Single domain antibodies targeting neuraminidase protect against an H5N1 influenza virus challenge

Influenza virus neuraminidase (NA) is an interesting target of small-molecule antiviral drugs. We isolated a set of H5N1 NAspecific single-domain antibodies (N1-VHHm) and evaluated their in vitro and in vivo antiviral potential. Two of them inhibited the NA activity and in vitro replication of clade 1 and 2 H5N1 viruses. We then generated bivalent derivatives of N1-VHHm by two methods. First, we made N1-VHHb by genetically joining two N1-VHHm moieties with a flexible linker. Second, bivalent N1-VHH-Fc proteins were obtained by genetic fusion of the N1-VHHm moiety with the crystallizable region of mouse IgG2a (Fc). The in vitro antiviral potency against H5N1 of both bivalent N1-VHHb formats was 30- to 240-fold higher than that of their monovalent counterparts, with 50% inhibitory concentrations in the low nanomolar range. Moreover, single-dose prophylactic treatment with bivalent N1-VHHb or N1-VHH-Fc protected BALB/c mice against a lethal challenge with H5N1 virus, including an oseltamivir-resistant H5N1 variant. Surprisingly, an N1-VHH-Fc fusion without in vitro NA-inhibitory or antiviral activity also protected mice against an H5N1 challenge. Virus escape selection experiments indicated that one amino acid residue close to the catalytic site is required for N1-VHHm binding. We conclude that single-domain antibodies directed against influenza virus NA protect against H5N1 virus infection, and when engineered with a conventional Fc domain, they can do so in the absence of detectable NA-inhibitory activity.Fil: Cardoso, Francisco Miguel. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Ibañez, Lorena Itatí. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencias y Tecnología "Dr. Cesar Milstein"; Argentina. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Van Den Hoecke, Silvie. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: De Baets, Sarah. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Smet, Anouk. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Roose, Kenny. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Schepens, Bert. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Descamps, Francis J.. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Fiers, Walter. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; BélgicaFil: Muyldermans, Serge. Structural Biology Research Center; Bélgica. Vrije Universiteit Brussel. Laboratory of Cellular and Molecular Immunology; BélgicaFil: Depicker, Ann. VIB. Department of Plant Systems Biology; Bélgica. Department of Biotechnology and Bioinformatics; BélgicaFil: Saelens, Xavier. VIB Inflammation Research Center; Bélgica. Ghent University. Department for Biomedical Molecular Biology; Bélgic

Cell Culture-Induced Gradual and Frequent Epigenetic Reprogramming of Invertedly Repeated Tobacco Transgene Epialleles1[W]

Using a two-component transgene system involving two epiallelic variants of the invertedly repeated transgenes in locus 1 (Lo1) and a homologous single-copy transgene locus 2 (Lo2), we have studied the stability of the methylation patterns and trans-silencing interactions in cell culture and regenerated tobacco (Nicotiana tabacum) plants. The posttranscriptionally silenced (PTGS) epiallele of the Lo1 trans-silences and trans-methylates the target Lo2 in a hybrid (Lo1/Lo2 line), while its transcriptionally silenced variant (Lo1E) does not. This pattern was stable over several generations in plants. However, in early Lo1E/Lo2 callus, decreased transgene expression and partial loss of Lo1E promoter methylation compared with leaf tissue in the parental plant were observed. Analysis of small RNA species and coding region methylation suggested that the transgenes were silenced by a PTGS mechanism. The Lo1/Lo2 line remained silenced, but the nonmethylated Lo1 promoter acquired partial methylation in later callus stages. These data indicate that a cell culture process has brought both epialleles to a similar epigenetic ground. Bisulfite sequencing of the 35S promoter within the Lo1 silencer revealed molecules with no, intermediate, and high levels of methylation, demonstrating, to our knowledge for the first time, cell-to-cell methylation diversity of callus. Regenerated plants showed high interindividual but low intraindividual epigenetic variability, indicating that the callus-induced epiallelic variants were transmitted to plants and became fixed. We propose that epigenetic changes associated with dedifferentiation might influence regulatory pathways mediated by trans-PTGS processes

T-DNA Integration in Arabidopsis Chromosomes. Presence and Origin of Filler DNA Sequences

To investigate the relationship between T-DNA integration and double-stranded break (DSB) repair in Arabidopsis, we studied 67 T-DNA/plant DNA junctions and 13 T-DNA/T-DNA junctions derived from transgenic plants. Three different types of T-DNA-associated joining could be distinguished. A minority of T-DNA/plant DNA junctions were joined by a simple ligation-like mechanism, resulting in a junction without microhomology or filler DNA insertions. For about one-half of all analyzed junctions, joining of the two ends occurred without insertion of filler sequences. For these junctions, microhomology was strikingly combined with deletions of the T-DNA ends. For the remaining plant DNA/T-DNA junctions, up to 51-bp-long filler sequences were present between plant DNA and T-DNA contiguous sequences. These filler segments are built from several short sequence motifs, identical to sequence blocks that occur in the T-DNA ends and/or the plant DNA close to the integration site. Mutual microhomologies among the sequence motifs that constitute a filler segment were frequently observed. When T-DNA integration and DSB repair were compared, the most conspicuous difference was the frequency and the structural organization of the filler insertions. In Arabidopsis, no filler insertions were found at DSB repair junctions. In maize (Zea mays) and tobacco (Nicotiana tabacum), DSB repair-associated filler was normally composed of simple, uninterrupted sequence blocks. Thus, although DSB repair and T-DNA integration are probably closely related, both mechanisms have some exclusive and specific characteristics