Cross-Neutralizing mAb to Influenza Viruses Figure 5. Sequence analysis of H3N2 HA1-hemagglutinins of viral isolates neutralized and not neutralized by PN-SIA28. (A) Aminoacidic sequences of H3N2 strains studied in the paper and in proximity to the region identified on H1N1 hemagglutinin were aligned. Differences among solvent exposed residues close to PN-SIA28 epitope core are boxed. The yellow circle on Lys57Ala mutant indicate 50–75% PN- SIA28 binding compared to wild-type HA. (B) (H3N2) A/Aichi/2/68 hemagglutinin trimer: grey areas indicate the HA1 domains whereas white areas indicate HA2 domains. Red regions indicate the residues able to inhibit PN-SIA28 binding to H3-HA. Yellow circle define the residues involved in the interaction of PN-SIA28 with both, H1 and H3 HAs. doi:10.1371/journal.pone.0028001.g005 by this human mAb. As described for a few other mAbs [25,26,27,28,29,30,31,32,41], PN-SIA28 is mainly directed against the highly conserved HA2 domain whose use in novel anti-influenza vaccinal approaches is being widely investigated. However, PN-SIA28 demonstrates that not all antibodies directed against the stem region have the same characteristics, and that their neutralizing activity is modulated by the specifically recognized epitope. Indeed, the data presented in this paper show that in the design of such innovative approaches (i.e. the so-called ‘‘headless approach’’ [16]) it is important to identify also residues belonging to HA1 domain to be preserved to elicit the widest immunity. Several immunogens are usually obtained on the basis of in silico analysis, but their real protective potential can only be ascertained after time-consuming and expensive in vivo studies. Under this perspective, PN-SIA28 could therefore be an extremely important reagent to identify immunogens more likely to stimulate antibodies with a similar broad heterosubtypic neutralizing activity that will constitute the best candidates for further in vivo studies. Materials and Methods Monoclonal antibodies The isolation of PN-SIA28 has been previously described [19,20]. In brief, PN-SIA28 was isolated from a 55 year old patient with a negative clinical history of infection from influenza viruses during the past ten years, and with a detectable serum neutralizing titre (half maximal inhibitory dilution 2 ID50 . = 1:20) against two reference strains belonging to two distinct HA-based phylogenetic groups: the H1N1 strain A/Puerto Rico/8/1934 for group 1 and the H3N2 strain A/Port Chalmers/1/1973 for group 2. It was then expressed in E. coli as Fab fragment to define its features. To obtain the whole IgG described in this study, the BD BaculoGold System (BD Biosciences Pharmingen, San Diego, CA, USA) was used. Briefly, nucleotide sequences codifying heavy and light chains of PN-SIA28 Fab fragment were sub-cloned into the baculovirus expression vector pAc-k-Fc (PROGEN Biotechnik GmbH, Heidelberg, Germany). Sf-9 insect cells (Invitrogen, Carlsbad, CA, USA) were co-transfected with the linearized baculovirus DNA (BD Biosciences Pharmingen, San Diego, CA, USA) and the pAc-k-Fc/PN-SIA28. The obtained virus was employed at a molteplicity of infection (MOI) of 5 to infect 16109 H5 insect cells (Invitrogen, Carlsbad, CA, USA) in a final volume of 1 liter. After an incubation of 96 hours the culture media was collected, clarified by centrifugation and filtered with 0.2 mm filter (Millipore, Billerica, MA, USA). The media was loaded into a protein G column (Amersham Biosciences GE Healthcare, Zurich, Switzerland), the antibody was eluted with citric acid 0.1 M, pH 3, and immediately neutralized with Tris Base 1 M, pH 9. The PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e28001