells . Then wecrophage . GEPIA20crophage .We further analyzed the correlation of NR1H3 expression and monocyte/macrophage markers in tumor tissues using TIMER2. We adjusted these results based on tumor purity, and revealed significant correlations between NR1H3 expression and monocyte markers , macrophage markers , M2 macrophage markers  and M1 macrophage markers (IRF5), whereas the M1 macrophage markers INOS/NOS2 and COX2/PTGS2 showed no correlation with NR1H3 expression .In order to verify the findings from the database, we detected the protein level of NR1H3 and macrophage marker CD68 in paraffin tissue microarrays from breast cancer patients by IHC. The slides showed that NR1H3 and CD68 protein were expressed in interstitial cells of breast tumor tissues. A typical staining pattern is shown in p = 0.0311), compared with lower macrophage infiltration level. On the contrary, the low M2 macrophage infiltration level predicted favorable prognosis under the high expression of NR1H3 using the CIBERSORT algorithm in BRCA , compared with the high M2 macrophage infiltration level. In BRCA-LumA, under low NR1H3 expression, higher monocyte level had a worse outcome . The statistically different scatterplot data of the above tumors produced using different algorithms was presented in As mentioned above, we observed a statistical positive correlation between the immune infiltration of macrophages and NR1H3 expression in BRCA. Then we evaluated the prognostic efficiency of the combination of infiltrated macrophages and NR1H3 expression patterns for breast cancer . The lowp = 0.002), stage 3 , stage 4  and NR1H3 expression  were prognostic variables for the prognosis of OS in BRCA patients. After adjustments of age, stage, gender, race, and tumor purity, the level of macrophage infiltration  and the expression of NR1H3  were independent prognostic factors in BRCA. These results suggest that NR1H3 is an independent prognostic biomarker and combining its expression level with the macrophage would help to play a more effective role in the prognosis prediction of BRCA.Additionally, to evaluate whether NR1H3 expression level and macrophage infiltration are independent risk factors for prognosis of BRCA, we conducted the univariate and multivariate analysis included seven variables: macrophage infiltration level, age, stage, gender, race, tumor purity and expression of NR1H3 . The resThe TISIDB database was used to infer the correlations between expression of NR1H3 and immunomodulators/chemokines across human cancers. As shown in Homo sapiens. The network defined the neighborhood-based entity set centered by NR1H3 and containing 19 interaction nodes and 22 physical entity nodes  and grade 1 subtype breast cancer patients with Tamoxifen treatment  based on relapse-free survival (RFS) at 5 years  family members are key regulators of macrophage function, controlling transcriptional programs involved in inflammation and lipid homeostasis . As an iBreast cancer is a heterogeneous disease and is divided into different subtypes based on the expression status of ER/PR and HER-2 . Basal tIn vitro, culture medium from NR1H3 activated macrophages causes growth inhibition and apoptosis of breast tumor cells (Our data are in line with experimental results previously published.NR1H3 was reported to be an onco-suppressor gene in various cancers . In vitror cells . In mousor cells . These f+ T Cells, CD8+ T Cells, neutrophils, macrophages and DCs. These results indicate that expression of NR1H3 is related to the immune activation of TME.To further explore the underlying mechanisms of NR1H3 in breast carcinogenesis, we investigated the correlation of NR1H3 expression with tumor-infiltrating immune cells of breast cancer. Our results revealed the important role of NR1H3 in TME as well as providing a potential relationship between NR1H3 and tumor-immune interactions in breast cancer. As we all known, activated immune cells attacks tumor cells to prevent the development of cancer in the early phase of carcinogenesis. Here, we provide evidence that high expression of NR1H3 is strongly correlated with multiple immune infiltration in breast cancer tissues, including B Cells, CD4Existing studies have shown that tumor-infiltrating immune cells play important roles in the initiation, progression, metastasis and therapeutic resistance of cancers . Among vWe found the association between NR1H3 expression and mutated FBXW7, which is one of the most frequently mutated genes in human cancers and its functional inactivation can lead to tumorigenesis. FBXW7α, the most abundant isoform in proliferating cells, attenuates the LPS response through inhibition of C/EBPδ and TLR4 expression and that FBXW7α-depletion alone is sufficient to activate inflammatory signaling . ImportaWe also integrated the information on NR1H3-binding components and NR1H3 expression-related genes for a series of enrichment analyses. We identified a potential impact of NR1H3 in regulation of macrophage activation and inflammatory response regulation. Gene interaction network and functional enrichment analysis revealed the molecular mechanism by which low expression of NR1H3 gene leads to poor prognosis of breast cancer patients. However, the limitations in current study are also lies in the lack of experimental verification. Moreover, the detailed mechanisms of NR1H3 in regulating activation of TME in breast cancer needs further study.In summary, our study showed that low NR1H3 expression was correlated with worse survival, especially for basal subtype, HER2 positive subtype and grade 3 breast cancer patients. NR1H3 was related to immune cells infiltration and regulation of macrophage activation. Importantly, the expression of NR1H3 and macrophage infiltration level were independent risk factors for prognosis of breast cancer patients. Therefore, NR1H3 could be a useful biomarker in breast cancer patients and activation of NR1H3 might be a potential therapeutic antitumor strategy of breast cancer.


The systematic review by Coelho-Junior et al. found that frail older adults consumed significantly less animal-derived protein than healthy people . One rea2 concentration of 27 nmol/L  and a 25(OH)D3 concentration of 31 nmol/L ; meanwhile, non-vegetarians had a mean 25(OH)D2 concentration of 2 nmol/L  and a 25(OH)D3 concentration of 90 nmol/L   concentration among 2107 white men and women in the UK reported that the amount of animal products in the diet significantly affected serum 25(OH)D concentrations . Daily mAnimal protein is primarily muscles. Vitamin D is stored in muscles as 25(OH)D. A study using primary rat muscle fibers found that 25(OH)D is absorbed in mature muscle cells and held there by vitamin D-binding protein ,7.Vitamin D deficiency is an important risk factor for frailty. A review discussed the genomic and nongenomic mechanisms whereby vitamin D increases muscle strength and reduces risk of frailty . In in 23 to raise serum 25(OH)D concentrations to above 30 or 40 ng/mL , in contrast to vitamin D3 supplementation, which did reduce mortality rate  [2 or vitamin D3 found vitamin D3 is approximately 87% more potent in raising and maintaining serum 25(OH)D concentrations and produces 2–3-fold greater storage of vitamin D than equimolar vitamin D2 [3 intervention was more efficacious than vitamin D2 in improving vitamin D status , and regulating PTH levels, irrespective of the participant demographics, dosage, and vehicle of supplementation [Vegans do not consume animal products, so are unlikely to take vitamin Dtamin D2 . A systeentation .


CX3CL1) is critically involved in the pathophysiology of different inflammatory diseases and myocardial ischemia-reperfusion (I/R). This study aimed to analyze the role of CX3CL1 in the activation of platelets and leukocytes during hepatic I/R.The chemokine fractalkine . The inflammatory response and sinusoidal perfusion failure were evaluated by intravital microscopy. Hepatic transaminases plasma levels and histopathological tissue damage were determined as markers of hepatocellular injury.Under inhalation anesthesia, C57BL6 mice were subjected to warm hepatic I/R (90 min/240 min). The animals were pretreated either with a function-blocking anti-mouse CX3CL1 antibody. We demonstrate that inhibition of CX3CL1 signaling attenuates leukocyte adhesion in the postischemic liver but does not significantly ameliorate overall perfusion failure and hepatocellular injury.Sinusoidal perfusion failure, leukocyte recruitment to the liver, and transaminase activities were sharply increased upon I/R compared to sham-operated mice. Firm adhesion of platelets and concordantly leukocytes to endothelial cells is reduced significantly by a function-blocking anti-CX3CL1 blockade on platelet and leukocyte, but not CD4+ T cell accumulation and activation in hepatic I/R injury. We report a significant effect of blocking chemokine CX3CL1 on sinusoidal perfusion failure without considerably improving overall hepatocellular injury during early reperfusion.Our in vivo data demonstrate a mild attenuating effect of  Ischemia-reperfusion (I/R) injury causes significant morbidity and mortality in organ transplantation, trauma, and septic or hemorrhagic shock. It is one of the main reasons for early organ failure after liver transplantation. The complex cascade of I/R-induced injury includes different mechanisms of cell death and the activation of immune cells in a sterile inflammatory response. Hepatic microcirculation is considered one of the primary targets of I/R injury of the liver. The injured endothelial lining upregulates adhesion molecules, allowing the interaction of neutrophils, CD4+ T cells, and platelets . The accCX3CL1) is a chemokine of the CX3C family [CX3CR1 is profusely expressed on various cell types, such as circulating leukocytes [Endothelial leukocyte capture with subsequent rolling and firm adhesion is followed by extravasation into the perivascular tissue , 3. SmalC family . IncreasC family , 6. The ukocytes , 8.CX3CL1 and its receptor mediates chemoattraction and promotes leukocyte adhesion in an integrin-independent way [CX3CL1-coated surfaces in venules or sinusoids [CX3CR1, are strictly required for CX3CL1-induced leukocyte adhesion [CX3CL1 also induces activation of surface-adherent platelets with consecutive platelet degranulation and exposure of the adhesion molecule P-selectin. In the liver, CX3CR1 is most abundantly expressed on Kupffer cells (KCs) [CX3CL1 [CX3CL1 has been shown to mediate lymphocyte recruitment to the liver [CX3CL1/CX3CR1 axis attenuates postischemic recruitment of leukocytes, CD4+ T cells, and platelets, resulting in protection from I/R injury without increasing the risk for hemorrhage.Interaction between endothelial dent way , 9, 10. adhesion . Endothels (KCs)  and endo [CX3CL1 . In primhe liver . In thisFemale 5- to 7-week-old C57BL/6 mice were used in all experiments. The mice were purchased from Charles River®  and were housed for at least 1 week in our animal facility to recover from transportation before experiments. All experiments were carried out according to German animal safety regulations and the ARRIVE guidelines.n = 7 each): (1) sham, (2) anti-CX3CL1 mAb + labeled platelets, (3) isotype control + labeled platelets. (4) anti-CX3CL1 mAb + labeled CD4+ T lymphocytes, and (5) isotype control + CD4+ T lymphocytes. A function-blocking anti-mouse CX3CL1 antibody  or rabbit IgG was administered 24 h before experiments  and 10 min before reperfusion (50 μg intravenously via a jugular catheter).The surgical procedure was described elsewhere . Brieflywww.karger.com/doi/10.1159/000524024).A modified Leitz-Orthoplan microscope was used for intravital fluorescence microscopy, as described previously 17). Leukocytes were stained in vivo by rhodamine 6G . Fluorescein isothiocyanate-conjugated dextran  was used as plasma marker for sinusoidal perfusion. Platelets or CD4+ T cells were isolated from syngeneic mice with a magnetic cell separation kit according to the manufacturer's instructions and labeled ex vivo with rhodamine 6G before intravenous application and intravital microscopy. For detailed protocol of intravital microscopy, see Figure 7. LeukocSerum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity was determined with an automated analyzer using standardized test systems, see online supplementary protocol p values less than 0.05 were considered significant.ANOVA on ranks followed by the Student-Newman-Keuls test was used to estimate stochastic probability in intergroup comparison . Mean values ± SEM are given. 2) were observed in postsinusoidal venules, whereas I/R led to a significant increase (7.7 ± 0.6/mm/s and 231.4 ± 15.7/mm2). Platelets were rarely observed in sinusoids of sham-operated mice (0.7 ± 0.1/acinus) but accumulated in postischemic sinusoids (4.8 ± 0.4/acinus). In contrast, pretreatment with anti-CX3CL1 antibody significantly attenuated I/R-induced platelet-endothelial cell interactions in sinusoids (1.4 ± 0.3/acinus) and postsinusoidal venules (5.7 ± 0.5/mm/s and 176 ± 14.8/mm2)  in postsinusoidal venules were very low in sham-operated mice in contrast to 90 min of ischemia followed by 120 min of reperfusion in vehicle-treated mice . Blocking CX3CL1 by using function-blocking anti-mouse CX3CL1 antibody reduced the postischemic number of rolling and adherent leukocytes by about ∼20% respective ∼50% , (data not shown).Leukocyte-endothelial cell interactions were analyzed in postsinusoidal venules as an indicator of microvascular injury and hepatic inflammation after I/R. As shown in Figure CX3CL1 antibody and control antibody. Similar findings were observed after both 30 min and 120 min of reperfusion.As shown in Figure CX3CL1 treatment significantly improved post-I/R perfusion failure (14 ± 2% nonperfused sinusoids).Sinusoidal perfusion failure was determined using in vivo microscopy as a recognized parameter of microvascular I/R injury. In the vehicle-treated I/R group, about 29 ± 1% of all sinusoids were not perfused Fig. . Anti-CXCX3CL1 did not have a protective effect  increased the activity of AST and ALT considerably in the vehicle-treated group compared to the sham-operated group. In contrast to the data on sinusoidal perfusion, blocking 0 min incCX3CL1 antibody did not improve the postischemic tissue injury  effector T cells by rapidly capturing them from the blood and promoting tissue migration [CX3CL1 to act as a chemoattractant for CD4+ T cells suggests an important role in the recruitment of these cells to the postischemic liver. Moreover, allograft rejection, a process also characterized by an intense cellular immune response with an influx of circulating leukocytes into the transplanted organ [CX3CR1-specific blocking antibodies. However, the accumulation of CD4+ T cells in the postischemic liver was not significantly affected by anti-CX3CL1 antibody treatment. Recent findings of our group show that T cells directly interact with hepatic dendritic cells [CX3CL1 in their activation, especially in the early phase of hepatic I/R analyzed in this study.igration . T cellsigration . CD4+ T ed organ , could bic cells . TherefoCX3CL1 on platelet and neutrophil activation and sinusoidal failure. However, AST/ALT levels and histopathologic damage as markers of overall hepatocellular integrity and liver necrosis were not significantly affected. The decisive mechanism why platelet and leukocyte interaction are blocked but migration and liver injury are not affected may be found in the activity of KCs. Experimental data from a murine model of liver injury shows that KCs as tissue-resident liver macrophages play an essential role in initiating and maintaining inflammatory responses. They release proinflammatory cytokines and chemokines and activate other nonparenchymal liver cells, such as endothelial or HSCs [CX3CL1/CX3CR-axis [CX3CR1/CX3CL1 on the inflammatory activity of dendritic cells. Sutti et al. [CX3CL1/CX3CR1 axis affects the IL-10 mediated anti-inflammatory activity of type 2 myeloid liver DCs. Moreover, blockade of CX3CL1 reduced monocyte-derived proinflammatory DC-mediated liver damage in a hepatotoxic model of inflammatory liver injury [We aimed to investigate whether the treatment with a chemokine inhibitor is protective against I/R injury. Such an effect seemed consequential as platelets, neutrophils, and T cells are known to mediate necrotic and apoptotic injury in the postischemic liver and play a critical role in sinusoidal perfusion failure , 22, 23. or HSCs . Therefo or HSCs , 26. As 3CR-axis , we hypo3CR-axis . Anotheri et al.  show thar injury . ContrarCX3CL1 blockade on platelet and leukocyte, but not CD4+ T cell accumulation and activation in hepatic I/R injury. We report a significant effect of blocking chemokine CX3CL1 on sinusoidal perfusion failure without considerably improving overall hepatocellular injury during early reperfusion.Taken together, our in vivo data show a mild attenuating effect of Our experiments were reviewed by the Government of Upper Bavaria before they were performed and approved as applications 8-13 and 23-13.The authors have no conflicts of interest to declare.This study was supported by the German Research Foundation DFG (KH92-3/1).D.F.: concept and design, experiments and procedures, and writing of the article; A.B.: experiments and procedures and writing of the article; K.M.: concept and design, and experiments and procedures; M.L.: concept and design and writing of the article; M.R.: concept and design; A.K.: concept and design and writing of the article.All data generated or analyzed during this study are included in this article and/or its supplementary material files. Further inquiries can be directed to the corresponding author.Supplementary dataClick here for additional data file.


Collariella virescens is only available as a recombinant enzyme (rCviUPO), which is produced in Escherichia coli for protein engineering and analytical-scale optimization of plant lipid oxygenation. Engineering the active site of rCviUPO  improved the epoxidation of individual 18-C unsaturated fatty acids and hydrolyzed sunflower oil. The double mutation at the heme channel (F88A/T158A) enhanced epoxidation of polyunsaturated linoleic and α–linolenic acids, with the desired diepoxides representing > 80% of the products (after 99% substrate conversion). More interestingly, process optimization increased (by 100-fold) the hydrolyzate concentration, with up to 85% epoxidation yield, after 1 h of reaction time with the above double variant. Under these conditions, oleic acid monoepoxide and linoleic acid diepoxide are the main products from the sunflower oil hydrolyzate.Vegetable oils are valuable renewable resources for the production of bio-based chemicals and intermediates, including reactive epoxides of industrial interest. Enzymes are an environmentally friendly alternative to chemical catalysis in oxygenation reactions, epoxidation included, with the added advantage of their potential selectivity. The unspecific peroxygenase of  The oxirane ring of epoxides has been termed the “Lord of the chemical rings”  because 2O2 reactions on oils, free fatty acids, and their methyl esters [Fatty acid epoxidation is industrially performed by the Prileschajew reaction  via percl esters ,12,13,14l esters , cytochrl esters , and funl esters  catalyzel esters ,19. UPOsl esters , but, afl esters ,22, numel esters  and the Recent studies on structure–function relationships in fatty-acid oxygenation by UPOs have revealed that the different peroxygenation patterns (enzyme regioselectivity) are ruled by the structure of the heme channel ,25,26. ACollariella virescens  has been heterologously expressed in Escherichia coli and the recombinant enzyme (rCviUPO), isolated without any purification tag [CviUPO shows good conversion of unsaturated fatty acids and, in contrast to other UPOs, generates epoxides as main products [The UPO from the ascomycete tion tag . The obtproducts . CviUPO was already engineered by site-directed mutagenesis [Chaetomium globosum, which efficiently epoxidizes unsaturated fatty acids [CviUPO promoted diepoxidation [Marasmius rotula enzyme [E. coli (rMroUPO) [MroUPO has the disadvantage, compared to rCviUPO, of its much lower expression in E. coli as a soluble enzyme. ragenesis  in an inty acids . Then, ixidation . This waa enzyme , the firrMroUPO) . HoweverCviUPO via two different strategies: (i) enzyme engineering to achieve two epoxy groups per molecule, and (ii) analytical scale optimization of the reaction parameters to epoxidize hydrolyzed vegetable oil.In the present work, we aimed to improve the conversion yield and selectivity of plant lipid epoxidation by rCviUPO sequence [E. coli expression (using the software Optimizer) [The sequence  was optitimizer) , synthestimizer) . The nonCviUPO structure was obtained at the Swiss-Model server  [Agrocybe aegerita (AaeUPO) as a template (PDB entry 2YP1). This model was used for the design of four mutated variants with progressively enlarged heme access channels. The F88A and T158A single mutations were introduced in the CviUPO gene cloned in pET23a using the Expand Long Template PCR kit from Roche  and the following oligonucleotides as primers (direct sequences with the mutated codons underlined): F88A: 5′-ACT TAC ACC GTT CAG CAG CGT ATC GCG AGT TAC GGT GAA ACG-3′, T158A: 5′-AAC CGC CAT AAC CTG GCG GAA CAT GAT GCA TCT C-3′. For double mutation, the vector containing the CviUPO gene with the first mutation was used as a template. The PCR products were digested with DpnI and transformed into E. coli DH5α for propagation. The gene of the 6Ala sextuple variant—including the L64A, I61A, F88A, T157A, T158A, and T165A mutations—was synthesized by ATG-biosynthetics GmbH . All the variants were produced in E. coli as active cytosolic enzymes, and purified as described above for the native rCviUPO.A molecular model of the ch 2022)  with thech 2022)  and crysch 2022)  UPO of A2S2O4 and CO flushing. UPO concentrations were calculated using the rCviUPO molar extinction coefficient of ℇ420 = 114.2 mM−1·cm−1 [Enzyme purity was confirmed under denaturing conditions by 12% polyacrylamide gel electrophoresis (PAGE) in the presence of 0.1% sodium dodecyl sulfate (SDS) and 1% mercaptoethanol . Proper M−1·cm−1 .CviUPO and its variants were analyzed in the range of pH 2–10 using 0.2 M Britton–Robinson buffer, at 24 °C, in the presence of 1 mM H2O2. Optimal pH values for oxidation of several UPO substrates—namely veratryl and benzyl alcohols (10 mM) and naphthalene (1 mM) from Merck  and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)  from Boehringer Mannheim —by the native r310 = 9300 M−1·cm−1), benzyl alcohol  at 280 nm (ℇ280 = 1400 M−1·cm−1), naphthalene  at 303 nm (ℇ303 = 2030 M−1·cm−1), and ABTS  at 436 nm . Reactions (1 mL) were triggered by the addition of 1 mM or 24 mM H2O2. Kinetic curves for H2O2 were obtained with different peroxide concentrations  in the presence of 2.5 mM ABTS whose (one-electron) oxidation was monitored at 436 nm (as explained above) for activity estimation (note that two moles of ABTS are oxidized by each mole of peroxide), and the H2O2 Km values were obtained.Kinetic curves for the above enzyme-reducing substrates were obtained (with spectrophotometric assays) from the initial (10–30 s) increase of product absorbance, using a Thermo Spectronic UV–visible spectrophotometer  at the optimal pH for each enzyme and substrate. Oxidation of veratryl alcohol  was followed at 310 nm (ℇv/v). Reactions (1 mL) were triggered by the addition of 24 mM H2O2 and stopped after 1 min by vigorous shaking with 100 μL of 0.1 M sodium azide. The oxygenated products were extracted, analyzed by gas chromatography–mass spectrometry (GC-MS) as described below, and their total abundance was used for the calculation of kinetic constants. All reactions were carried out in triplicate.Kinetic curves for oleic acid oxidation  were obtained by varying the concentration from 12.5 μM to 1.6 mM substrate, in 50 mM phosphate (pH 7) containing 20% acetone (Km) and turnover number , and their standard errors were obtained by non-linear fitting the kobs values to Equation (1) (Michaelis–Menten model) except for (i) ABTS oxidation by rCviUPO and T158A using 1 mM H2O2; (ii) ABTS oxidation by F88A (using either 1 mM or 24 mM H2O2); (iii) benzyl alcohol oxidation by F88A and F88A/T158A using 1 mM H2O2; (iv) oleic acid oxidation by the F88A, F88A/T158A, and 6Ala variants; (v) H2O2 reduction (in presence of 2.5 mM ABTS) by rCviUPO and T158A variant, where enzyme inhibition was observed, being therefore adjusted to Equation (2) ; and (vi) benzyl alcohol oxidation by rCviUPO and T158A using 1 mM H2O2 that was adjusted to Equation (3) .Curve-fit and data analysis for kinetic constant estimation were carried out using Sigma Plot 11.0. Michaelis–Menten constant , linoleic , and α-linolenic  acids. Reactions running for 30 min were performed using 0.1 mM substrate, 0.25 or 0.4 µM enzyme , and 1.25 mM H2O2 in 50 mM phosphate (pH 7), at 30 °C in the presence of 20% acetone (for better substrate solubilization). For more complete characterization, epoxyoleic acid was prepared in larger scale (50 mL) reactions and purified by silica gel (60–200 µm) column chromatography using hexane–EtOAc (10:1 → 5:1) as the mobile phase.For evaluating the UPO epoxidation ability, the following 18-C unsaturated fatty acids  were used as substrates: oleic  was saponified and the hydrolyzed fatty acids extracted at acidic pH as previously described [2O2 in 50 mM phosphate buffer (pH 7) containing 20% acetone . In further reactions, up to 10 mM oil hydrolyzate was used, and the effect of several variables was studied including: pH 5.5/7.0, 20/30% acetone cosolvent, 0.25/0.50 µM enzyme (resulting in 100–400 substrate/enzyme (S/E) molar ratio), 1–100 mM H2O2  added with a syringe pump, and 30/60 min reaction time. Given the results obtained with individual fatty acids, the F88A/T158A variant was selected for hydrolyzate reactions, compared with native rCviUPO. Epoxidation yields were calculated taking into account the epoxidation degree, the number of unsaturations, and the reaction conversion for each substrate. In all cases, control experiments were carried out under the same conditions (H2O2 included), but in the absence of enzyme. Oil hydrolyzate was assayed for epoxide production as a more economical and sustainable substrate than pure fatty acids. Sunflower oil , which was evaporated under a N2 stream. N,O-Bis(trimethylsilyl)trifluoroacetamide  was used to prepare trimethylsilyl derivatives. The GC-MS analyses were performed with an Agilent  GC-MS QP2020 Ultra equipment using a fused-silica DB-5HT 30 m capillary column from J&W Scientific . The oven was heated from 120 °C (1 min) to 300 °C (15 min) at 5 °C min−1. The injector and transfer line were kept at 300 °C. Compounds were identified by mass fragmentography and comparison of their mass spectra with those of authentic standards. Quantifications were obtained from total-ion peak areas  and molar response factors of the same or similar compounds.Products (and unreacted substrates) from the above reactions were liquid–liquid extracted with methyl iPrOH-AcOH (99.65:0.3:0.05), at a flow rate of 0.8 mL·min−1 and monitored at 202 nm. Enantiomer assignment was done following the reported literature [Chiral analyses of epoxides of oleic acid were performed after derivatization into methyl esters  with a Shimadzu  i-Prominence 2030C high-performance liquid chromatography (HPLC) equipment using an OB-H  column from Daicel . Compounds were eluted with hexane–terature .CviUPO variants [Previous results with rvariants  led us tvariants . In all CviUPO and variants (2O2 as the enzyme-oxidizing substrate (2O2 were determined using 2.5 mM ABTS as co-substrate. It was observed that the native rCviUPO and its T158A variant are inhibited by high H2O2 concentrations (ki) of 5.6 mM and 1.8 mM, respectively . Therefore, the kcat values for the different enzyme-reducing substrates would be often undervalued under the above conditions.First, the optimal pH for oxidation of four peroxidase/peroxygenase substrates was determined for the native rvariants . Kinetictrations , with inectively . The rCv2O2 (CviUPO) were found for naphthalene, veratryl alcohol/ABTS, and benzyl alcohol, respectively. The broadest channel variant 6Ala showed improved affinity with all substrates, but often also lower reaction rates, resulting in lower catalytic efficiency.For this reason, second sets of kinetic curves and constants were obtained using 24 mM HCviUPO and variants  to α-linolenic acid (32%). However, the changes introduced in the mutated variants improved the epoxidation yields, reaching 93% with oleic acid (T158A), 90% with linoleic acid (F88A/T158A), and 60% with α-linolenic acid (F88A/T158A).GC-MS analyses revealed different oxygenation products in the reactions of oleic, linoleic, and α-linolenic acids with rConcerning the products, chromatograms in CviUPO for comparative purposes). The results obtained (ee 0–40%), although an inversion of the configuration of the main enantiomer was produced between the F88A/T158A and 6Ala variants .Finally, encouraged by the recently reported enantioselectivity of fatty acid mono-epoxidations with UPOs ,25, reacobtained  revealedCviUPO variants on individual unsaturated fatty acids, the goal was to accomplish the epoxidation of hydrolyzed vegetable oil. For this purpose, a sunflower oil hydrolyzate was used as substrate, and several parameters—including substrate, enzyme, and H2O2 concentrations, among others—were optimized. After testing the rCviUPO and its F88A/T158A variant (anti and syn) from linoleic acid, more abundant in the F88A/T158A reaction; and (iii) small amounts of hydroxy- and hydroxy-epoxy derivatives of oleic acid and linoleic acids, together with some minor products.Firstly, reactions under the same conditions used with pure fatty acids were performed on hydrolyzed sunflower oil (0.1 mM) using native rCviUPO dose did not result in improved epoxidation yield, but doubling the amount of F88A/T158A  yielded higher amounts of diepoxides from linoleic acid (the epoxidation of oleic acid was retained); consequently, a better epoxidation yield (72%) was attained. Nevertheless, a 1 μM dose of the double variant (S/E ratio 100) produced virtually the same epoxidation profile. Therefore, optimal 400 and 200 S/E ratios were fixed for further scale up with rCviUPO and its double variant, respectively.Then, differences in the epoxidation yields were observed when varying the enzyme dose, using different substrate/enzyme (S/E) molar ratios. As shown in However, when the substrate loading was increased to 5 mM  led to a decrease in epoxidation yield (to only 44%).With the enzyme dose, amount of co-solvent, and reaction time already optimized, assays were conducted to determine the lowest dose of HCviUPO double variant for epoxidizing sunflower oil hydrolyzate, demonstrating a much higher conversion yield than the native enzyme.The pattern of oxygenation products produced under optimized conditions C,D was sCviUPO heme access channel allowed us to improve the epoxidation activity of this enzyme, selected for protein engineering in E. coli. In this way, the variant with the widest channel, after a sextuple mutation, yielded the highest epoxidation of oleic acid to the corresponding monoepoxide (with less than 5% of other oxygenation products). In contrast, a double variant (F88A/T158A) produced the best conversion of both linoleic and α–linolenic acids with diepoxides, of interest as crosslinking molecules, representing > 80% of products. Under the same conditions used for individual fatty acids, the double variant also converted hydrolyzed sunflower oil with a higher epoxidation yield than the native enzyme. Moreover, process optimization permitted us to increase (×100) the hydrolyzate concentration, epoxidizing 85% of double bonds after 1 h of reaction time with the mutated double variant. UPO engineering, strongly limited in the past by difficulties in heterologous expression of these enzymes in adequate hosts, is a requirement for developing the large repertoire of reactions of industrial relevance suggested in recent reviews [Opening the r reviews ,43,44,45 reviews .


Influenza outbreaks caused by A/H7N9 viruses have occurred since 2013. After 2016, A/H7N9 influenza viruses underwent evolutionary changes. In this study, we examined the antigenic properties of influenza neuraminidase (NA) of A/H7N9 viruses as part of a live influenza vaccine (LAIV). It was shown that neuraminidase inhibiting (NI) antibodies obtained after A/Anhui/1/2013(H7N9)-based LAIV vaccination did not inhibit A/Hong Kong/125/2017(H7N9) NA and vice versa. The A/Hong Kong/125/2017(H7N9)-based LAIV elicited higher levels of NI antibodies compared to the A/Anhui/1/2013(H7N9)-based LAIV after two doses. Thelow degree of coincidence of the antibody response to hemagglutinin (HA) and NA after LAIV vaccination allows us to consider an enzyme-linked lectin assay (ELLA) as an additional measure for assessing the immunogenicity of influenza vaccines. In mice, N9-reactive monoclonal antibodies (mABs) for the A/environment/Shanghai/RL01/2013(H7N9) influenza virus partially protected against lung infection from the A/Guangdong/17SF003/2016 IDCDC-RG56N(H7N9) virus, thus showing the cross-protective properties of monoclonal antibodies against the drift variant. During the period from March 2013 to February 2015, 571 laboratory-confirmed cases of infection with the avian influenza A/H7N9 virus were registered in China, of which 212 (37.1%) were fatal , accessed on 21 October 2021. A number of A/H7N9 vaccines have been developed, including subunit, adjuvanted, and live influenza vaccines (LAIVs) [As a part of its pre-pandemic preparedness efforts, the WHO has developed a number of avian influenza A/H7N9 candidate vaccine viruses [ (LAIVs) ,7,8,9. TThe aim of this study was to compare the antigenic properties of the NA of A/H7N9 viruses isolated before and after 2016. For these purposes, we studied the archival sera of volunteers obtained during clinical trials of two A/H7N9 LAIVs ,9.The molecular genetic analysis showed that the A/H7N9 influenza viruses isolated from humans in 2017 and 2018 form a separate phylogenetic branch, in contrast to viruses isolated before 2016 .The multiple alignment of the amino acid sequences of the N9 viruses showed that the NA of A/Anhui/1/2013(H7N9) and A/Hong Kong/125/2017(H7N9) LAIV viruses differed by 8 amino acid substitutions B: (I16T)We developed two A/H6N9 recombinant viruses in order to avoid non-specific reaction with antibodies to HA when analyzing neuraminidase inhibiting (NI) antibodies to A/H7N9 viruses. It was shown that the NA of A/Anhui/1/2013(H7N9) and A/Hong Kong/125/2017(H7N9) in recombinant A/H6N9 viruses differ in enzymatic activity with large molecular substrate fetuin  Figure. This foFirst, the formation of hemagglutination inhibiting (HI) and NI antibodies was determined by an HI test and enzyme linked lectin assay (ELLA). It was shown that a significant increase in mean antibody titers of HI antibodies to the A/Anhui/1/2013(H7N9) virus was observed after the first dose, and the second dose 28 days later caused a statistically significant increase in antibodies compared with the first dose A. Mean NIt was shown that after the first vaccination, there were no combined conversions of HI and NI antibodies. After the second vaccination, NI antibody seroconversions did not occur without HI antibody conversions, and the number of combined conversions was 17.2% B. Thus, p = 0.01).Next, we studied the formation of serum antibodies after immunization with LAIV based on the A/Hong Kong/125/2017(H7N9) influenza virus. The results are presented in As in the case of A/Anhui/1/2013(H7N9)-based LAIV, after the first dose of A/Hong Kong/125/2017(H7N9)-based LAIV, there was no overlap in the conversions of HI and NI antibodies B. ConverIn general, immunization with A/H7N9 LAIV caused an increase in antibodies to HA and NA, on average, in more than 60% of vaccinated individuals. As was reported earlier, A/H7N9 LAIV also stimulated neutralizing antibodies and cellular immunity, so an increase in at least one parameter of the immune response was achieved in a much larger percentage of the vaccinated individuals ,9. The cNext, we tried to study whether there is a possibility of NI activity of antibodies acquired to the A/H7N9 virus isolated in 2013 against the A/H7N9 virus isolated in 2017 year and vice versa. To accomplish this, we took positive sera with different levels of antibodies to A/H7N9 viruses and tested them in the ELLA with a different antigenic variant. The data in We also studied the increase in antibodies to A/H6N9-13 and A/H6N9-17 in the sera of vaccinated people using ELISA. The results are shown in It was shown that after immunization with two doses of A/Hong Kong/125/2017(H7N9)-based LAIV, the levels of ELISA antibodies were higher than after immunization with two doses of A/Anhui/1/2013(H7N9)-based LAIV . A statiConsidering that ELISA antigens contained the H6 subtype of HA, the detected antibodies were not directed against HA of vaccine viruses, but were specific to the second surface antigen, NA, or, to a lesser extent, to other proteins, such as a nucleoprotein or a conserved HA stalk region ,13,14.When comparing the average level of OD450 of serial dilutions of mABs in 96-well panels sensitized with 20 HAU of A/H6N9-13 or A/H6N9-17 viruses, a more pronounced affinity of mAB#38 and mAB#40 for the A/H7N9 virus isolated before 2016 compared to the virus isolated after 2016 was observed .At the same time, mABs did not inhibit the enzymatic activity of N9 NA (data not shown).50 of the A/Guangdong/17SF003/2016 IDCDC-RG56N(H7N9) influenza virus, the infection was not lethal. After infection, the immunized mice lost weight relatively less compared to mock-immunized animals, but the differences were not statistically significant  and A/Hong Kong/125/2017(H7N9) have a similarity of 97% in NA amino acid sequence. At the same time, the enzymatic properties on A/Anhui/1/2013(H7N9) and A/Hong Kong/125/2017(H7N9) NA were different, in which the A/Hong Kong/125/2017(H7N9) virus NA demonstrated higher enzyme activity with high molecular substrate fetuin. This was also noticeable in terms of immunogenicity, since the titers of NI antibodies to the A/Hong Kong/125/2017(H7N9) influenza viruses were higher than those to A/Anhui/1/2013(H7N9) . Thus, iIt was noted that a significant increase in NI antibodies was obtained after the second immunization with LAIV based on both A/Anhui/1/2013(H7N9) and A/Hong Kong/125/2017(H7N9). The use of ELISA revealed thatlevels of antibodies to N9 was higher after the first and second vaccine doses of A/Hong Kong/125/2017(H7N9)-based LAIV compared to the A/Anhui/1/2013(H7N9)-based LAIV . It was In the event of a pandemic, the most effective way to fight influenza, as well as other viral infections, is vaccination. In this regard, it is important to develop, test, and deposit vaccines against emerging viruses. The created reserve will allow us, if necessary, to start production of vaccine preparations and the large-scale immunization of the population as soon as possible. It should be noted that the development of new vaccines cannot be accomplished without expanding the range of analyzed factors of the adaptive immune response in vaccinated people. Antibodies to NA—the second surface glycoprotein of the influenza virus—also have a protective effect, reducing the frequency of infection and the proportion of clinically pronounced forms in natural infection with drift and even shift variants of the influenza A virus .One of the priorities of the WHO is to prepare for a new influenza pandemic, which includes the creation of collections of vaccine strains in order to start the production of a vaccine as soon as possible and immunize the population in the event of a pandemic threat A/H7N9 and assess the degree of susceptibility of the population to a potentially pandemic strain of influenza A/H7N9. The first stage of preparation involves a comprehensive, complete characterization of the immunogenicity and protective efficacy of vaccine preparations of a potentially pandemic subtype, including the use of methods for detecting a humoral immune response to influenza virus NA. The second stage includes the study of heterosubtypic anti-influenza immunity mediated by a number of factors, including NI antibodies.Previously, it was shown that broadly neutralizing antibodies to seasonal influenza may be effective against pathogenic H7 strains and may contribute to the development of treatments against A/H7N9 strains . MoreoveIn this study, we used archived blood serum samples from volunteers aged 18–49 taken as part of clinical trials in 2014  [The A/H7N9 LAIV vaccine strains were a 6:2 reassortant developed in the Institute of Experimental Medicine  based on A/Leningrad/134/17/57(H2N2) master donor strain and human A/Anhui/1/2013(H7N9) and A/Hong Kong/125/2017(H7N9) strains ,9. To asAmino-acid sequences used in this study are available under the following accession numbers: AGQ88512, AGN69476, AGT41969, APR73187, AHZ60099, AGY41895, AGJ73505, AGL44440, AGR43209, AJJ91923, AIK26586, AJU15326, APR73167, AKI82234, MG214221, MF370248, MW142383, AIS16351, ISL249269, ARG43209, KY643848, ISL280902, MN170547, AID70635.Multiple alignment was carried out using UGENE software from Unipro  . The evoTo assess NI antibodies in ELLA, we used two A/H6N9 viruses containing HA from the A/herring gull/Sarma/51c/2006 (H6N1) strain and NA from A/Anhui/1/2013(H7N9) or A/Hong Kong/125/2017(H7N9). The A/H6N9 viruses were concentrated and purified on a stepwise gradient of sucrose. Egg-propagated viruses were clarified by centrifugation at 3000 rpm for 20 min. The supernatants were transferred to ultracentrifuge vials  and centrifuged at 17,000 rpm for 3 h in a Beckman coulter Optima™ L-100 XP Ultracentrifuge, Type 19 rotor . The supernatant was removed, and the pellet was resuspended in 2.0 mL of ice-cold calcium borate buffer (pH = 7.2). The resuspended virus was applied to a stepwise 30/60% sucrose gradient prepared in calcium borate buffer, followed by centrifugation at 20,000 rpm for 2.5 h (SW 40 Ti rotor). The virus-containing layer was taken and purified from sucrose in calcium-borate buffer by ultracentrifugation at 20,000 rpm for 2.5 h (SW 40 Ti rotor). The pellet was resuspended in 1.0 mL calcium borate buffer. Afterwards, 96-well plates  were coated with 50 µg/mL fetuin  overnight at +4 °C. Next, 60 μL of each serum was heated at 56 °C for 30 min, serially diluted with phosphate-buffered saline containing bovine serum albumin (PBS-BSA), and then incubated with an equal volume of pre-diluted virus at a working dose of 64–128 hemagglutinating units (HAU) per 0.1 mL for 30 min at 37 °C. After incubation, 100 μL of the mixtures were applied to the fetuin-coated wells which were washed 3 times. After incubation for 1 h at 37 °C, the plates were washed, before assessing NA activity by incubating with peroxidase-labeled peanut lectin  for 1 h at room temperature, followed by washing and addition of 100 μL of the peroxidase substrate 3,3′, 5,5′-tetramethylbenzidine (TMB). The reaction was stopped after 5 min by adding 100 μL of 1 N sulfuric acid. Optical density (OD) values were measured at 450 nm using a universal microplate reader . Titers of serum antibodies against NA were determined as the reverse dilution of the sample with 50% inhibition of NA activity. A two-fold increase in NI antibody titer after vaccination was considered significant.Vibrio cholerae NA extract  and heated at 56 °C for 30 min. Each serum (in duplicates) was serially diluted starting from the initial dilution at 1:5 or 1:10 and mixed with 8 HAU of testing influenza virus. Antibody titers were expressed as the reciprocal of the highest serum dilution at which inhibition of agglutination was observed. A four-fold increase in HI antibody titer after vaccination was considered significant.HI test was performed using a 0.75% suspension of human erythrocytes (group “0”) in 96-well U-bottomed polymer plates as described previously . The serSerum IgG levels were determined using ELISA in 96-well ELISA plates  coated with 20 HAU per 0.1 mL of the whole purified A/H6N9 viruses as previously described  in which9 plaque-forming units (PFU) per shot) at two-week intervals. Two weeks after the last immunization, splenocytes were harvested from mice, and monoclonal anti-N9 antibodies were produced by hybridoma technology. Three mABs with high binding affinity to N9 protein  were selected and amplified by the mouse ascites method. The final concentrations of these mABs are mAB#8-0.47 mg/mL; mAB#38-1 mg/mL; and mAB#40-1 mg/mL. All animal experiment procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Academia Sinica, Taiwan.Anti-N9 NA antibodies were prepared at the Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan. Specifically, cDNA sequence encoding the NA of A/environment/Shanghai/RL01/2013(H7N9) influenza virus was synthesized, and a recombinant baculovirus NA9-Bac displaying the NA protein was generated according to previous methods ,35,36. I50) of A/Guangdong/17SF003/2016 IDCDC-RG56N(H7N9) influenza virus. The animals were euthanized under ether anesthesia and cervical dislocation in order to determine the viral load in the lungs. To determine the viral titer in the lungs, the samples were homogenized in PBS containing 100 U/mL penicillin and 100 μg/mL streptomycin, and centrifuged for 10 min at 6000× g. To determine lung virus titers, the samples were titrated in CE starting from a dilution of 1:10. Virus titers were expressed as log10 50% embryo infective dose (EID50) as described previously [In the experiments of passive immunization of mice with NA9-Bac mABs, all animal procedures were carried out according to the “Rules of Laboratory Practice” . The female 8–10-week-old CBA mice were purchased from the breeding laboratory . The mice (20 per group) were lightly anesthetized with ether and immunized using a mix of mABs (15 μg of each per mouse in 0.1 mL). Animals in the control group were inoculated with PBS using the same route. Blood samples were obtained from the submandibular plexus 5 h after immunization. At the same time, mice were infected intranasally with 500 50% mouse infectious dose  was used as conjugate.p value < 0.05 was considered statistically significant.Data were analyzed using Statistica software, version 6.0  and graphics were generated using Prism 8 . Antibody levels were presented as geometric mean titers (GMT). For statistical analysis, antibody titers were expressed as reciprocal final dilutions of log2. Means and standard deviations of means (SD) were calculated to represent viral titers. Two dependent variables were compared using Wilcoxon matched pair test. Comparisons of two independent groups were performed using the nonparametric Mann–Whitney U-test. A It was shown that there was no cross interaction of NI antibodies to the A/Anhui/1/2013(H7N9) and A/Hong Kong/125/2017(H7N9) influenza viruses. The A/Hong Kong/125/2017(H7N9)-based LAIV elicited higher levels of NI antibodies compared to the A/Anhui/1/2013(H7N9)-based LAIV after the second dose. The revealed low degree of coincidence of the serum immune response to HA and NA after LAIV vaccination allows us to consider ELLA as an additional criterion for assessing the immunogenicity of influenza vaccines. In mice, mABs to NA of the 2013 A/H7N9 virus partially protected against lung infection with the A/H7N9 virus isolated after 2016. Thus, it can be assumed that the presence of NA-reactive antibodies that do not inhibit NA activity can still limit influenza infection.


A growing number of studies have indicated that H2S plays a key role in the pathophysiology of vascular diseases and physiological vascular homeostasis. Alteration in H2S biogenesis has been reported in a variety of vascular diseases and H2S supplementation exerts effects of vasodilation. Accumulating evidence has shown vascular potassium channels activation is involved in H2S-induced vasodilation. This review aimed to summarize and discuss the role of H2S in the regulation of vascular tone, especially by interaction with different vascular potassium channels and the underlying mechanisms.Hydrogen sulfide (H Endogenous H2S is mainly produced by enzyme pathways in vivo, including cystathionine γ-lyase (CES), cystathionine β-synthase (CBS), cysteine aminotransferase and 3-mercaptopyruvate sulfurtransferase (3-MST)  . In the d by CES . Biogenertension . H2S pladilation . The mecV), Ca2+-activated potassium channels (KCa), ATP-sensitive potassium channels (KATP), inward rectifier potassium channels (Kir), and tandem two-pore potassium channels (K2P)  . Accumuldilation . However2S or downregulation of H2S has been found in a variety of vascular diseases. Plasma H2S levels were significantly lower in ever-treated hypertensive patients compared with normal subjects, and were higher in patients with well-controlled blood pressure than in patients with grade 2 and 3 hypertension, suggesting that the reduction of endogenous H2S plays a role in the pathophysiology of hypertension   . Tain etrtension . The intzed rats . The prenditions . NaHS prnditions , coronarnditions , carotidnditions , and mesnditions . GYY4137arteries  and boviarteries . N-phenyic rings . Novel H vessels . L-cyste vessels . However2S-induced vasodilation via activation of potassium channels in different vascular beds. Modulation of potassium channel activity by H2S donors may be a novel treatment for vascular diseases. This review presents the effects and mechanisms of H2S-induced vasodilation via activation of potassium channels, including ATP-sensitive potassium channels (KATP), calcium activated potassiumchannels (KCa) and voltage dependent potassium channels (KV).Potassium channels play important roles in regulating of membrane potential and vascular tone. There are many studies having been carried out on HATP channel structurally is a hetero-octamer of four Kir6 subunits at the center and four sulfonylurea receptor (SUR) subunits surrounded    and GYY4 donors)  respectia leaves  and sulfa leaves , and the2S directly activated KATP channel. H2S increased the whole-cell currents and the single channel open probability of KATP channels. Inhibition of endogenous H2S production reduced whole-cell KATP currents in mesenteric artery smooth muscle cells (ATP channel can be sulfhydrated by H2S. H2S-mediated sulfhydration and hyperpolarization are absent in cells overexpressing C43S mutant Kir 6.1. Mutating the site of sulfhydration (Kir 6.1 C43S) inhibited H2S-induced hyperpolarization. Sulfhydration augments KATP activity by enhancing Kir 6.1-PIP2 binding and reducing Kir 6.1-ATP binding , the intermediate conductance calcium-activated potassium channels  and the small conductance calcium-activated potassium channels  . BK chan2S produced dilation of mouse coronary arteries   . Each KVdilation . And thesel beds .V channel blocker, significantly inhibited the relaxant effect of NaHS, suggesting a role of KV in H2S induce vasodilation in diabetes (V channels in mouse mesenteric arteries (NaHS relaxed carotid arteries precontracted by phenylephrine of diabetic rabbits, while 4-amynopiridine, a Kdiabetes . Vasodilarteries , but onlarteries .V7 channel, encoded by KCNQ gene, is thought as a powerful mechanism to H2S induced vasorelaxation. NaHS produced concentration-dependent vasorelaxation, which was blocked by XE991, a KV7 channel inhibitor. Perivascular adipose tissue released H2S, which play a major role in control of vascular tone via KV7 channel (V7 blocker (V7 channels by CBS-derived H2S (K channel . XE991 a channel  and muri channel . GYY4137 blocker . The mecived H2S .2S is involved in vascular diseases, such as hypertension, diabetes, pulmonary hypertension and so on. H2S causes vasodilation in numerous vascular beds and regulates vascular tone by interaction with different vascular potassium channels, including KATP, KCa, and KV channels via the following different mechanisms. H2S can directly activated vascular KATP and BK channels and increased sulfhydration of KATP and KV7 channels. H2S promoted vasodilation in partly through upregulating the expression of Kir6.1 and SUR2B subunits of KATP, or BK channel activation by increased Ca2+influx. Thus, H2S may present a potential therapeutic target on vascular diseases. However, the vasorelaxant effect of H2S in different vessel beds is complex. More studies are needed to focus on the activation mechanisms of H2S on potassium channels, and novel, low-releasing H2S donors are required for treatment in the future.Taken together, H


As a consequence, the majority of antibodies elicited by infection or vaccination protect only against closely related strains. The immunodominance of the globular head of the main glycoprotein has been shown to mask the immunogenicity of the conserved regions located within the hemagglutinin (HA) protein. It has been shown that the broadly neutralizing universal antibodies recognize the HA2 domain in headless hemagglutinin . Therefore, the HA-stalk is a highly conserved antigen, which makes it a good candidate to be used in universal vaccine development against AIVs. (2) Methods: Sf9 insect cells were used to produce triple H5N1/NA-HA-M1 influenza virus-like particles (VLPs) via co-expression of neuraminidase, hemagglutinin and matrix proteins from a tricistronic expression cassette. Purified influenza VLPs were used to immunize broiler hens. An in-depth characterization of the immune response was performed with an emphasis on the pool of elicited universal antibodies. (3) Results: Our findings suggest, that after vaccination with triple H5N1/NA-HA-M1 VLPs, hens generate a pool of broad-spectrum universal anti-HA-stalk antibodies. Furthermore, these universal antibodies are able to recognize the mammalian-derived HA-stalk recombinant proteins from homologous H5N1 and heterologous H7N9 AIVs as well as from the heterosubtypic human H1N1 influenza strain. (4) Conclusions: Our findings may suggest that highly pathogenic avian influenza H5 HA protein contain functional epitopes that are attractive targets for the generation of broad-spectrum antibodies against AIVs in their native hosts. Avian influenza virus (AIV) belongs to the genus Alphainfluenzavirus in the family Orthomyxoviridae . AIVs are divided into subtypes based on the antigenic differences found in the two major surface glycoproteins—hemagglutinin (HA) and neuraminidase (NA)  viruses.The technology of virus-like particles (VLPs) paved a new road in vaccinology when they were used for vaccine formulation. VLPs are self-assembled viral protein complexes whose structures closely mimic their parent viruses in conformation and organization . VLPs arMost strategies for designing AIV vaccines rely on the main glycoprotein of the influenza virus. HA is an essential multifunctional protein for virus pathogenicity. The HA protein allows for the binding of the virus to the host cell receptor and is considered to be the primary antigen that induces the neutralizing antibodies during infection . Each HAHens are highly susceptible to the H5N1 HPAI virus infection and play a critical role in the spread of the virus. Chickens provide a natural model to determine the contributions of HA1 and HA2 subunits of the H5N1 HPAI virus for the induction of neutralizing antibody production and protection. There are several studies that use the intact HA protein of the H5N1 HPAI virus to produce viral vector vaccines for chickens ,24. To aAntibodies: Anti-H5N1 virus A/Ck/Scot/59 polyclonal chicken antibodies ; anti-H5N2 virus A/Ost/Den/72420/96 polyclonal chicken antibodies ; anti-M1 influenza mouse monoclonal ; anti-avian influenza A neuraminidase antibody ; anti-H1N1 mouse monoclonal antibodies ; FI6—highly specific humanized synthetic universal neutralizing monoclonal antibodies selected from plasma cells that bind group 1 and 2 influenza A HA (kind gift from Dr. Krzysztof Lacek and Alfredo Nicosia); anti-HA H5N1 mouse monoclonal antibodies ; anti-HA H3N2 mouse monoclonal antibodies ; anti-HA H7N9 mouse monoclonal antibodies .Peptides: LAH peptide, representing the long alpha-helix within HA-stalk from H3 subtype: biotinylated RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENA LAH peptide .2. Madin–Darby canine kidney cells  and human embryonic kidney 293 cells  were cultured in Dulbecco’s Modified Eagle’s Medium , supplemented with a 10% heat-inactivated fetal bovine serum , 2 mM L-glutamine , antibiotic antimitotic solution 1× , at 37 °C under 5% COThe AIV A/ostrich/Denmark/725/96 (H5N2) and A/turkey/Poland/95/1995 (H7N2) were kindly provided by the Department of Poultry Diseases, National Veterinary Research Institute, Pulawy, Poland. Inactivated H5N1 virus A/Ck/Scot/59 and inactivated H5N2 virus A/Ost/Den/72420/96 were purchased from the Animal Health and Veterinary Laboratories . The human influenza virus (the pandemic human influenza A/Virginia/ATCC3/2009/H1N1 virus) from ATCC (cat. No VR-1738) was also used in this study. HPAI H5N1 A/swan/Poland 305-135V08-2006 RNA for cDNA synthesis was kindly provided by the Department of Poultry Diseases, National Veterinary Research Institute, Pulawy, Poland.All the active influenza A viruses were propagated in MDCK.2 cells in the presence of 2 µg/mL TPCK (L-1-tosylamide-2-phenylethyl chloromethyl ketone)—trypsin .Spodoptera frugiperda insect cells  were cultured in synthetic HyClone SFX-Insect cell culture media  supplemented with antibiotic antimitotic solution 1x , at 27 °C. Cells were grown in suspension or adherent culture depending on the experiment. The cDNA synthesis of the full length of HA, NA and M1 genes from the H5N1 A/swan/Poland 305-135V08-2006 strain of the HPAI virus was carried out using universal primers and the Improm-II reverse transcription system . The full length of genes coding structural proteins was amplified using Taq polymerase . The HA gene was amplified using primers For (5′- ATAGGATC CAAAATAGTGCTTCTTTTTGC-3′) and Rev (5′-AATACTATGACTCTGAACTCACAA ATTT-3′). The NA gene was amplified using primers For (5′- CTCGAGAGCAAAAGCAGGAGTTCAAA ATG-3′) and Rev (5′- AAGCTTCTACTTGTCAATGGTGAATGG-3′). The M1 gene was amplified using primers For (5′- GAATTC AGCAAAAGCAGGTAGATGTTG-3′) and Rev (5′- GGAGTAAAAAACTACCTTGTTTCTACTGAATTC-3′). The 1700 bp (HA), 1400 bp (NA) and 1000 bp (M1) PCR products were ligated as blunt fragments into a pGem-Teasy plasmid , followed by sequencing and subcloning into the pFastBac1 vector downstream of the ACMPV polyhedrin promoter. For pHA BamHI and SpeI, for pNA XhoI and HindIII, for pM1 EcoRI, recognition sites were used. All enzymes were purchased from Thermo Scientific (USA).Tricistronic construct: First, the bacmid transfer vector expressing HA and M1 genes (pHA/M1) was obtained by subcloning the DNA fragment of pM1 plasmid containing M1 under the polyhedrin promoter, using SnaBI and HpaI restriction enzymes into the HpaI site of pHA plasmid. A bacmid transfer vector expressing all three structural proteins of the influenza virus (pNA/HA/M1) was constructed by subcloning the bicistronic cassette containing the HA and M1 genes from the pHA/M1 plasmid using SnaBI and AvrII restriction enzymes in the HpaI and AvrII sites of pNA plasmid. HA-stalk: All HA-stalk sequences were designed as headless, lacking the HA1 domain of hemagglutinin. To preserve proper conformation according to Steel et al., a glycine linker flanked with cysteines replaced the head region of the HA1 domain ,26. SyntEscherichia coli DH10 Bac competent cells were transformed with the recombinant pFastBac vector . The isolated bacmid DNA was purified and transfected into Sf9 cells using Lipofectamine . After 6 days, recombinant baculoviruses (rBVs) were harvested, amplified, and titrated using the plaque assay method.Recombinant bacmids containing tricistronic expression cassettes and the HA-stalk coding sequence were generated using the Bac-to-Bac system . Briefly, g. Concentrated VLPs were resuspended in NTE buffer containing EDTA and then purified on a 10%–60% sucrose gradient for 16 h at 4 °C, 82,000× g. Purified VLPs were further concentrated through ultracentrifugation for 2 h at 4 °C, 82,000× g. The proteins of each fraction were analyzed by SDS-PAGE and Western analysis. Purified influenza VLPs and viruses were visualized using transmission electron microscopy (TEM) and stored at 4 °C for further analysis. To produce influenza VLPs, Sf9 cells were infected with recombinant baculovirus at an MOI of 5 and harvested 96 h post-infection. The supernatant was collected after centrifugation in a microfuge at 8500 rpm for 10 min followed by ultracentrifugation for 2 h at 4 °C, 82,000× For SDS-PAGE, proteins/VLPs were dissolved in the denaturing loading 4X Bolt™ LDS Sample Buffer  and heated for 10 min at 82 °C. Aliquots of 40 μL of each sample were loaded on 10%–20% precast Wedge Well gel  and run at a constant voltage of 200 V. Polyacrylamide gel was stained with Coomassie brilliant blue solution . To perform a Western blot analysis, a semi-dry immunotransfer was performed using a Bio-Rad kit . After the transfer, PVDF membranes were blocked for 1 h in a 5% semi-skimmed milk solution and incubated for 1 h with primary antibodies  in a 5% milk solution. Next, membranes were washed and incubated with appropriate secondary HRP/AP-conjugated antibody solution. The reaction was developed with ECL Plus Chemiluminescent substrate for horseradish peroxidase  or NBT/BCIP substrates .2 buffer). VLPs and viral particles were adsorbed onto carbon-coated grids, stained with 2% uranyl acetate, and examined immediately in a Philips CM 100 electron microscope. Influenza VLPs purified from the supernatant of infected Sf9 cells were diluted in TM buffer  assay kit, cat. No A22178, Life Technologies, Waltham, MA, USA). An aliquot of influenza VLPs and positive control virus were incubated with fetuin for 16 h at 37 °C. The amount of sialic acid released was determined chemically with thiobarbituric acid which produces a pink color in proportion to the amount of free sialic acid, which was measured by spectrophotometry at 560 nm. The hemagglutination test was performed according to the standard protocol . Briefly, a twofold dilution of the H5 antigens in a form of VLPs was incubated with 1% chicken erythrocytes for 30 min at room temperature. The positive control was the inactivated H5N1 virus A/Ck/Scot/59 and inactivated H5N2 virus A/Ost/Den/72420/96 from the Animal Health and Veterinary Laboratories Agency . The negative control was the medium collected from uninfected Sf9 cells.H5N1 avian influenza VLPs obtained in the baculovirus system in insect cells were used to immunize chickens. A group of six 3 week old broiler hens (Ross 308) were housed in cages in an experimental poultry house under standard commercial conditions. The vaccine was administered intramuscularly into the breast muscle with a dose of 200 µL of the vaccine preparation containing 15 µg of VLPs and incomplete Freund’s adjuvant (ICF). The second group of six 3 week old broiler hens, vaccinated with 1:1 PBS/ICF, served as the control. After 17 days, a booster shot was performed with the same dose of the vaccine. Blood was collected for analysis on days 0, 17, 35 and 41. The immune response of the chickens was tested with ELISA and hemagglutination inhibition (HI) tests. The experiment was approved by the II Local Ethical Committee for Animal Experiments at the Medical University of Warsaw, permit number 17/2009. All efforts were made to minimize animal suffering. At the end of the experiment, the birds were humanely euthanized by decapitation.Sera collected from triple H5N1/NA-HA-M1 VLP vaccinated birds on day 41 were pooled and tested in the hemagglutinin inhibition (HI) test. The HI test was performed according to a standard procedure . Briefly, 25 µL of sera in serial twofold dilutions were incubated for 25 min with four HA units of the inactivated H5N1 virus A/Ck/Scot/59 from the Animal Health and Veterinary Laboratories Agency . Next, 25 µL of suspension of 1% hen erythrocytes was added and incubated for 30 min at room temperature. The HI titer was determined as the reciprocal of the highest dilution in which hemagglutination is inhibited. Samples were assigned as positive when their titer was ≥16. The negative control was the serum of hens vaccinated with a 1:1 PBS/ICF solution. H5N1 A/Ck/Scot/59 and H5N2 A/Ost/Den/72420/96 reference antibodies were used as a positive control. The ELISA test was used to detect and quantify the immune response toward triple H5N1/NA-HA-M1 VLP vaccination in hens. A 96-well plate  was coated with the 50 ng/well reference antigen inactivated H5N1 virus, purchased from the Animal Health and Veterinary Laboratories Agency reference laboratory . The coated plate was incubated overnight at 4 °C. Then, the plate was washed 4 × 5 min with 200 µL/well of washing buffer (PBS/0.05%Tween20), blocked for 1 h at 37 °C with 250 µL/well of blocking buffer (3%BSA/PBS/0.05%Tween20) and washed as previously. Next, 100 µL/well of chicken sera diluted 1:300 from immunized hens collected on days 0, 17, 34, 41 after the first immunization was added and incubated for 1 h at 37 °C, and the plate was washed as previously. Next, 100 µL/well of AP-conjugated goat anti-chicken IgY antibodies  (1:2000 in 3%BSA/PBS/0.05%Tween20) were added and incubated for 1 h at room temperature. Finally, following the last plate-washing step (6 × 5 min with 200 µL/well) 100 µL/well of TMB chromogen solution  was added. The reaction was stopped with 0.5 M sulphuric acid. The signal intensity was measured at 450 nm using a plate reader .Sera from immunized broiler hens were collected and pooled on day 41 after immunization. A 96-well ELISA plate  was coated with 50 ng/well reference antigen inactivated H5N1 virus, purchased from the Animal Health and Veterinary Laboratories Agency reference laboratory . The coated plate was incubated overnight at 4 °C. Then, the plate was washed 4 × 5 min with 200 µL/well of washing buffer (PBS/0.05%Tween20), blocked for 1 h at 37 °C with 250 µL/well of blocking buffer (3%BSA/PBS/0.05%Tween20) and washed as previously. After washing, serial dilutions of pooled chicken sera (in 3%BSA/PBS/0.05%Tween20) were added to the wells and incubated for 1 h at room temperature. Serial dilutions (in 3%BSA/PBS/0.05%Tween20) of chicken polyclonal antibody A/H5N1/HPAI from the Veterinary Laboratories Agency  served as a positive control. After incubation, the plate was washed as previously, and a secondary antibody solution  in 3%BSA/PBS/0.05%Tween20 was used for detection. Finally, following the last plate-washing step (6 × 5 min with 200 µL/well), 100 µL/well of TMB chromogen solution  was added. The reaction was stopped with 0.5 M sulphuric acid. The signal intensity was measured at 450 nm using a plate reader .A 96-well streptavidin-coated ELISA plate  was coated with 100 µL/well of biotinylated RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLRENA LAH peptide (synthesized by JPT Peptide Technologies) adjusted to 10 µg/mL. The coated plate was incubated for 2 h with shaking at room temperature. Then, the plate was washed 4 × 5 min with 200 µL/well of washing buffer . Next, 100 µL/well of chicken sera diluted 1:300 from vaccinated hens was added. The plate was incubated for 2 h with shaking at room temperature and washed as previously. Next, 100 µL/well of HRP-conjugated goat anti-chicken IgY secondary antibody  was added, and the plate was incubated for 1 h at room temperature. Finally, following the last plate-washing step (6 × 5 min with 200 µL/well), 100 µL/well of TMB-substrate solution  was added. The reaction was stopped with 0.5 M sulphuric acid. The signal intensity was measured at 450 nm using a plate reader (TECAN).5) plated in a 2.5-cm culture dish in a synthetic SFX medium were infected with the H5N1 HA-stalk baculovirus at an MOI of 5. At 48 h post-infection, cells were washed with PBS and fixed with 70% cold methanol for 20 min. Next, fixed Sf9 cells were washed twice with PBS buffer and incubated for 1 h at room temperature with 500 µL/well of anti-H1N1 Mab antibodies diluted 1:1000 or with FI6 hMab antibodies diluted 1:1000 with 1% Tween 80 in PBS. Next, 500 µL/well of secondary antibodies—HRP-conjugated goat anti-mouse IgG  and HRP conjugated goat anti-human IgG , diluted 1:2000 in PBS and 1% Tween 80 was used. The plate was incubated for 1 h and washed 3 times with 1 mL of washing buffer . The reaction was developed with the NovaRED substrate kit . The cells were visualized using a Nikon PCM2000 confocal microscope. Sf9 cells (8 × 105) plated in a 2.5-cm culture dish in complete DMEM were transfected with pcDNA3.1 H1N1 HA-stalk, pcDNA3.1 H5N1 HA-stalk or pcDNA3.1 H7N9 HA-stalk using the JetPrime transfection reagent according to the manufacturer’s instructions . Forty-eight hours post-transfection cells were washed with PBS, frozen at −70 °C for 15 min, and fixed with 4% paraformaldehyde. Next, cells were incubated with 500 µL/well of chicken sera from day 41 of the immunization experiment diluted 1:300 in PBS with 1% Tween 80. The plate was incubated for 1 h and washed 3 times with 1 mL of washing buffer . Next, 500 µL/well of secondary antibodies—HRP-conjugated goat anti-chicken IgY antibodies , diluted 1:2000 in PBS and 1% Tween 80 was used. The plate was incubated for 1 h and washed 3 times with 1 mL of washing buffer . The reaction was developed with the NovaRED substrate kit . The cells were visualized using a Nikon PCM2000 confocal microscope.HEK293 cells  and Excel . The analysis was carried out with the use of nonparametric tests due to the limited number of samples, which were insufficient to confirm the normal distribution of obtained results.p = 0.05). In the next step, to control the false discovery rate, the Benjamini, Krieger and Yekutieli multiple comparison test (p = 0.05) was conducted. Each serum was tested in duplicates and average measurements of absorbance were used for the statistical analysis.Statistical analysis of antibody titers in sera collected at different time points was carried out in GraphPad Prism with the use of the nonparametric Kruskal–Wallis test (p = 0.05) for paired groups. Statistical analyses were conducted for sera collected from each hen and also between the groups of unvaccinated and vaccinated animals. Each serum was tested in duplicates in two technical repeats.A comparison of antibody titer for sera collected before and after vaccination was carried out with the use of a nonparametric Wilcoxon test , AGL44438 A/Shanghai/02/2013 (H7N9), ABU50579 A/Texas/01/2007 (H3N2), EpiFlu database, accession number: EPI156789 A/swan/Poland 305-135V08-2006 (H5N1)) were compared with Geneious Prime software using the Geneious alignment algorithm.To achieve simultaneous expression of all three structural proteins of influenza  from one expression vector, a tricistronic expression cassette was generated. Genes coding NA, HA and M1 were amplified by PCR from cDNA of the H5N1 A/swan/Poland 305-135V08-2006 strain of the HPAI virus. PCR products were ligated into the pFastBac1 vector downstream of the ACMPV polyhedrin promoter to obtain monocistronic constructs—pNA, pHA and pM1. Next, a bicistronic vector expressing HA and M1 genes (pHA/M1) was obtained by subcloning the DNA fragment of the pM1 plasmid containing M1 under the polyhedrin promoter into a plasmid. A bacmid transfer vector expressing all three structural proteins of the influenza virus (pNA/HA/M1) was constructed by subcloning the bicistronic cassette containing HA and M1 gene from the pHA/M1 plasmid into the pNA plasmid. A schematic representation of the tricistronic expression cassette is shown in The final pNA/HA/M1 construct was then used to generate recombinant baculovirus using the Bac-to-Bac system. In order to produce influenza VLPs, Sf9 cells were infected with NA/HA/M1 recombinant baculovirus. The medium from infected cells containing VLPs was collected, concentrated and purified on a 10%–60 % sucrose gradient. The proteins of each fraction were analyzed by SDS-PAGE and Western blotting, confirming the presence of all three proteins B. The scPurified triple H5N1/NA-HA-M1 VLPs were visualized using transmission electron microscopy, revealing a spherical structure similar to the AIV A. To conIn the next part of this study, purified triple H5N1/NA-HA-M1 VLPs were used to immunize chickens. To assess the immune response to the potential vaccine in the animal model of the natural poultry industry, broiler hens were used. Birds were vaccinated intramuscularly into the breast muscle with 15 µg of VLPs and ICF. After 17 days, chickens were vaccinated again with the same formulation of the vaccine. Blood was collected for analysis at 0, 17, 34 and 41 days of the experiment. A schematic timeline of the immunization is shown in To assess the immune response to the potential vaccine in broiler hens, the end-point titration of sera via ELISA assay was performed. The end-point serum titrations show that the final antibody titer obtained after immunization with triple H5N1/NA-HA-M1 VLPs reached 1:2700 . The antTo further evaluate the humoral response of broiler hens to the immunization with triple H5N1/NA-HA-M1 VLPs, antibody production kinetics were evaluated . An analysis of antibody titers in blood collected at 0, 17, 34 and 41 days of the experiment revealed a steady increase in the humoral response after broiler immunization with triple H5N1/NA-HA-M1 VLPs. Robust production of the antibodies was detected after the first dose of the vaccine. The second dose of triple H5N1/NA-HA-M1 VLPs did not significantly increase the number of antibodies produced. Additionally, to evaluate the efficacy of the vaccination, an HI assay was performed. The HI titer of pooled chicken sera reached the level of 128 HI . It is eTo further characterize the repertoire of antibodies obtained after triple H5N1/NA-HA-M1 VLP vaccination in the hen model, their ability to bind the universal influenza antigens was tested. For this part of the study, the conserved stalk of the hemagglutinin protein  was chosen as a universal influenza antigen. The recombinant HA-stalk antigen from the homologous H5N1 avian influenza strain (A/H5N1 HPAI virus) was designed according to Uranowska et al. and Krammer & Palese ,28. BrieFurther, to confirm the broad spectrum of universal recognition of the obtained hens’ sera, HA-stalk constructs from both 1 and 2 HA groups were designed and produced in mammalian cells. The mammalian-derived HA-stalk recombinant protein from heterologous H7N9/Shanghai AIVs as well as from the heterosubtypic human pH1N1 influenza strain was designed with the same strategy as described above A. The syNext, hens’ sera after vaccination with triple H5N1/NA-HA-M1 VLPs were used to detect mammalian-derived HA-stalk recombinant protein from heterologous H7N9/Shanghai AIVs as well as from heterosubtypic human pH1N1 and homologous H5N1 influenza strains. IPMA showed reactivity of the obtained sera with different HA-stalk universal antigens. These results indicate that immunization with triple H5N1/NA-HA-M1 VLPs was able to elicit a pool of universal antibodies .The least variable region within HA-stalk is the LAH region. The alignment of amino acid sequences coding LAH regions from H5N1, pH1N1, H7N9 and H3N2 HA protein shows over a 45% sequence identity between them .To further analyze the pool of universal antibodies elicited after vaccination of chickens with triple H5N1/NA-HA-M1 VLPs, peptide ELISA was performed on a plate coated with LAH peptide. Sera from day 0 and day 41 from five chickens were chosen to analyze the ability to bind LAH peptides. Obtained results suggest that sera from day 41 contained substantially higher amounts of universal IgY antibodies, specifically recognizing LAH epitopes . HoweverThe development of new technologies for the production of influenza vaccines is one of the biggest concerns of the poultry industry and public health. Vaccines containing complete, attenuated or inactivated viruses require the handling of infectious virus’ (or virus components) at BSL3 and/or BSL2-enhanced safety levels during the vaccine production process. Consequently, studies are focused on developing the next generation of vaccines containing recombinant antigens in the form of VLPs. These nanostructures seem to be a perfect vaccine since they not only imitate a native viral particle and do not contain genetic material, but also have the ability to stimulate both types of an immune response ,29. VLPsIn 2005, Pushko et al. described for the first time, that VLPs built with the HA, NA and M1 proteins of the H9N2 influenza virus induce protective immune responses in mice . From thDepending on the structural components of the influenza virus, different types of extracellular particles can be described: sub-viral particles composed of HA, NA and M2 proteins that lack the matrix protein M1; VLPs containing M1 protein and other influenza glycoproteins or M2 structural proteins; and capsid-like particles containing only the M1 protein, forming extracellular nanostructures. Diverse VLPs were obtained for different types of influenza  through different strategies. The most effective way of VLP production in insect cells is by the co-expression of influenza genes from one vector instead of co-infection with several baculoviruses expressing singular AIV genes (data not shown) ,40. The It is worth mentioning that the H5N1 subtype crossed the species barrier for the first time in 1997, and from that time still, there are several challenges in designing an effective vaccine. The phylogenetic characterization and sequence homology in the highly pathogenic H5N1 HA groups (viruses of 10 clades) are defined by the WHO/OIE/FAO H5N1 Evolution Working Group. Moreover, many of these clades have additional subclades and sub-subclades ,44. CurrOur triple H5N1/NA-HA-M1 VLPs served as a surrogate model of highly pathogenic avian influenza strain to characterize the immune response in the natural model after vaccination. Hens were vaccinated twice with H5N1/NA-HA-M1 VLPs formulated with ICF. To evaluate the kinetics of humoral response of hens after vaccination with triple H5N1/NA-HA-M1 VLPs, direct ELISA with inactivated H5N1 virus was performed. The end-point titration of IgY antibodies after vaccination reached a high and comparable level to other data ,46,47,48The efficacy of the triple H5N1/NA-HA-M1 VLP-based vaccine was evaluated via HI assay. The virus used in this assay was homosubtypic H5N1 from clade 0. The results show that the antigen in the form of VLPs from clade 2.2.2 elicits antibodies that possess the ability to inhibit the hemagglutination of homosubtypic antigen H5N1 on a high level of 128 HI units, which is above the cut-off of the protective level 40 HI unit . In compMany studies describe the efficacy of the VLP immunization in order to define the HA dominant and NA dominant immune response in the chicken model. Here, we present a first-time in-depth characterization of the immune response toward a generation of universal anti-HA-stalk antibodies after vaccination of broiler hens with HPAI H5N1 VLPs. Our results suggest that after vaccination with triple H5N1/NA-HA-M1 VLPs, hens generate a pool of broad-spectrum universal anti-HA-stalk antibodies. To evaluate the immune response toward the headless hemagglutinin, we designed the HA-stalk antigens from different influenza strains. Our previous results by Uranowska et al. show the characterization of avian HA-stalk antigen as a potentially universal one . Based oLAH is the least variable region within the HA-stalk. It is considered to be the most universal epitope. To analyze whether the pool of universal antibodies elicited after vaccination of chickens with triple H5N1/NA-HA-M1 VLPs is able to recognize LAH, we performed an ELISA assay on plates coated with LAH peptide from the heterosubtypic H3N2 influenza strain. Our results suggest that hen sera from day 41 is cross-reactive with the heterosubtypic H3N2, and contains substantially higher amounts of universal IgY antibodies, specifically recognizing the LAH epitope. Statistical analyses of the results did not reveal significant differences between samples obtained before and after vaccination in each chicken or between grouped data. However, it is important to note that obtained absorbance values were at a low level due to the antibody dilutions used, which at the same time resulted in a decrease in the differences between the two types of tested samples. Moreover, a nonparametric test had to be used for the analysis. This test is characterized by lower statistical power. In the case of having a larger test group, more powerful parametric tests could be used for analysis, the use of which would give a chance to detect statistically significant differences. In the case of nonparametric tests, there is a risk of staying with a faulty hypothesis that the data groups are not statistically different.Moreover, Kim et al. suggested that the chimeric cHA H9/H5N2 vaccination strategy provides robust protection against homologous, heterologous, and heterosubtypic viruses of both subtypes . FurtherOn the other hand, an H5N1 vaccine construct, incorporating either HA1 or HA2 subunit, may not provide protection against an HPAI virus challenge in chickens, according to Shirvani et al. . Their rWe strongly believe that further analysis towards the characterization of antibodies that are able to recognize the universal HA-stalk antigens is of great importance, thus helping to design a broad-spectrum vaccine against poultry threats.Vaccination of broiler hens with triple HPAI H5N1 VLPs triggers the generation of broad-spectrum anti-HA-stalk antibodies. Furthermore, these universal antibodies are able to recognize the mammalian-derived form of HA-stalk recombinant proteins from homologous H5N1 and heterologous H7N9 AIVs as well as from the heterosubtypic human H1N1 influenza strain. To further evaluate the effectiveness of the prepared VLP vaccine, an in vivo challenge should be performed.