h13  (Sino Biological)

Journal: EMBO Molecular Medicine

Article Title: Adeno‐associated virus‐vectored influenza vaccine elicits neutralizing and Fcγ receptor‐activating antibodies

doi: 10.15252/emmm.201910938

Figure Lengend Snippet: Induction of distinct antibody profiles after vaccination with AAV ‐vectored vaccines or WIV Phylogenetic tree of the complete HA sequences of the four H1N1 viruses used for immunoblot analysis with mouse serum. The table shows the amino acid identities of the H1N1 viruses HA1 or HA2 subdomain compared to Cal/7/9 HA1 and HA2, respectively, as determined with Geneious 11.1.5 software. Quantification of immunoblot data as shown in Fig 3 A. Intensities of bands were analyzed using ImageJ. Dots indicate individual experiments, bars mean ± SE ( n = 3). Numbers in the AAV‐HA panel indicate fold‐change of signal of the HA2 band of the AAV‐HA compared to the AAV‐cHA group. In‐cell ELISA with transfected MDCKII cells expressing either Cal/7/9 full‐length HA (pAAV‐HA), the HA‐stalk (pAAV‐mHL1 + transmembrane region), or chimeric HA consisting of the Cal/7/9 HA stalk and the H13 HA head (pcHA3). Detection of the HA constructs was done with indicated mouse serum at a 1:250 dilution or with the conformational stalk antibody C179. Dots indicate individual experiments, bars mean ± SE ( n = 3, technical triplicates). Numbers indicated the fold‐change of signal between AAV‐HA and AAV‐cHA groups tested against the indicated HA construct. Results of 15‐mer Cal/7/9 peptide screen with AAV‐HA pooled pre‐challenge serum ( n = 18 mice). Peptides were coated in 96‐well plates, incubated with serum before binding was detected with a HRP‐coupled secondary antibody. Data are shown as fold induction over AAV‐GFP signal intensity ( n = 3, technical duplicates). Statistical significance between each peptide signal and the baseline value 1 (dotted line) was determined using one‐sample t ‐test (* P

Article Snippet: Cells were fixed, permeabilized, blocked, and immunostained with anti‐V5‐tag (1:250; SV5‐Pk1; Bio‐Rad, München, Germany), C179 (1:250; M145; TaKaRa, Kusatsu, Japan) antibody, rabbit hyperimmune serum against the influenza subtypes H2, H10, and H13 (1:500; 11688‐RP01‐100, 11693‐RP01‐100, 11721‐RP01‐100 Sino Biological Inc., Eching, Germany), or animal serum followed by suitable secondary antibody coupled to AlexaFluor488 (1:1,000; Thermo Fisher Scientific).

Techniques: Software, In-Cell ELISA, Transfection, Expressing, Construct, Mouse Assay, Incubation, Binding Assay

Journal: EMBO Molecular Medicine

Article Title: Adeno‐associated virus‐vectored influenza vaccine elicits neutralizing and Fcγ receptor‐activating antibodies

doi: 10.15252/emmm.201910938

Figure Lengend Snippet: (H1N1)pdm‐based AAV ‐vectored antigens are strongly expressed in vitro 3D structure of a HA trimer (PDB 3UBE generated with PyMol). Each monomer consists of a HA1 (dark gray) and HA2 (light gray) subunit. The trimer can be divided into a membrane distal head which contains the RBS (yellow) and a proximal stalk region. HA and NP represent the Cal/7/9 wild‐type proteins. Chimeric HA (cHA) 1 contains the head regions of H2 HA, cHA2 of H10 HA, and cHA3 of H13 HA, while they all contain the Cal/7/9 HA‐stalk region. Headless HA (HL) contains a deletion in the HA‐head region (dashed line). Modified headless HA (mHL1 and mHL2) contain stabilizing mutations (black boxes) and lack additional internal parts. All constructs were codon‐optimized and carry a V5‐tag at their C‐terminus. Frequency of C179 + or V5‐tag + 293T cells 24 h after transfection of AAV vector plasmids as measured by flow cytometry. Symbols represent single experiments and bars the mean ± SE ( n = 3). Immunoblot of 293T cells 72 h after transduction with AAV vectors at a MOI of 10 6 . Antigen expression was detected with an anti‐V5‐tag antibody. Equal loading was controlled with a GAPDH antibody ( n = 3). Source data are available online for this figure.

Article Snippet: Cells were fixed, permeabilized, blocked, and immunostained with anti‐V5‐tag (1:250; SV5‐Pk1; Bio‐Rad, München, Germany), C179 (1:250; M145; TaKaRa, Kusatsu, Japan) antibody, rabbit hyperimmune serum against the influenza subtypes H2, H10, and H13 (1:500; 11688‐RP01‐100, 11693‐RP01‐100, 11721‐RP01‐100 Sino Biological Inc., Eching, Germany), or animal serum followed by suitable secondary antibody coupled to AlexaFluor488 (1:1,000; Thermo Fisher Scientific).

Techniques: In Vitro, Generated, Modification, Construct, Transfection, Plasmid Preparation, Flow Cytometry, Transduction, Expressing