Peptide panning A phagemidic 12-mer peptide library (Ph.D.12TM Phage Display Peptide Library, New England Biolabs Inc., Boston, MA, USA) was screened against PN-SIA 28 Fab fragment. Briefly, 300 ng of PN- SIA 28 were coated on four wells (96-wells plate, COSTAR), and the peptide library was amplified transforming electrocompetent E. coli XL1 Blue. 70 ml of the obtained phage preparation was incubated with PN-SIA 28 for 1 hour at 37uC and subsequently the unbound phages were removed by washing with PBS 1X-Tween20 using progressive Tween20 concentration starting from 0.1% to 0.5%. The phage that bound PN-SIA 28 was then eluted using low pH and, once neutralized, used to infect E. coli XL1 Blue for a subsequent ELISA screening on 100 ng/well coated PN-SIA 28. Positive clones were then sequenced using the kit-provided primers (New England Biolabs Inc., Boston, MA, USA). Positive clones were then sequenced. An in silico analysis was performed, using Mimox and Pepitope servers, comparing the selected peptides to the available crystal structure of A/PR/8/34 and A/South Carolina/1918 haemagglutinins (1RU7.pdb and 1RD8.pdb). Selection and characterization of antibody escape mutants under selective pressure of antibody PN-SIA28 The experiment was performed on 90% confluent MDCK cells growing in T25 flasks (Nunc, Rochester, NY, USA) in MEM supplemented with 10% FBS. PN-SIA28 as well as e137 mock control, were diluted in 1.5 mL MEM supplemented with TPCK trypsin (2 mg/ml) to obtain final concentrations of 2 mg/ml, 10 mg/ml and 20 mg/ml of antibody. 100 TCID50 of A/PR/8/34 were prepared in 1.5 mL of the same medium. The two solutions, containing antibodies and virus, were mixed in order to obtain a final concentration of 1 mg/ml, 5 mg/ml and 10 mg/ml for the mAbs in a final volume of 3 mL. The mixes were then incubated 1 h at 37uC. An infection positive control (virus without PN- SIA28) was included as well as non-infected cells. After two washes with sterile PBS 16 (Gibco Invitrogen, Carlsbad, CA, USA), 1 ml of each neutralization mix was added to the flasks containing MDCK cells and the infection was performed for 1 h at 34uC in 5% CO2. After absorption, the medium was removed and the monolayer washed twice with sterile PBS. 3 mL of MEM supplemented with trypsin (2 mg/ml) was added to the infection positive control and to non-infected cells. Antibody PN-SIA28 and e137 were added to the previously treated infected cells, maintaining the concentrations used during the infection step. Cells were incubated at 34uC in 5% CO2 and regularly checked for 48 hours for the presence of cytopathic effect (CPE) by comparing the positive infection control to the treated infected cells. The supernatant was then centrifuged (2000 rcf for 10 minutes), collected and stored at 280uC. All the viral stocks were titrated and used to infect new cell preparations, increasing, when it was possible, the concentration of PN-SIA28. After ten passages, the cells of the infection positive control and mock control were compared with infected cells under selective pressure of PN-SIA28. Once a strong cytopathic effect was evident in the positive and mock controls, the presence or absence of CPE in the PN-SIA28 treated infected cells was evaluated. All the superna- tants were collected, centrifuged, stored and used to perform a full length DNA sequence of influenza genomic fragment 4 coding for viral HA. HA cloning and mutagenesis A/PR/8/34 (H1N1) HA was amplified as previously described [19,20] using the following PCR-oligonucleotides: APR834_s: 59- Cross-Neutralizing mAb to Influenza Viruses CACCATGAAGGCAAACCTACTGGTCCTGTTATGTG-39; APR834_as: 59-TCAGATGCATATTCTGCACTGCAAAGAT- CCATTAGA-39. A/Aichi/2/1968 (H3N2) HA was amplified using the following PCR-oligonucleotides: Aichi/2/68H3N2f: 59- CACCATGAAGACCATCATTGCTTTG-39; Aichi/2/68H3- N2r: 59-TCAAATGCAAATGTTGCACCTAATG-39. The PCR products were cloned into the pcDNA 3.1D/V5-His-TOPO vector (Invitrogen, Carlsbad, CA, USA). Subsequently, alanine mutants for H1N1 and H3N2 HA were generated using Gene Tailor Site- Directed Mutagenesis System (Invitrogen, Carlsbad, CA, USA). A total of 20 HA mutants were generated for H1N1 and 7 for H3N2 (Table S1). Cytofluorimetric binding assays The binding activity of PN-SIA28 was assayed using full-length wild type and mutants HAs cloned as described above. In brief, 16106 human epithelial kidney (HEK) 293T cells (ATCC CRL- 1573) were transfected in 6 wells plate (Corning, Corning, NY, USA) with 4 mg of pcDNA 3.1D/V5-His-TOPO vector contain- ing the HA nucleotide sequences. After centrifugation and fixation with paraformaldehyde 4%, the transfected cells were incubated for 30 minutes at room temperature with PN-SIA28 or a conformational control for H1N1 (RB62) or a control for H3N2 (12D1, kindly provided by P.Palese) at 1 mg/ml and 10 mg/ml. Additionally, the isotype control, e137 (1 and 10 mg/ml) was introduced as well as untransfected cells and a mouse anti-H1 subtype monoclonal antibody directed against a linear epitope (anti-influenza A hemagglutinin [12D1102] GeneTex Inc., Irvine, CA, USA) to evaluate the transfection efficiency for each HA. The cells were then washed and incubated for 30 minutes at room temperature with FITC-conjugated anti-human (Sigma Aldrich) or anti-mouse (Argene, Shirley, NY, USA) monoclonal antibodies. Afterwards, the cells were washed and analysed by FACS. The FACS data were analyzed using the software Weasel w 2.5 (Waler+Eliza Hall, Institute of Medical Research, Parkville Victoria, Australia). The binding of PN-SIA28 to the different HA-mutants was expressed as a binding percentage compared to wild-type. Software For sequences analysis the following software packages were used: SeqScape (Applied Biosystems), ClustalX (Toby Gibson), Bio Edit (Tom Hall, Ibis Therapeutics), and Treeview (GubuSoft). For molecular visualization and rendering UCSF Chimera package from the Resource for Biocomputing Visualization and Informat- ics at University of California, RasMol (Roger Sayle), Jmol (Jmol: an open-source Java viewer for chemical structures in 3D. http:// www.jmol.org/), Cn3D (United States National Library of Medicine, NLM), Pepitope server (Pepitope: epitope mapping from affinity-selected peptides. Bioinformatics 2007 23(23):3244- 3246.), Mimox (BMC Bioinformatics 2006, 7:451 doi:10.1186/1471- 2105-7-451) were used. Finally for data analysis and graphical editing GraphPad Prism was used. Supporting Information Figure S1 Neutralization assays against viruses from group 1 subtypes. Dose–response curve fit nonlinear regression is reported for PN-SIA28 against H1N1, H5N1, H2N2 and H9N2 strains studied in this paper. Data from two different experiments for each strain are reported. Each point was performed in triplicate. (TIF) Figure S2 Neutralization assays against H3N2 viruses. Dose–response curve fit nonlinear regression is reported only the PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e28001