Cross-Neutralizing mAb to Influenza Viruses Figure 4. Comparison of the core of PN-SIA28 epitope on the surface of H1 and H3 trimers. (A) (H1N1) A/PR/8/34 HA: white areas indicate the HA1 domains of H1 trimer whereas cyan areas indicate its HA2 domains. (B) (H3N2) A/Aichi/2/68 hemagglutinin: grey areas indicate the HA1 domains of H3 trimer whereas white areas indicate its HA2 domains. On both pictures, red regions indicate the residues 25 and 45 on HA1 and residues 361 and 362 on HA2 that, when mutated, were able to inhibit PN-SIA28 binding both to H1 and H3 HAs. doi:10.1371/journal.pone.0028001.g004 viruses belonging to this subtype (Figure 2). The testing of HA alanine mutants for position His45 on H1N1 and Asn54 on H3N2, and the increased binding of PN-SIA28 to Asn54His mutant on H3N2, confirm that this key residue is particularly important for the interaction of PN-SIA28 with HA. However, other residues, including Lys57 and others not yet identified, certainly play an important role in the PN-SIA28 binding to HA, as shown by the lack of activity observed against the recent H3N2 isolates tested, as well as against the H7N2 isolate, despite a substantial identity in the studied region (Figure 2). In conclusion, the molecule described in this paper is a singular human mAb featuring a broad heterosubtypic neutralizing activity encompassing group 1 and group 2 influenza A subtypes, and therefore recognizing a broad-range neutralizing human B epitope in the stem region of HA. Indeed, PN-SIA28 neutralizes influenza viruses belonging to all subtypes that have caused pandemics in humans, as well as other subtypes with pandemic potential. The data presented may therefore be crucial not only for the improvement of classical passive anti-influenza prophylactic and therapeutic strategies [33,34], but also for the correct understand- ing of mechanisms leading to in vivo protective anti-influenza immunity and therefore for the design of more effective vaccinal strategies. Indeed, several pharmacokinetic studies have shown that the extremely low IC50 featured by PN-SIA28 against most of the isolates tested can easily be reached in humans after systemic administration [35,36]. Given the frequent correlation between a low IC50 value and in vivo efficacy observed for other anti-influenza mAbs [25,26,27,28,29,30,31,37,38,39], PN-SIA28 could be a really promising compound to be used in passive immunization strategies. More importantly, PN-SIA28 represents the molecular evidence that an extremely wide unusual neutralizing immunity although, uncommon, may be elicited during the course of a natural infection and, potentially, even after a new-generation vaccinal approach focused on its epitope [40]. As a consequence, PN- SIA28 could be crucial in the identification of promising HA- based vaccinal strategies aimed at the elicitation of a broadly heterosubtypic protective immunity similar to the one represented PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e28001