Nation between pIX genes, right ITR DNA sequences present at the ends of linearized PanAd3-EGFP shuttle and viral genomic DNA allowed its insertion in the plasmid vector, simultaneously replacing the E1 region with a human cytomegalovirus (HCMV) promoter-driven EGFP Title Loaded From File expression cassette containing the bovine growth hormone polyadenylation signal (BGH polyA), generating pPanAd3DE1-EGFP. The E3 region (nucleotides 28684 to 32640) was then deleted through several cloning and homologous recombination steps to generate the pPanAd3DE1DE3 backbone, which was propagated in HEK 293 cells. Expression cassettes containing consensus sequences of NP and M1 plus the human cytomegalovirus promoter and bovine growth hormone polyadenylation signal were constructed. The influenza expression cassette contains consensus sequences of NP and M1. Influenza A NP and M1 sequences were obtained from the NCBI Influenza Virus Resource database (http://www.ncbi.nlm.nih. gov/genomes/FLU/FLU.html). Protein sequences were chosen from among different subtype strains isolated between 1990 and 2009 that caused infection in humans worldwide. The NP consensus sequence was derived by alignment of all non-identical sequences (H1N1: 88 of 629 sequences, H1N2: 5 of 26, H3N2: 244 of 1557, H5N1: 50 of 121) using MUSCLE version 3.6, and applying the majority rule. Further, the NP sequence used in the PanAd3 vaccine lacks the Nuclear Localization Signal residing in aa 6? (TKR mutated to AAA), which results in increased cytoplasmic accumulation. The M1 consensus sequence was similarly derived by the alignment of non-identical sequences (H1N1: 51 of 808 sequences, H1N2: 3 of 34, H3N2: 115 of 2150, H5N1: 23 of 145). NP and M1 sequences were spaced by a flexible linker (GGGSGGG). The resulting NPM1 sequence was codonoptimized for expression in eukaryotic cells. A diagram of the insert and its full sequence are given in Figure 1. The NPM1 expression cassette was inserted into the PanAd3DE1DE3 backbone via homologous recombination in E.coli. Sequences for HIV gag protein or a Title Loaded From File respiratory syncytial virus (RSV) fusion protein of F protein, nucleoprotein N and transcription factor M21 were inserted in constructs to be used as specificity controls. Expression cassettes were inserted into a pNEB shuttle vector and then transferred into the SnaBI linearized pPanAd3DE1DE3EGFP plasmid by homologous recombination in E. coli, exploiting the homology between the HCMV promoter and BGH polyA sequences. The PanAd3 vectors were produced in Procell 92 cells, which were derived from the HEK 293 cell line originally banked at the University of Leiden in 1973 [36] and obtained from Frank Graham at MacMaster University (Hamilton, Canada), and further adapted at Okairos to be suitable for manufacturing by ` incorporation of a plasmid carrying a Tet repressor expression cassette and G418-resistance gene. The protocol for generating the Procell 92 cell line followed essentially that published by Matthews et al. [37]. Briefly, HEK 293 cells were transfected with an expression vector containing a cassette encoding the Tet repressor under control of the human phosphoglycerate kinase-1 (PGK) promoter, and the G418-resistance gene. Single clones were selected by growing the transfected cells in the presence of 1 mg/Highly Immunogenic Simian Adenovirus VectorFigure 1. NPM1 fusion protein insert. a) Design of the insert showing CMV promoter, NPM1 transgene, and BGH-polyadenylation cassettes. b) Complete amino acid se.Nation between pIX genes, right ITR DNA sequences present at the ends of linearized PanAd3-EGFP shuttle and viral genomic DNA allowed its insertion in the plasmid vector, simultaneously replacing the E1 region with a human cytomegalovirus (HCMV) promoter-driven EGFP expression cassette containing the bovine growth hormone polyadenylation signal (BGH polyA), generating pPanAd3DE1-EGFP. The E3 region (nucleotides 28684 to 32640) was then deleted through several cloning and homologous recombination steps to generate the pPanAd3DE1DE3 backbone, which was propagated in HEK 293 cells. Expression cassettes containing consensus sequences of NP and M1 plus the human cytomegalovirus promoter and bovine growth hormone polyadenylation signal were constructed. The influenza expression cassette contains consensus sequences of NP and M1. Influenza A NP and M1 sequences were obtained from the NCBI Influenza Virus Resource database (http://www.ncbi.nlm.nih. gov/genomes/FLU/FLU.html). Protein sequences were chosen from among different subtype strains isolated between 1990 and 2009 that caused infection in humans worldwide. The NP consensus sequence was derived by alignment of all non-identical sequences (H1N1: 88 of 629 sequences, H1N2: 5 of 26, H3N2: 244 of 1557, H5N1: 50 of 121) using MUSCLE version 3.6, and applying the majority rule. Further, the NP sequence used in the PanAd3 vaccine lacks the Nuclear Localization Signal residing in aa 6? (TKR mutated to AAA), which results in increased cytoplasmic accumulation. The M1 consensus sequence was similarly derived by the alignment of non-identical sequences (H1N1: 51 of 808 sequences, H1N2: 3 of 34, H3N2: 115 of 2150, H5N1: 23 of 145). NP and M1 sequences were spaced by a flexible linker (GGGSGGG). The resulting NPM1 sequence was codonoptimized for expression in eukaryotic cells. A diagram of the insert and its full sequence are given in Figure 1. The NPM1 expression cassette was inserted into the PanAd3DE1DE3 backbone via homologous recombination in E.coli. Sequences for HIV gag protein or a respiratory syncytial virus (RSV) fusion protein of F protein, nucleoprotein N and transcription factor M21 were inserted in constructs to be used as specificity controls. Expression cassettes were inserted into a pNEB shuttle vector and then transferred into the SnaBI linearized pPanAd3DE1DE3EGFP plasmid by homologous recombination in E. coli, exploiting the homology between the HCMV promoter and BGH polyA sequences. The PanAd3 vectors were produced in Procell 92 cells, which were derived from the HEK 293 cell line originally banked at the University of Leiden in 1973 [36] and obtained from Frank Graham at MacMaster University (Hamilton, Canada), and further adapted at Okairos to be suitable for manufacturing by ` incorporation of a plasmid carrying a Tet repressor expression cassette and G418-resistance gene. The protocol for generating the Procell 92 cell line followed essentially that published by Matthews et al. [37]. Briefly, HEK 293 cells were transfected with an expression vector containing a cassette encoding the Tet repressor under control of the human phosphoglycerate kinase-1 (PGK) promoter, and the G418-resistance gene. Single clones were selected by growing the transfected cells in the presence of 1 mg/Highly Immunogenic Simian Adenovirus VectorFigure 1. NPM1 fusion protein insert. a) Design of the insert showing CMV promoter, NPM1 transgene, and BGH-polyadenylation cassettes. b) Complete amino acid se.
