Supplementary MaterialsTable S1: Sera from healthy human individuals from different geographical areas in Europe and the United States had been screened previously for neutralizing activity to Ad5 . strain BJ5183 by co-transformation with PanAd3 purified viral DNA and a PanAd3-EGFP shuttle vector. Homologous recombination 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 pPanAd3E1-EGFP. The E3 region (nucleotides 28684 to 32640) was then deleted through several cloning and homologous recombination steps to generate the pPanAd3E1E3 backbone, which was propagated in HEK 293 cells. Expression Procyanidin B3 cost 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 ITM2A 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 6C8 (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 ( em class=”gene” GGGSGGG /em ). The resulting NPM1 sequence was codon-optimized 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 PanAd3E1E3 backbone via homologous recombination in em E.coli /em . Sequences for HIV gag protein or a respiratory syncytial virus (RSV) fusion protein of F protein, nucleoprotein N and transcription factor M2-1 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 pPanAd3E1E3-EGFP plasmid by homologous recombination in em E. coli /em , 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  and obtained from Frank Graham at MacMaster University (Hamilton, Canada), and further adapted at Okairs 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. . Briefly, HEK 293 cells were transfected with an expression Procyanidin B3 cost 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/ml G418 in culture medium. Single clones were amplified and tested Procyanidin B3 cost for Tet repressor expression by Western Blot analysis. The stability of Tet repressor expression in the selected clone was tested up to passage 63. PanAd3 vectors grown in these cells were purified by cesium chloride gradients and stored in buffer A195 . Open in a separate window Procyanidin B3 cost Figure 1 NPM1 fusion protein insert.a) Design of the insert showing CMV promoter, NPM1 transgene, and BGH-polyadenylation cassettes. b) Complete amino acid sequence of the consensus NPM1 fusion protein. NP is indicated in red, linker sequence Procyanidin B3 cost is shown in black, and M1 is green. The deletion of the nuclear localization signal by mutation of TKR to AAA in NP is indicated in bold text. Viral particle (vp) measurements of adenovirus stocks were made by measurement of absorbance at 260 nm as described . Peptides and proteins Peptides NP147C155 (TYQRTRALV) and SARS M209C221 (HAGSNDNIALLVQ) were synthesized by the CBER core facility. An MHC-I restricted peptide of adenovirus DNA-binding protein (Dbp419C427: FALSNAEDL), present in PanAd3  and recombinant M1 (rM1) protein from strain.