pr8 h1n1 influenza virus strain  (Sino Biological)


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    Name:
    Influenza A H1N1 Nucleoprotein NP Protein
    Description:
    A DNA sequence encoding the Influenza A virus A Puerto Rico 8 34 Mount Sinai H1N1 nucleoprotein AAM75159 1 Met1 Gly490 was fused with a polyhistidine tag at the C terminus
    Catalog Number:
    11675-V08B
    Price:
    None
    Category:
    recombinant protein
    Product Aliases:
    NP Protein H1N1
    Host:
    Baculovirus-Insect Cells
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    Structured Review

    Sino Biological pr8 h1n1 influenza virus strain
    CD4 and CD8 T cells are required for protective immunity to influenza A virus. C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in ADJ+GLA. At 70 days after booster vaccination, mice were challenged intranasally with <t>H1N1/PR8</t> strain of influenza A virus; unvaccinated mice were challenged as controls. Cohorts of vaccinated virus-challenged mice were treated (intravenously and intranasally) with isotype control IgG, anti-CD4 or anti-CD8 antibodies at days -5, -3, -1 and 1, 3 and 5, relative to viral challenge. On the 6 th day after viral challenge, virus-specific T cells and viral titers were quantified in lungs. (A) FACS plots are gated on live lymphocytes and numbers are percentages among live lymphocytes. (B) FACS plots are gated on CD8 T cells and numbers are percentages of D b /NP366 tetramer-binding CD8 T cells among CD8 T cells. (C) FACS plots are gated on CD4 T cells and numbers are percentages of I-A b /NP311 tetramer-binding CD4 T cells among CD8 T cells. (D) Viral titers in lungs were quantified by a plaque assay. Data are from two independent experiments. *, **, and *** indicate significance at P
    A DNA sequence encoding the Influenza A virus A Puerto Rico 8 34 Mount Sinai H1N1 nucleoprotein AAM75159 1 Met1 Gly490 was fused with a polyhistidine tag at the C terminus
    https://www.bioz.com/result/pr8 h1n1 influenza virus strain/product/Sino Biological
    Average 95 stars, based on 1 article reviews
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    pr8 h1n1 influenza virus strain - by Bioz Stars, 2021-07
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    Images

    1) Product Images from "Programming Multifaceted Pulmonary T-Cell Immunity by Combination Adjuvants"

    Article Title: Programming Multifaceted Pulmonary T-Cell Immunity by Combination Adjuvants

    Journal: bioRxiv

    doi: 10.1101/2020.07.10.197459

    CD4 and CD8 T cells are required for protective immunity to influenza A virus. C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in ADJ+GLA. At 70 days after booster vaccination, mice were challenged intranasally with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged as controls. Cohorts of vaccinated virus-challenged mice were treated (intravenously and intranasally) with isotype control IgG, anti-CD4 or anti-CD8 antibodies at days -5, -3, -1 and 1, 3 and 5, relative to viral challenge. On the 6 th day after viral challenge, virus-specific T cells and viral titers were quantified in lungs. (A) FACS plots are gated on live lymphocytes and numbers are percentages among live lymphocytes. (B) FACS plots are gated on CD8 T cells and numbers are percentages of D b /NP366 tetramer-binding CD8 T cells among CD8 T cells. (C) FACS plots are gated on CD4 T cells and numbers are percentages of I-A b /NP311 tetramer-binding CD4 T cells among CD8 T cells. (D) Viral titers in lungs were quantified by a plaque assay. Data are from two independent experiments. *, **, and *** indicate significance at P
    Figure Legend Snippet: CD4 and CD8 T cells are required for protective immunity to influenza A virus. C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in ADJ+GLA. At 70 days after booster vaccination, mice were challenged intranasally with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged as controls. Cohorts of vaccinated virus-challenged mice were treated (intravenously and intranasally) with isotype control IgG, anti-CD4 or anti-CD8 antibodies at days -5, -3, -1 and 1, 3 and 5, relative to viral challenge. On the 6 th day after viral challenge, virus-specific T cells and viral titers were quantified in lungs. (A) FACS plots are gated on live lymphocytes and numbers are percentages among live lymphocytes. (B) FACS plots are gated on CD8 T cells and numbers are percentages of D b /NP366 tetramer-binding CD8 T cells among CD8 T cells. (C) FACS plots are gated on CD4 T cells and numbers are percentages of I-A b /NP311 tetramer-binding CD4 T cells among CD8 T cells. (D) Viral titers in lungs were quantified by a plaque assay. Data are from two independent experiments. *, **, and *** indicate significance at P

    Techniques Used: Mouse Assay, FACS, Binding Assay, Plaque Assay

    Histopathological analysis of lungs following viral challenge of vaccinated mice. Groups of C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in various adjuvants. At 100 days after booster vaccination, vaccinated mice were challenged intranasally with H1N1/PR8 strain of influenza A virus. On the 6 th day after viral challenge, lungs were collected in neutral-buffered formailin, and tissue sections were stained with Hematoxylin and Eosin (H E). H E stained lung sections were evaluated by a board-certified pathologist (Dr. Gasper); he was blinded to the identity of sections. In each image (40X magnification), asterisks indicate similarly sized large bronchioles, arrow heads indicate regions in which bronchial lesions extend in to the adjacent alveoli, and arrows indicate perivascular lymphoid nodules. A. Adjuplex-vaccinated mouse: there is mild necrotizing bronchitis asterisks). B. CPG-vaccinated mouse: there is obliteration of two bronchioles by inflammation that extends far into the surrounding alveoli (arrowheads). C. GLA-vaccinated mouse: there is bronchiolitis affecting 1 of the larger bronchioles, with minimal extension into the adjacent alveoli. D. ADJ+CPG-vaccinated mouse. Broncholitis is similar to that in A, but alveolar regions around the affected bronchiole (center) are infiltrated by inflammatory cells. E. ADJ+GLA vaccinated mouse: bronchiolitis is of intermediate severity between B and C, and regionally extends into the adjacent alveolar tissue (arrowhead). Each lung section was scored individually, and lesion scores from 0-3 were assigned for bronchial lesions, alveolar lesions, and specific disease patterns, with 0 = absent, 1 = mild, 2 = moderate, 3 = severe. Bronchioloar Lesions: Epithelial degeneration/necrosis; Intraepithelial neutrophils; Intraepithelial eosinophils; Intraepithelial lymphocytes; Luminal dislodged epithelial cells/debris; Luminal cellular exudate; Peribronchiolar neutrophils; Pavementing/Subendothelial leukocytes. Alveolar Lesions: Alveolar wall thickening; Interstitial macrophages; Interstitial lymphocytes; Interstitial granulocytes; Epithelial necrosis; Luminal edema; Luminal hemorrhage; Luminal cellular exudate; Luminal alveolar macrophages; Luminal neutrophils; Luminal sloughed epithelial cells.
    Figure Legend Snippet: Histopathological analysis of lungs following viral challenge of vaccinated mice. Groups of C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in various adjuvants. At 100 days after booster vaccination, vaccinated mice were challenged intranasally with H1N1/PR8 strain of influenza A virus. On the 6 th day after viral challenge, lungs were collected in neutral-buffered formailin, and tissue sections were stained with Hematoxylin and Eosin (H E). H E stained lung sections were evaluated by a board-certified pathologist (Dr. Gasper); he was blinded to the identity of sections. In each image (40X magnification), asterisks indicate similarly sized large bronchioles, arrow heads indicate regions in which bronchial lesions extend in to the adjacent alveoli, and arrows indicate perivascular lymphoid nodules. A. Adjuplex-vaccinated mouse: there is mild necrotizing bronchitis asterisks). B. CPG-vaccinated mouse: there is obliteration of two bronchioles by inflammation that extends far into the surrounding alveoli (arrowheads). C. GLA-vaccinated mouse: there is bronchiolitis affecting 1 of the larger bronchioles, with minimal extension into the adjacent alveoli. D. ADJ+CPG-vaccinated mouse. Broncholitis is similar to that in A, but alveolar regions around the affected bronchiole (center) are infiltrated by inflammatory cells. E. ADJ+GLA vaccinated mouse: bronchiolitis is of intermediate severity between B and C, and regionally extends into the adjacent alveolar tissue (arrowhead). Each lung section was scored individually, and lesion scores from 0-3 were assigned for bronchial lesions, alveolar lesions, and specific disease patterns, with 0 = absent, 1 = mild, 2 = moderate, 3 = severe. Bronchioloar Lesions: Epithelial degeneration/necrosis; Intraepithelial neutrophils; Intraepithelial eosinophils; Intraepithelial lymphocytes; Luminal dislodged epithelial cells/debris; Luminal cellular exudate; Peribronchiolar neutrophils; Pavementing/Subendothelial leukocytes. Alveolar Lesions: Alveolar wall thickening; Interstitial macrophages; Interstitial lymphocytes; Interstitial granulocytes; Epithelial necrosis; Luminal edema; Luminal hemorrhage; Luminal cellular exudate; Luminal alveolar macrophages; Luminal neutrophils; Luminal sloughed epithelial cells.

    Techniques Used: Mouse Assay, Staining

    Vaccine-induced protective immunity to H1N1 and H5N1 influenza viruses. (A-C) Groups of C57BL/6 mice were vaccinated twice at 3-week intervals with NP protein formulated in various adjuvants.. (A) At 100 days after the booster vaccination, mice were challenged intranasally with H1N1/PR8 strain of influenza A virus. Viral tiers and virus-specific T cell responses in lungs were quantified on the 6 th day after virus challenge. (A) Viral titers in the lungs on the 6 th day after virus challenge. (B) Percentages of NP366-specific IFN-γ and IL-17 producing cells among CD8 T cells (bar graphs) and calculated proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ+IL-7-producing peptide-stimulated NP366-specific T cells (Pie charts). (C) Percentages of NP311-specific IFN-γ and IL-17 producing cells among CD4 T cells and calculated proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ+IL-7-producing peptide-stimulated NP311-specific T cells. (D) C57BL/6 mice were vaccinated with NP+ADJ+GLA twice at an interval of 3 weeks. One hundred and eighty days after the last vaccination, mice were challenged intransally with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged with virus as controls. Cohorts of vaccinated virus-challenged mice were treated with isotype control IgG or anti-IL-17A antibodies (intravenously and intranasally) at -1, 0, 1, 3 and 5 days relative to virus challenge. On the 6 th day after viral challenge, viral titers and virus-specific T cell responses were quantified in lungs. (E) Groups of C57BL/6 mice were vaccinated twice with NP protein alone or formulated in various adjuvants. Fifty days after booster vaccination, vaccinated and unvaccinated mice were challenged intranasally with the highly pathogenic H5N1 avian influenza A virus; weight loss and survival was monitored until day 14. Data are pooled from 2 independent experiments or representative of two independent experiments. *, **, and *** indicate significance at P
    Figure Legend Snippet: Vaccine-induced protective immunity to H1N1 and H5N1 influenza viruses. (A-C) Groups of C57BL/6 mice were vaccinated twice at 3-week intervals with NP protein formulated in various adjuvants.. (A) At 100 days after the booster vaccination, mice were challenged intranasally with H1N1/PR8 strain of influenza A virus. Viral tiers and virus-specific T cell responses in lungs were quantified on the 6 th day after virus challenge. (A) Viral titers in the lungs on the 6 th day after virus challenge. (B) Percentages of NP366-specific IFN-γ and IL-17 producing cells among CD8 T cells (bar graphs) and calculated proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ+IL-7-producing peptide-stimulated NP366-specific T cells (Pie charts). (C) Percentages of NP311-specific IFN-γ and IL-17 producing cells among CD4 T cells and calculated proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ+IL-7-producing peptide-stimulated NP311-specific T cells. (D) C57BL/6 mice were vaccinated with NP+ADJ+GLA twice at an interval of 3 weeks. One hundred and eighty days after the last vaccination, mice were challenged intransally with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged with virus as controls. Cohorts of vaccinated virus-challenged mice were treated with isotype control IgG or anti-IL-17A antibodies (intravenously and intranasally) at -1, 0, 1, 3 and 5 days relative to virus challenge. On the 6 th day after viral challenge, viral titers and virus-specific T cell responses were quantified in lungs. (E) Groups of C57BL/6 mice were vaccinated twice with NP protein alone or formulated in various adjuvants. Fifty days after booster vaccination, vaccinated and unvaccinated mice were challenged intranasally with the highly pathogenic H5N1 avian influenza A virus; weight loss and survival was monitored until day 14. Data are pooled from 2 independent experiments or representative of two independent experiments. *, **, and *** indicate significance at P

    Techniques Used: Mouse Assay

    Kinetics and durability of influenza viral control in vaccinated mice. B6 mice were vaccinated twice (at 3 weeks intervals) intranasally with NP protein formulated with the indicated adjuvants. Unvaccinated mice and mice vaccinated with NP only (without adjuvants) served as controls. At 100 and 180 days after booster vaccination, mice were challenged intranasally with PR8/H1N1 influenza virus. (A) Body weight loss was assessed by calculating bodyweight at different days after challenge, relative to bodyweight before challenge at 100 days after vaccination. (B) Vaccinated mice were challenged with PR8/H1N1at 100 days after vaccination and viral titers in lungs were quantified at day 2 and 4 after challenge, using a plaque assay. (C) At day 180 after booster vaccination, mice were challenged with PR8/H1N1 virus, and viral titers in lungs were assessed at day 6 after challenge. (D) Percentages and numbers of NP366-specific CD8 T cells and NP311-specific CD4 T cells in lungs and percentage of these cells in the vascular and non-vascular compartment at day 6 after challenge (challenged at 100 days after vaccination). *, **, and *** indicate significance at P
    Figure Legend Snippet: Kinetics and durability of influenza viral control in vaccinated mice. B6 mice were vaccinated twice (at 3 weeks intervals) intranasally with NP protein formulated with the indicated adjuvants. Unvaccinated mice and mice vaccinated with NP only (without adjuvants) served as controls. At 100 and 180 days after booster vaccination, mice were challenged intranasally with PR8/H1N1 influenza virus. (A) Body weight loss was assessed by calculating bodyweight at different days after challenge, relative to bodyweight before challenge at 100 days after vaccination. (B) Vaccinated mice were challenged with PR8/H1N1at 100 days after vaccination and viral titers in lungs were quantified at day 2 and 4 after challenge, using a plaque assay. (C) At day 180 after booster vaccination, mice were challenged with PR8/H1N1 virus, and viral titers in lungs were assessed at day 6 after challenge. (D) Percentages and numbers of NP366-specific CD8 T cells and NP311-specific CD4 T cells in lungs and percentage of these cells in the vascular and non-vascular compartment at day 6 after challenge (challenged at 100 days after vaccination). *, **, and *** indicate significance at P

    Techniques Used: Mouse Assay, Plaque Assay

    Regulation of vaccine-induced CD8 T-cell memory and protective immunity by CD4 T cells. Groups of C57BL/6 mice were vaccinated twice at 3-week intervals with NP protein formulated in ADJ+GLA. Cohorts of vaccinated mice were treated with isotype control antibodies (Non Depleted) or anti-CD4 antibodies (CD4 Depleted) intravenously and intranasally on days −1, 0, and 1 relative to prime and boost vaccination with NP+ADJ+GLA. T-cell memory in lungs (A-F) and protective immunity to influenza A virus (G-P) was determined at 80 days after booster vaccination. (A-F) T-cell memory in lungs at day 80 after booster vaccination. To stain for vascular cells, mice were injected intravenously with anti-CD45.2 antibodies, 3 minutes prior to euthanasia. Lung cells were stained directly ex vivo with D b /NP366 or I-A b /NP311 tetramers along with the indicated antibodies for cell surface markers. For cytokine analysis, lung cells were stimulated with NP366 or NP311 peptide for 5 hours before intracellular staining. (A) FACS plots are gated on total CD4 T cells and show NP311-specific tetramer-binding memory CD4 T cells only in non-depleted mice. (B) NP366-specific tetramer-binding memory CD8 T cells in lungs of non-d epleted and CD4 T cell-depleted mice. (C) Expression of tissue residency markers on NP366-specific tetramer-binding memory CD8 T cells in lungs. (D) Percentage of vascular (CD45.2 +ve ) and non-vascular (CD45.2 -ve ) cells among NP366-specific tetramer-binding memory CD8 T cells in lungs. (E) Percentages of IFN-γ- and IL-17-producing NP366-specific cells among CD8 T cells in lungs. (F) Calculated proportions of IFN-γ and/or IL-17-producing cells among cytokine-producing peptide-stimulated IFN-γ+IL-17 NP366-specific CD8 T cells. (G-P) At day 80 after booster vaccination, non-depleted and CD4 T cell-depleted mice were challenged intranasally with PR8/H1N1 influenza A virus; recall virus-specific CD8/CD4 T cell responses and viral load in lungs were assessed at day 6 after challenge. (G) Percentages of NP366-specific tetramer-binding cells among CD8 T cells in lungs. (H) Percentages of NP366-specific tetramer-binding CD8 T cells in vascular and nonvascular lung compartment. (I) Percentages of NP311-specific tetramer-binding cells among CD4 T cells in lungs. (J) Expression of tissue residency markers on NP366-specific tetramer-binding CD8 T cells. (K) Chemokine receptor and transcription factor expression in NP366-specific CD8 T cells in lungs. (L) Granzyme B expression by NP366-specific CD8 T cells directly ex vivo. (M) Percentages of IFN-γ and IL-17 producing NP366-specific CD8 T cells. (N) Relative proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ plus IL-17-producing peptide-stimulated NP366-specific CD8 T cells. (O) Viral titers in lungs at day 6 after challenge. (P) Body weight, measured as a percentage of starting body weight prior to challenge. Data are pooled from two independent experiments. *, **, and *** indicate significance at P
    Figure Legend Snippet: Regulation of vaccine-induced CD8 T-cell memory and protective immunity by CD4 T cells. Groups of C57BL/6 mice were vaccinated twice at 3-week intervals with NP protein formulated in ADJ+GLA. Cohorts of vaccinated mice were treated with isotype control antibodies (Non Depleted) or anti-CD4 antibodies (CD4 Depleted) intravenously and intranasally on days −1, 0, and 1 relative to prime and boost vaccination with NP+ADJ+GLA. T-cell memory in lungs (A-F) and protective immunity to influenza A virus (G-P) was determined at 80 days after booster vaccination. (A-F) T-cell memory in lungs at day 80 after booster vaccination. To stain for vascular cells, mice were injected intravenously with anti-CD45.2 antibodies, 3 minutes prior to euthanasia. Lung cells were stained directly ex vivo with D b /NP366 or I-A b /NP311 tetramers along with the indicated antibodies for cell surface markers. For cytokine analysis, lung cells were stimulated with NP366 or NP311 peptide for 5 hours before intracellular staining. (A) FACS plots are gated on total CD4 T cells and show NP311-specific tetramer-binding memory CD4 T cells only in non-depleted mice. (B) NP366-specific tetramer-binding memory CD8 T cells in lungs of non-d epleted and CD4 T cell-depleted mice. (C) Expression of tissue residency markers on NP366-specific tetramer-binding memory CD8 T cells in lungs. (D) Percentage of vascular (CD45.2 +ve ) and non-vascular (CD45.2 -ve ) cells among NP366-specific tetramer-binding memory CD8 T cells in lungs. (E) Percentages of IFN-γ- and IL-17-producing NP366-specific cells among CD8 T cells in lungs. (F) Calculated proportions of IFN-γ and/or IL-17-producing cells among cytokine-producing peptide-stimulated IFN-γ+IL-17 NP366-specific CD8 T cells. (G-P) At day 80 after booster vaccination, non-depleted and CD4 T cell-depleted mice were challenged intranasally with PR8/H1N1 influenza A virus; recall virus-specific CD8/CD4 T cell responses and viral load in lungs were assessed at day 6 after challenge. (G) Percentages of NP366-specific tetramer-binding cells among CD8 T cells in lungs. (H) Percentages of NP366-specific tetramer-binding CD8 T cells in vascular and nonvascular lung compartment. (I) Percentages of NP311-specific tetramer-binding cells among CD4 T cells in lungs. (J) Expression of tissue residency markers on NP366-specific tetramer-binding CD8 T cells. (K) Chemokine receptor and transcription factor expression in NP366-specific CD8 T cells in lungs. (L) Granzyme B expression by NP366-specific CD8 T cells directly ex vivo. (M) Percentages of IFN-γ and IL-17 producing NP366-specific CD8 T cells. (N) Relative proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ plus IL-17-producing peptide-stimulated NP366-specific CD8 T cells. (O) Viral titers in lungs at day 6 after challenge. (P) Body weight, measured as a percentage of starting body weight prior to challenge. Data are pooled from two independent experiments. *, **, and *** indicate significance at P

    Techniques Used: Mouse Assay, Staining, Injection, Ex Vivo, FACS, Binding Assay, Expressing

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    Article Snippet: Antibody purity was examined by SDS-PAGE, and concentration was determined by the Bradford dye-binding method using mouse IgG as the standard. .. Recombinant HA proteins of various influenza virusesThe recombinant HA proteins of A/Puerto Rico/8/1934(H1N1), A/California/07/2009(H1N1), A/Victoria/361/2011(H3N2), A/Hong Kong/483/97(H5N1), A/chicken/Netherlands/1/03(H7N7), and A/Shanghai/1/2013(H7N9) were purchased from Sino Biological Inc. (Catalog Number: 11684-V08B, 11085-V08B, 40145-V08B, 11689-V08B, 11212-V08B, and 40104-V08H, respectively). .. Site-directed mutagenesis All of the pGEX-4T-3 plasmids for expression of the H7N9 rHA mutants in Escherichia coli (E. coli) were generated by the PCR-based QuikChange II Site-Directed Mutagenesis Kit (Stratagene, Agilent Technologies) according to the manufacturer’s instruction.

    Article Title: Programming Multifaceted Pulmonary T-Cell Immunity by Combination Adjuvants
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    Article Title: Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung
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    Article Title: Inducible Bronchus-Associated Lymphoid Tissues (iBALT) Serve as Sites of B Cell Selection and Maturation Following Influenza Infection in Mice
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    Article Title: Preexisting Antibody-Dependent Cellular Cytotoxicity–Activating Antibody Responses Are Stable Longitudinally and Cross-reactive Responses Are Not Boosted by Recent Influenza Exposure
    Article Snippet: .. Briefly, U-bottomed enzyme-linked immunosorbent assay (ELISA) plates are coated overnight with recombinant influenza virus proteins, H7 HA (H7N9; A/Anhui/01/2013), H1 HA (H1N1; A/California/04/2009), and NP (H7N9; A/Anhui/01/2013; > 95% homology with H1N1-derived NP; 400 ng/well; SinoBiological) in phosphate-buffered saline (PBS). .. Background ADCC activity by NK cells was determined by analysis of paired serum specimens with plate-bound nonspecific protein (allantoic fluid), with positive controls including purified CD16 antibody (Biolegend) for coating and pooled human sera (n = 4 donors pooled) tested against each protein.

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    Sequencing:

    Article Title: Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung
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    Derivative Assay:

    Article Title: Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung
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    Labeling:

    Article Title: Inducible Bronchus-Associated Lymphoid Tissues (iBALT) Serve as Sites of B Cell Selection and Maturation Following Influenza Infection in Mice
    Article Snippet: Prior to use, biotinylated HA proteins were labeled by the sequential addition of streptavidin (SA) conjugated to BV421, phycoerythrin (PE), or allophycocyanin (APC), and stored at 4°C. .. Recombinant influenza A H1N1 NP protein (11675-V08B; Sino Biological) was labeled with PE or APC fluorochromes using commercial conjugation kits, as per manufacturer's protocol (AB102918, AB 201807; Abcam). .. Flow CytometryFor experiments requiring discrimination of blood and tissue populations, mice were intravenously labeled with 3 μg of CD45.2 antibody (104; eBioscience) in a 200 μL volume prior to tissue collection.

    Conjugation Assay:

    Article Title: Inducible Bronchus-Associated Lymphoid Tissues (iBALT) Serve as Sites of B Cell Selection and Maturation Following Influenza Infection in Mice
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    Enzyme-linked Immunosorbent Assay:

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    Mouse Assay:

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  • 95
    Sino Biological pr8 h1n1 influenza virus strain
    CD4 and CD8 T cells are required for protective immunity to influenza A virus. C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in ADJ+GLA. At 70 days after booster vaccination, mice were challenged intranasally with <t>H1N1/PR8</t> strain of influenza A virus; unvaccinated mice were challenged as controls. Cohorts of vaccinated virus-challenged mice were treated (intravenously and intranasally) with isotype control IgG, anti-CD4 or anti-CD8 antibodies at days -5, -3, -1 and 1, 3 and 5, relative to viral challenge. On the 6 th day after viral challenge, virus-specific T cells and viral titers were quantified in lungs. (A) FACS plots are gated on live lymphocytes and numbers are percentages among live lymphocytes. (B) FACS plots are gated on CD8 T cells and numbers are percentages of D b /NP366 tetramer-binding CD8 T cells among CD8 T cells. (C) FACS plots are gated on CD4 T cells and numbers are percentages of I-A b /NP311 tetramer-binding CD4 T cells among CD8 T cells. (D) Viral titers in lungs were quantified by a plaque assay. Data are from two independent experiments. *, **, and *** indicate significance at P
    Pr8 H1n1 Influenza Virus Strain, supplied by Sino Biological, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pr8 h1n1 influenza virus strain - by Bioz Stars, 2021-07
    95/100 stars
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    96
    Sino Biological influenza a virus h1n1
    CD4 and CD8 T cells are required for protective immunity to influenza A virus. C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in ADJ+GLA. At 70 days after booster vaccination, mice were challenged intranasally with <t>H1N1/PR8</t> strain of influenza A virus; unvaccinated mice were challenged as controls. Cohorts of vaccinated virus-challenged mice were treated (intravenously and intranasally) with isotype control IgG, anti-CD4 or anti-CD8 antibodies at days -5, -3, -1 and 1, 3 and 5, relative to viral challenge. On the 6 th day after viral challenge, virus-specific T cells and viral titers were quantified in lungs. (A) FACS plots are gated on live lymphocytes and numbers are percentages among live lymphocytes. (B) FACS plots are gated on CD8 T cells and numbers are percentages of D b /NP366 tetramer-binding CD8 T cells among CD8 T cells. (C) FACS plots are gated on CD4 T cells and numbers are percentages of I-A b /NP311 tetramer-binding CD4 T cells among CD8 T cells. (D) Viral titers in lungs were quantified by a plaque assay. Data are from two independent experiments. *, **, and *** indicate significance at P
    Influenza A Virus H1n1, supplied by Sino Biological, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/influenza a virus h1n1/product/Sino Biological
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    influenza a virus h1n1 - by Bioz Stars, 2021-07
    96/100 stars
      Buy from Supplier

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    CD4 and CD8 T cells are required for protective immunity to influenza A virus. C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in ADJ+GLA. At 70 days after booster vaccination, mice were challenged intranasally with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged as controls. Cohorts of vaccinated virus-challenged mice were treated (intravenously and intranasally) with isotype control IgG, anti-CD4 or anti-CD8 antibodies at days -5, -3, -1 and 1, 3 and 5, relative to viral challenge. On the 6 th day after viral challenge, virus-specific T cells and viral titers were quantified in lungs. (A) FACS plots are gated on live lymphocytes and numbers are percentages among live lymphocytes. (B) FACS plots are gated on CD8 T cells and numbers are percentages of D b /NP366 tetramer-binding CD8 T cells among CD8 T cells. (C) FACS plots are gated on CD4 T cells and numbers are percentages of I-A b /NP311 tetramer-binding CD4 T cells among CD8 T cells. (D) Viral titers in lungs were quantified by a plaque assay. Data are from two independent experiments. *, **, and *** indicate significance at P

    Journal: bioRxiv

    Article Title: Programming Multifaceted Pulmonary T-Cell Immunity by Combination Adjuvants

    doi: 10.1101/2020.07.10.197459

    Figure Lengend Snippet: CD4 and CD8 T cells are required for protective immunity to influenza A virus. C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in ADJ+GLA. At 70 days after booster vaccination, mice were challenged intranasally with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged as controls. Cohorts of vaccinated virus-challenged mice were treated (intravenously and intranasally) with isotype control IgG, anti-CD4 or anti-CD8 antibodies at days -5, -3, -1 and 1, 3 and 5, relative to viral challenge. On the 6 th day after viral challenge, virus-specific T cells and viral titers were quantified in lungs. (A) FACS plots are gated on live lymphocytes and numbers are percentages among live lymphocytes. (B) FACS plots are gated on CD8 T cells and numbers are percentages of D b /NP366 tetramer-binding CD8 T cells among CD8 T cells. (C) FACS plots are gated on CD4 T cells and numbers are percentages of I-A b /NP311 tetramer-binding CD4 T cells among CD8 T cells. (D) Viral titers in lungs were quantified by a plaque assay. Data are from two independent experiments. *, **, and *** indicate significance at P

    Article Snippet: Recombinant nucleoprotein (NP) of the PR8/H1N1 influenza virus strain was purchased from Sino Biological Inc (Beijing, China).

    Techniques: Mouse Assay, FACS, Binding Assay, Plaque Assay

    Histopathological analysis of lungs following viral challenge of vaccinated mice. Groups of C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in various adjuvants. At 100 days after booster vaccination, vaccinated mice were challenged intranasally with H1N1/PR8 strain of influenza A virus. On the 6 th day after viral challenge, lungs were collected in neutral-buffered formailin, and tissue sections were stained with Hematoxylin and Eosin (H E). H E stained lung sections were evaluated by a board-certified pathologist (Dr. Gasper); he was blinded to the identity of sections. In each image (40X magnification), asterisks indicate similarly sized large bronchioles, arrow heads indicate regions in which bronchial lesions extend in to the adjacent alveoli, and arrows indicate perivascular lymphoid nodules. A. Adjuplex-vaccinated mouse: there is mild necrotizing bronchitis asterisks). B. CPG-vaccinated mouse: there is obliteration of two bronchioles by inflammation that extends far into the surrounding alveoli (arrowheads). C. GLA-vaccinated mouse: there is bronchiolitis affecting 1 of the larger bronchioles, with minimal extension into the adjacent alveoli. D. ADJ+CPG-vaccinated mouse. Broncholitis is similar to that in A, but alveolar regions around the affected bronchiole (center) are infiltrated by inflammatory cells. E. ADJ+GLA vaccinated mouse: bronchiolitis is of intermediate severity between B and C, and regionally extends into the adjacent alveolar tissue (arrowhead). Each lung section was scored individually, and lesion scores from 0-3 were assigned for bronchial lesions, alveolar lesions, and specific disease patterns, with 0 = absent, 1 = mild, 2 = moderate, 3 = severe. Bronchioloar Lesions: Epithelial degeneration/necrosis; Intraepithelial neutrophils; Intraepithelial eosinophils; Intraepithelial lymphocytes; Luminal dislodged epithelial cells/debris; Luminal cellular exudate; Peribronchiolar neutrophils; Pavementing/Subendothelial leukocytes. Alveolar Lesions: Alveolar wall thickening; Interstitial macrophages; Interstitial lymphocytes; Interstitial granulocytes; Epithelial necrosis; Luminal edema; Luminal hemorrhage; Luminal cellular exudate; Luminal alveolar macrophages; Luminal neutrophils; Luminal sloughed epithelial cells.

    Journal: bioRxiv

    Article Title: Programming Multifaceted Pulmonary T-Cell Immunity by Combination Adjuvants

    doi: 10.1101/2020.07.10.197459

    Figure Lengend Snippet: Histopathological analysis of lungs following viral challenge of vaccinated mice. Groups of C57BL/6 mice were vaccinated twice (at 3 weeks interval) with NP protein formulated in various adjuvants. At 100 days after booster vaccination, vaccinated mice were challenged intranasally with H1N1/PR8 strain of influenza A virus. On the 6 th day after viral challenge, lungs were collected in neutral-buffered formailin, and tissue sections were stained with Hematoxylin and Eosin (H E). H E stained lung sections were evaluated by a board-certified pathologist (Dr. Gasper); he was blinded to the identity of sections. In each image (40X magnification), asterisks indicate similarly sized large bronchioles, arrow heads indicate regions in which bronchial lesions extend in to the adjacent alveoli, and arrows indicate perivascular lymphoid nodules. A. Adjuplex-vaccinated mouse: there is mild necrotizing bronchitis asterisks). B. CPG-vaccinated mouse: there is obliteration of two bronchioles by inflammation that extends far into the surrounding alveoli (arrowheads). C. GLA-vaccinated mouse: there is bronchiolitis affecting 1 of the larger bronchioles, with minimal extension into the adjacent alveoli. D. ADJ+CPG-vaccinated mouse. Broncholitis is similar to that in A, but alveolar regions around the affected bronchiole (center) are infiltrated by inflammatory cells. E. ADJ+GLA vaccinated mouse: bronchiolitis is of intermediate severity between B and C, and regionally extends into the adjacent alveolar tissue (arrowhead). Each lung section was scored individually, and lesion scores from 0-3 were assigned for bronchial lesions, alveolar lesions, and specific disease patterns, with 0 = absent, 1 = mild, 2 = moderate, 3 = severe. Bronchioloar Lesions: Epithelial degeneration/necrosis; Intraepithelial neutrophils; Intraepithelial eosinophils; Intraepithelial lymphocytes; Luminal dislodged epithelial cells/debris; Luminal cellular exudate; Peribronchiolar neutrophils; Pavementing/Subendothelial leukocytes. Alveolar Lesions: Alveolar wall thickening; Interstitial macrophages; Interstitial lymphocytes; Interstitial granulocytes; Epithelial necrosis; Luminal edema; Luminal hemorrhage; Luminal cellular exudate; Luminal alveolar macrophages; Luminal neutrophils; Luminal sloughed epithelial cells.

    Article Snippet: Recombinant nucleoprotein (NP) of the PR8/H1N1 influenza virus strain was purchased from Sino Biological Inc (Beijing, China).

    Techniques: Mouse Assay, Staining

    Vaccine-induced protective immunity to H1N1 and H5N1 influenza viruses. (A-C) Groups of C57BL/6 mice were vaccinated twice at 3-week intervals with NP protein formulated in various adjuvants.. (A) At 100 days after the booster vaccination, mice were challenged intranasally with H1N1/PR8 strain of influenza A virus. Viral tiers and virus-specific T cell responses in lungs were quantified on the 6 th day after virus challenge. (A) Viral titers in the lungs on the 6 th day after virus challenge. (B) Percentages of NP366-specific IFN-γ and IL-17 producing cells among CD8 T cells (bar graphs) and calculated proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ+IL-7-producing peptide-stimulated NP366-specific T cells (Pie charts). (C) Percentages of NP311-specific IFN-γ and IL-17 producing cells among CD4 T cells and calculated proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ+IL-7-producing peptide-stimulated NP311-specific T cells. (D) C57BL/6 mice were vaccinated with NP+ADJ+GLA twice at an interval of 3 weeks. One hundred and eighty days after the last vaccination, mice were challenged intransally with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged with virus as controls. Cohorts of vaccinated virus-challenged mice were treated with isotype control IgG or anti-IL-17A antibodies (intravenously and intranasally) at -1, 0, 1, 3 and 5 days relative to virus challenge. On the 6 th day after viral challenge, viral titers and virus-specific T cell responses were quantified in lungs. (E) Groups of C57BL/6 mice were vaccinated twice with NP protein alone or formulated in various adjuvants. Fifty days after booster vaccination, vaccinated and unvaccinated mice were challenged intranasally with the highly pathogenic H5N1 avian influenza A virus; weight loss and survival was monitored until day 14. Data are pooled from 2 independent experiments or representative of two independent experiments. *, **, and *** indicate significance at P

    Journal: bioRxiv

    Article Title: Programming Multifaceted Pulmonary T-Cell Immunity by Combination Adjuvants

    doi: 10.1101/2020.07.10.197459

    Figure Lengend Snippet: Vaccine-induced protective immunity to H1N1 and H5N1 influenza viruses. (A-C) Groups of C57BL/6 mice were vaccinated twice at 3-week intervals with NP protein formulated in various adjuvants.. (A) At 100 days after the booster vaccination, mice were challenged intranasally with H1N1/PR8 strain of influenza A virus. Viral tiers and virus-specific T cell responses in lungs were quantified on the 6 th day after virus challenge. (A) Viral titers in the lungs on the 6 th day after virus challenge. (B) Percentages of NP366-specific IFN-γ and IL-17 producing cells among CD8 T cells (bar graphs) and calculated proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ+IL-7-producing peptide-stimulated NP366-specific T cells (Pie charts). (C) Percentages of NP311-specific IFN-γ and IL-17 producing cells among CD4 T cells and calculated proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ+IL-7-producing peptide-stimulated NP311-specific T cells. (D) C57BL/6 mice were vaccinated with NP+ADJ+GLA twice at an interval of 3 weeks. One hundred and eighty days after the last vaccination, mice were challenged intransally with H1N1/PR8 strain of influenza A virus; unvaccinated mice were challenged with virus as controls. Cohorts of vaccinated virus-challenged mice were treated with isotype control IgG or anti-IL-17A antibodies (intravenously and intranasally) at -1, 0, 1, 3 and 5 days relative to virus challenge. On the 6 th day after viral challenge, viral titers and virus-specific T cell responses were quantified in lungs. (E) Groups of C57BL/6 mice were vaccinated twice with NP protein alone or formulated in various adjuvants. Fifty days after booster vaccination, vaccinated and unvaccinated mice were challenged intranasally with the highly pathogenic H5N1 avian influenza A virus; weight loss and survival was monitored until day 14. Data are pooled from 2 independent experiments or representative of two independent experiments. *, **, and *** indicate significance at P

    Article Snippet: Recombinant nucleoprotein (NP) of the PR8/H1N1 influenza virus strain was purchased from Sino Biological Inc (Beijing, China).

    Techniques: Mouse Assay

    Kinetics and durability of influenza viral control in vaccinated mice. B6 mice were vaccinated twice (at 3 weeks intervals) intranasally with NP protein formulated with the indicated adjuvants. Unvaccinated mice and mice vaccinated with NP only (without adjuvants) served as controls. At 100 and 180 days after booster vaccination, mice were challenged intranasally with PR8/H1N1 influenza virus. (A) Body weight loss was assessed by calculating bodyweight at different days after challenge, relative to bodyweight before challenge at 100 days after vaccination. (B) Vaccinated mice were challenged with PR8/H1N1at 100 days after vaccination and viral titers in lungs were quantified at day 2 and 4 after challenge, using a plaque assay. (C) At day 180 after booster vaccination, mice were challenged with PR8/H1N1 virus, and viral titers in lungs were assessed at day 6 after challenge. (D) Percentages and numbers of NP366-specific CD8 T cells and NP311-specific CD4 T cells in lungs and percentage of these cells in the vascular and non-vascular compartment at day 6 after challenge (challenged at 100 days after vaccination). *, **, and *** indicate significance at P

    Journal: bioRxiv

    Article Title: Programming Multifaceted Pulmonary T-Cell Immunity by Combination Adjuvants

    doi: 10.1101/2020.07.10.197459

    Figure Lengend Snippet: Kinetics and durability of influenza viral control in vaccinated mice. B6 mice were vaccinated twice (at 3 weeks intervals) intranasally with NP protein formulated with the indicated adjuvants. Unvaccinated mice and mice vaccinated with NP only (without adjuvants) served as controls. At 100 and 180 days after booster vaccination, mice were challenged intranasally with PR8/H1N1 influenza virus. (A) Body weight loss was assessed by calculating bodyweight at different days after challenge, relative to bodyweight before challenge at 100 days after vaccination. (B) Vaccinated mice were challenged with PR8/H1N1at 100 days after vaccination and viral titers in lungs were quantified at day 2 and 4 after challenge, using a plaque assay. (C) At day 180 after booster vaccination, mice were challenged with PR8/H1N1 virus, and viral titers in lungs were assessed at day 6 after challenge. (D) Percentages and numbers of NP366-specific CD8 T cells and NP311-specific CD4 T cells in lungs and percentage of these cells in the vascular and non-vascular compartment at day 6 after challenge (challenged at 100 days after vaccination). *, **, and *** indicate significance at P

    Article Snippet: Recombinant nucleoprotein (NP) of the PR8/H1N1 influenza virus strain was purchased from Sino Biological Inc (Beijing, China).

    Techniques: Mouse Assay, Plaque Assay

    Regulation of vaccine-induced CD8 T-cell memory and protective immunity by CD4 T cells. Groups of C57BL/6 mice were vaccinated twice at 3-week intervals with NP protein formulated in ADJ+GLA. Cohorts of vaccinated mice were treated with isotype control antibodies (Non Depleted) or anti-CD4 antibodies (CD4 Depleted) intravenously and intranasally on days −1, 0, and 1 relative to prime and boost vaccination with NP+ADJ+GLA. T-cell memory in lungs (A-F) and protective immunity to influenza A virus (G-P) was determined at 80 days after booster vaccination. (A-F) T-cell memory in lungs at day 80 after booster vaccination. To stain for vascular cells, mice were injected intravenously with anti-CD45.2 antibodies, 3 minutes prior to euthanasia. Lung cells were stained directly ex vivo with D b /NP366 or I-A b /NP311 tetramers along with the indicated antibodies for cell surface markers. For cytokine analysis, lung cells were stimulated with NP366 or NP311 peptide for 5 hours before intracellular staining. (A) FACS plots are gated on total CD4 T cells and show NP311-specific tetramer-binding memory CD4 T cells only in non-depleted mice. (B) NP366-specific tetramer-binding memory CD8 T cells in lungs of non-d epleted and CD4 T cell-depleted mice. (C) Expression of tissue residency markers on NP366-specific tetramer-binding memory CD8 T cells in lungs. (D) Percentage of vascular (CD45.2 +ve ) and non-vascular (CD45.2 -ve ) cells among NP366-specific tetramer-binding memory CD8 T cells in lungs. (E) Percentages of IFN-γ- and IL-17-producing NP366-specific cells among CD8 T cells in lungs. (F) Calculated proportions of IFN-γ and/or IL-17-producing cells among cytokine-producing peptide-stimulated IFN-γ+IL-17 NP366-specific CD8 T cells. (G-P) At day 80 after booster vaccination, non-depleted and CD4 T cell-depleted mice were challenged intranasally with PR8/H1N1 influenza A virus; recall virus-specific CD8/CD4 T cell responses and viral load in lungs were assessed at day 6 after challenge. (G) Percentages of NP366-specific tetramer-binding cells among CD8 T cells in lungs. (H) Percentages of NP366-specific tetramer-binding CD8 T cells in vascular and nonvascular lung compartment. (I) Percentages of NP311-specific tetramer-binding cells among CD4 T cells in lungs. (J) Expression of tissue residency markers on NP366-specific tetramer-binding CD8 T cells. (K) Chemokine receptor and transcription factor expression in NP366-specific CD8 T cells in lungs. (L) Granzyme B expression by NP366-specific CD8 T cells directly ex vivo. (M) Percentages of IFN-γ and IL-17 producing NP366-specific CD8 T cells. (N) Relative proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ plus IL-17-producing peptide-stimulated NP366-specific CD8 T cells. (O) Viral titers in lungs at day 6 after challenge. (P) Body weight, measured as a percentage of starting body weight prior to challenge. Data are pooled from two independent experiments. *, **, and *** indicate significance at P

    Journal: bioRxiv

    Article Title: Programming Multifaceted Pulmonary T-Cell Immunity by Combination Adjuvants

    doi: 10.1101/2020.07.10.197459

    Figure Lengend Snippet: Regulation of vaccine-induced CD8 T-cell memory and protective immunity by CD4 T cells. Groups of C57BL/6 mice were vaccinated twice at 3-week intervals with NP protein formulated in ADJ+GLA. Cohorts of vaccinated mice were treated with isotype control antibodies (Non Depleted) or anti-CD4 antibodies (CD4 Depleted) intravenously and intranasally on days −1, 0, and 1 relative to prime and boost vaccination with NP+ADJ+GLA. T-cell memory in lungs (A-F) and protective immunity to influenza A virus (G-P) was determined at 80 days after booster vaccination. (A-F) T-cell memory in lungs at day 80 after booster vaccination. To stain for vascular cells, mice were injected intravenously with anti-CD45.2 antibodies, 3 minutes prior to euthanasia. Lung cells were stained directly ex vivo with D b /NP366 or I-A b /NP311 tetramers along with the indicated antibodies for cell surface markers. For cytokine analysis, lung cells were stimulated with NP366 or NP311 peptide for 5 hours before intracellular staining. (A) FACS plots are gated on total CD4 T cells and show NP311-specific tetramer-binding memory CD4 T cells only in non-depleted mice. (B) NP366-specific tetramer-binding memory CD8 T cells in lungs of non-d epleted and CD4 T cell-depleted mice. (C) Expression of tissue residency markers on NP366-specific tetramer-binding memory CD8 T cells in lungs. (D) Percentage of vascular (CD45.2 +ve ) and non-vascular (CD45.2 -ve ) cells among NP366-specific tetramer-binding memory CD8 T cells in lungs. (E) Percentages of IFN-γ- and IL-17-producing NP366-specific cells among CD8 T cells in lungs. (F) Calculated proportions of IFN-γ and/or IL-17-producing cells among cytokine-producing peptide-stimulated IFN-γ+IL-17 NP366-specific CD8 T cells. (G-P) At day 80 after booster vaccination, non-depleted and CD4 T cell-depleted mice were challenged intranasally with PR8/H1N1 influenza A virus; recall virus-specific CD8/CD4 T cell responses and viral load in lungs were assessed at day 6 after challenge. (G) Percentages of NP366-specific tetramer-binding cells among CD8 T cells in lungs. (H) Percentages of NP366-specific tetramer-binding CD8 T cells in vascular and nonvascular lung compartment. (I) Percentages of NP311-specific tetramer-binding cells among CD4 T cells in lungs. (J) Expression of tissue residency markers on NP366-specific tetramer-binding CD8 T cells. (K) Chemokine receptor and transcription factor expression in NP366-specific CD8 T cells in lungs. (L) Granzyme B expression by NP366-specific CD8 T cells directly ex vivo. (M) Percentages of IFN-γ and IL-17 producing NP366-specific CD8 T cells. (N) Relative proportions of IFN-γ and/or IL-17 producing cells among total IFN-γ plus IL-17-producing peptide-stimulated NP366-specific CD8 T cells. (O) Viral titers in lungs at day 6 after challenge. (P) Body weight, measured as a percentage of starting body weight prior to challenge. Data are pooled from two independent experiments. *, **, and *** indicate significance at P

    Article Snippet: Recombinant nucleoprotein (NP) of the PR8/H1N1 influenza virus strain was purchased from Sino Biological Inc (Beijing, China).

    Techniques: Mouse Assay, Staining, Injection, Ex Vivo, FACS, Binding Assay, Expressing