Journal: PLoS ONE

Article Title: Hydrophobin Fusion of an Influenza Virus Hemagglutinin Allows High Transient Expression in Nicotiana benthamiana , Easy Purification and Immune Response with Neutralizing Activity

doi: 10.1371/journal.pone.0115944

Figure Lengend Snippet: Wild-type cells (a), cells expressing H1 (b) and cells expressing H1-HFBI (c–e) at the exponential phase (3 days after dilution) were submitted to in situ immunolocalization as described in the using an FITC-conjugated anti-influenza H1N1. Bars = 25 µm (a, b, c) and 5 µm (d, e).

Article Snippet: To obtain a rough estimation of the expression level of recombinant H1 and H1-HFBI proteins, an immunoblotting technique was applied using the extracellular domain of a recombinant Influenza A/Texas/05/2009(H1N1) hemagglutinin produced in human cells (11085-V08H, Sino Biologicals, China) as a standard.

Techniques: Expressing, In Situ

Journal: PLoS ONE

Article Title: Hydrophobin Fusion of an Influenza Virus Hemagglutinin Allows High Transient Expression in Nicotiana benthamiana , Easy Purification and Immune Response with Neutralizing Activity

doi: 10.1371/journal.pone.0115944

Figure Lengend Snippet: (a) Hemagglutination assay was performed as indicated in Material and methods using serial two-fold diluted samples of duplicate (R1, R2) TSP fractions extracted from leaves expressing H1-HFBI or untagged H1. The two bottom rows contain inactivated A/Texas/05/2009(H1N1) virus as a positive control or a GFP-HFBI extract as a negative control. (b) Hemagglutination assay using serial two-fold diluted samples of ATPS-purified H1-HFBI (triplicates, R1–R3). The two bottom rows contain bovine serum albumin as a negative control or inactivated A/Texas/05/2009(H1N1) virus as a positive control. The hemagglutination titer (HT) or the amount of hemagglutination units (HAU) was calculated according to the well with the highest dilution giving a complete hemagglutination. This test was also used to quantify inactivated virus concentration in terms of HAU for inhibition assay.

Article Snippet: To obtain a rough estimation of the expression level of recombinant H1 and H1-HFBI proteins, an immunoblotting technique was applied using the extracellular domain of a recombinant Influenza A/Texas/05/2009(H1N1) hemagglutinin produced in human cells (11085-V08H, Sino Biologicals, China) as a standard.

Techniques: Hemagglutination Assay, Expressing, Positive Control, Negative Control, Purification, Concentration Assay, Inhibition

Journal: PLoS ONE

Article Title: Hydrophobin Fusion of an Influenza Virus Hemagglutinin Allows High Transient Expression in Nicotiana benthamiana , Easy Purification and Immune Response with Neutralizing Activity

doi: 10.1371/journal.pone.0115944

Figure Lengend Snippet: (a) ATPS-purified H1-HFBI (200 µg) was injected onto a Superdex G200 size exclusion column as described in the . Elution was fractionated in 1 ml aliquots. The logarithmic size of the standards (black square) is plotted according to their elution volume and peaks corresponding to H1-HFBI are indicated (red dot). (b) Fractions eluted at 14 ml to 18 ml and 27 ml to 31 ml were analyzed by Western blotting with an anti-influenza H1N1 and an anti-goat HRP-conjugated secondary antibody, and the signal was quantified using the Kodak image station 4000R (Arbitrary unit; <: below detection level).

Article Snippet: To obtain a rough estimation of the expression level of recombinant H1 and H1-HFBI proteins, an immunoblotting technique was applied using the extracellular domain of a recombinant Influenza A/Texas/05/2009(H1N1) hemagglutinin produced in human cells (11085-V08H, Sino Biologicals, China) as a standard.

Techniques: Purification, Injection, Western Blot

Journal: PLoS ONE

Article Title: Hydrophobin Fusion of an Influenza Virus Hemagglutinin Allows High Transient Expression in Nicotiana benthamiana , Easy Purification and Immune Response with Neutralizing Activity

doi: 10.1371/journal.pone.0115944

Figure Lengend Snippet: (a) Ten mice were immunized with H1-HFBI as indicated in the . Anti-HA antibodies were assayed by ELISA in the pre-immune sera (open circle) and the sera collected after the 4 th (blue triangle) and 6 th (red square) boost. Plates were coated with 5 µg/ml of recombinant Influenza A/Texas/05/2009(H1N1) ectodomain expressed in mammalian cells (Sino Biologicals, 11085-V08H). HRP-conjugated anti-mouse secondary antibody was used for detection. HA titer was calculated as the highest dilution giving a signal higher than three times the signal coming from the negative control. (b) Box and whisker analysis of antibody titers obtained after endpoint ELISA titer analysis of the test groups. Each dot represents the antibody titer from an individual mouse. (p-values = 0.18 (boost 4/boost 6), 1.8.10 −4 (boost 4/pre-immune), 3.8.10 −4 (boost 6/pre-immune))

Article Snippet: To obtain a rough estimation of the expression level of recombinant H1 and H1-HFBI proteins, an immunoblotting technique was applied using the extracellular domain of a recombinant Influenza A/Texas/05/2009(H1N1) hemagglutinin produced in human cells (11085-V08H, Sino Biologicals, China) as a standard.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Negative Control, Whisker Assay

Journal: Frontiers in Immunology

Article Title: Vaccine-induced neutralizing antibody responses to seasonal influenza virus H1N1 strains are not enhanced during subsequent pandemic H1N1 infection

doi: 10.3389/fimmu.2023.1256094

Figure Lengend Snippet: Study protocol. Animals were immunized in weeks 0, 6, 12, and 21 (indicated by blue bar) with VLPs displaying HA of seasonal influenza A (H1N1) virus strains. Animals received monovalent VLPs displaying HA from a single HA variant (influenza A/Brisbane/59/2007; monovalent vaccine group) or pentavalent VLPs displaying a combination of HA from five different variants: A/Puerto Rico/8/34 (PR8/34), A/USSR/92/77, A/Texas/36/91, A/New Caledonian/20/99 (NC/20/99), A/Brisbane/59/07 (Bris/59/07) (pentavalent vaccine group). Antibody responses were measured 4 weeks after the first, second, and third immunization procedures (weeks 4, 10, and 16) and 3 weeks after the fourth immunization procedure (week 24). T cell responses were measured 2 weeks after the first, second, and third immunization procedures (weeks 2, 8, and 14). All immunized animals and 4 naive control animals were challenged (indicated in red) in week 25 with pandemic influenza A/California/04/2009 virus (Cal/04/09). Blood, nasal swabs, and throat swabs were collected on days 1, 2, 4, 6, 8, 10, 14, and 21 after infection; BAL fluid was collected on days 6 and 14 to monitor virus replication.

Article Snippet: The wells of a 96-well ELISA plate (Maxisorp, Nunc) were coated overnight at 4°C with 400 ng/well purified HA protein (influenza A/California/07/2009 (H1N1), Sino Biological Inc.) in PBS.

Techniques: Virus, Variant Assay, Control, Infection

Journal: Frontiers in Immunology

Article Title: Vaccine-induced neutralizing antibody responses to seasonal influenza virus H1N1 strains are not enhanced during subsequent pandemic H1N1 infection

doi: 10.3389/fimmu.2023.1256094

Figure Lengend Snippet: Antibody responses after challenge with influenza A/California/04/09 (H1N1pdm09). In all graphs, the antibody levels measured 3 weeks after the fourth immunization procedure are included for comparison (Imm). (A) IgG antibody responses to HA protein of Cal/07/09 measured 10 and 56 days after challenge (Ch d10 and Ch d56, respectively) among animals in the monovalent vaccine group (black circles), pentavalent vaccine group (black squares), and naive control group (white triangles). Antibody levels are expressed as arbitrary units (AU), defined as the dilution where the OD 450 value is 1 unit above background. Gray area indicates the average response observed in pre-immunization serum samples (157 AU). (B) Neutralizing antibody titers measured 10 and 56 days after challenge against Cal/04/09 virus. Symbols as indicated for panel (A) . Neutralizing antibody titers measured against H1N1 vaccine strains PR/8/34, USSR/92/77, Texas/36/91, NC/20/99, and Bris/59/07 on day 56 after challenge among animals in the monovalent vaccine group (C) and pentavalent vaccine group (D) . (E) Neutralizing antibody titers 10 days after challenge measured against influenza A/Swine/Iowa/15/30 (H1N1). (F) Neutralizing antibody titers 10 days after challenge measured against influenza A/New Jersey/8/76 (H1N1). Gray area in graphs (B–F) indicates lowest dilution tested. Significant differences between groups were determined using the Mann–Whitney test. Significant differences between pre- and post-challenge responses were determined using the paired Wilcoxon test. *p<0.05.

Article Snippet: The wells of a 96-well ELISA plate (Maxisorp, Nunc) were coated overnight at 4°C with 400 ng/well purified HA protein (influenza A/California/07/2009 (H1N1), Sino Biological Inc.) in PBS.

Techniques: Comparison, Control, Virus, MANN-WHITNEY