rga/vietnam/1203/04 x a/pr/8/34  (Sanofi)

Journal: Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

Article Title: Wide Prevalence of Heterosubtypic Broadly Neutralizing Human Anti–Influenza A Antibodies

doi: 10.1093/cid/cir121

Figure Lengend Snippet: Serological study of 77 paired pre- and postimmune serum samples from H5N1 vaccinees. A–D, Enzyme-linked immunosorbent assays (ELISAs). Serum samples were serially diluted and applied to H5-VN04– (A and B), H3-A2/68– (C), or H7-NL03– (D) coated ELISA plates and the binding of immunoglobulin (Ig) M or IgG in serum samples to hemagglutinin (HA) proteins were determined using secondary Ab HRP-anti-human IgM (A) or IgG (B - –D). The binding levels are shown as optical density at 450 nm (OD 450). E, microneutralization assay (MN) titer against H5N1 virus; y-axis shows the Log 2 MN titer. F, Competition ELISA. Pre- and post-immune serum samples from H5N1 vaccinees were tested for their competition activity against a Group 1-specific BnAb, F10, binding to H5-VN04. Serially diluted serum samples were mixed with 3 ng/mL Bio-F10 and applied to H5-coated ELISA plates. The serum competition for binding of Bio-F10 to H5 was determined by measuring the remaining binding of Bio-F10 using HRP-Streptavidin. The serum competition activity is shown as percentage of inhibition. For all panels except panel E, data at 1 representative serum dilution are shown, as follows: panel A, 1:270; panel B, 1:5120; panels C and D, 1:2430; and panel F, 1:90. For all panels, data are shown in a box and whiskers graph. The box extends from 25th percentile to the 75th percentile, with a line at the median. The whiskers above and below the box indicate the 95th and 5th percentiles, respectively. The dots above and below the whiskers are data points beyond the 95th and 5th percentiles.

Article Snippet: The vaccine (manufactured by Sanofi Pasteur) used in the trial was a monovalent, inactivated subvirion H5N1 vaccine (rgA/ Vietnam/1203/04 X A/PR/8/34).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Microneutralization Assay, Virus, Activity Assay, Inhibition

Journal: PLoS ONE

Article Title: A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells

doi: 10.1371/journal.pone.0017297

Figure Lengend Snippet: (A) To enhance HA protein expression from the FRT-CMV vector (Invitrogen), an RNA splicing sequence from HTLV-I gene and a cassette containing Not I and Pac I cloning sites was introduced after CMV promoter using Topo cloning system. (B) Schematic of HA1 (1-330) & HA0 (1-500) V5-His 6 tagged fusion proteins expressed in Flp-In system. The H5N1/Vietnam HA sequence coding for either HA1 (1-330) or HA0 (1-500) was inserted into the vector as a Not I -Pac I insert. (C–D) Expression and purification of H5N1 A/Vietnam/1203/2004 hemagglutinin proteins from 293 Flp-In stable cell lines. (C) Expression of HA proteins secreted into supernatant from 293 Flp-In cells [S], and in the cell lysates [C] was analyzed by western blot using anti-V5 MAb. (D) HA protein level from supernatant of 293 Flp-In cell culture in serum free medium, collected at different time points: 24, 48, 72 and 96 hours post culture splitting and was analyzed in SDS PAGE followed by western blot with anti-HA1 polyclonal sera.

Article Snippet: Inactivated subvirion H5N1 vaccine (rgA/Vietnam/1203/04 X A/PR/8/34) that contains 90 μg/ml HA was used as a vaccine ( Sanofi Pasteur ).

Techniques: Expressing, Plasmid Preparation, Sequencing, Cloning, Purification, Stable Transfection, Western Blot, Cell Culture, SDS Page

Journal: PLoS ONE

Article Title: A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells

doi: 10.1371/journal.pone.0017297

Figure Lengend Snippet: (A) Proper protein folding as demonstrated by steady-state binding equilibrium analysis of conformational dependent human H5N1 neutralizing MAb FLA5.10 (10 µg/ml) to purified Flp-In expressed H5N1 HA1 proteins immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. Purified mammalian cell derived H5N1 HA1 or the HA0 proteins obtained from Immune Technology Corp were also analyzed. Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA). (B–C) Analysis of purified H5N1 protein from Flp-In cells in SDS-PAGE under reducing conditions (B) and non-reducing conditions (C). Purified HA protein from Flp-In cell has higher order protein structure as analyzed by coomassie staining of the reducing SDS PAGE (B) and by coomassie stained non-reducing SDS PAGE (C). Subunit H5N1 vaccine (Sanofi Pasteur) was run as comparator. Western blot analysis of non-reducing SDS PAG using an anti-H5N1 HA1 antibody confirmed the identity of bands observed in coomassie stained gel in .

Article Snippet: Inactivated subvirion H5N1 vaccine (rgA/Vietnam/1203/04 X A/PR/8/34) that contains 90 μg/ml HA was used as a vaccine ( Sanofi Pasteur ).

Techniques: Binding Assay, Purification, Derivative Assay, SPR Assay, SDS Page, Staining, Western Blot

Journal: PLoS ONE

Article Title: A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells

doi: 10.1371/journal.pone.0017297

Figure Lengend Snippet: Superdex S-200 gel filtration chromatography of Flp-In derived H5N1 HA proteins. Purified H5N1 HA1 (1-330) protein (A) or the HA0 (1-500) protein (B) were subjected to gel filtration. The panels present superimposed elution profiles of purified HA proteins (blue line) overlaid with calibration standards (grey line). The elution volumes of protein species are shown in parenthesis. While purified HA 1-330 protein was presented in monomer, trimer and oligomer form (A), the HA1-500 protein was observed primarily in a monomeric form (B). Gel filtration profile of Subunit H5N1 vaccine (Sanofi Pasteur) (C) show predominance of oligomers.

Article Snippet: Inactivated subvirion H5N1 vaccine (rgA/Vietnam/1203/04 X A/PR/8/34) that contains 90 μg/ml HA was used as a vaccine ( Sanofi Pasteur ).

Techniques: Filtration, Chromatography, Derivative Assay, Purification

Journal: PLoS ONE

Article Title: A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells

doi: 10.1371/journal.pone.0017297

Figure Lengend Snippet: (A–D) Antibody kinetics following H5N1 HA1 & HA0 vaccination in rabbits. Steady-state binding equilibrium analysis of pre & post-H5N1 HA1 immune sera (rabbit K8) in (A–B) or pre- & post-H5N1 HA0 immune sera (rabbit K9) in (C–D) to Flp-In derived H5N1 HA1 and HA0 proteins were measured using SPR. Ten-fold diluted individual post-vaccinated sera from each time point, were injected simultaneously onto recombinant Flp-In H5N1 HA1 in (A and C) and H5N1 HA0 in (B and D), immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA). (E) Flp-In HA1 and HA0 elicit high cross-neutralizing antibody titers against homologous and heterologous H5N1 viruses. Animals were immunized with 100 mg proteins mixed every three weeks. Sera was collected at 8 th day after each vaccination and analyzed in a microneutralization assay against various H5N1 virus strains. Data is representative of three experiments.

Article Snippet: Inactivated subvirion H5N1 vaccine (rgA/Vietnam/1203/04 X A/PR/8/34) that contains 90 μg/ml HA was used as a vaccine ( Sanofi Pasteur ).

Techniques: Binding Assay, Derivative Assay, Injection, Recombinant, SPR Assay, Microneutralization Assay, Virus

Journal: PLoS ONE

Article Title: A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells

doi: 10.1371/journal.pone.0017297

Figure Lengend Snippet: Mean Reciprocal Neutralizing Titers of Post-H5n1 Human Sera.

Article Snippet: Inactivated subvirion H5N1 vaccine (rgA/Vietnam/1203/04 X A/PR/8/34) that contains 90 μg/ml HA was used as a vaccine ( Sanofi Pasteur ).

Techniques: