1. Beigel J, Bray M. Current and future antiviral therapy of severe seasonal and avian influenza. Antiviral Res. 2008;78:91. [PMC free article] [PubMed] 2. Bright RA, Shay DK, Shu B, Cox NJ, Klimov AI. Adamantane resistance among influenza A viruses isolated early during the 2005–2006 influenza season in the United States. JAMA. 2006;295:891. [PubMed] 3. Kiso M, et al. Resistant influenza A viruses in children treated with oseltamivir: descriptive study. Lancet. 2004;364:759. [PubMed] 4. Lowen AC, Palese P. Influenza virus transmission: basic science and implications for the use of antiviral drugs during a pandemic. Infect Disord Drug Targets. 2007;7:318. [PubMed]
5. The most common vaccine formulations include influenza A H1N1 and H3N2 and influenza B components.
7. Interim within-season estimate of the effectiveness of trivalent inactivated influenza vaccine--Marshfield, Wisconsin, 2007–08 influenza season. MMWR Morb Mortal Wkly Rep. 2008;57:393. [PubMed] 8. Fleury D, Daniels RS, Skehel JJ, Knossow M, Bizebard T. Structural evidence for recognition of a single epitope by two distinct antibodies. Proteins. 2000;40:572. [PubMed] 9. Barbey-Martin C, et al. An antibody that prevents the hemagglutinin low pH fusogenic transition. Virology. 2002;294:70. [PubMed] 10. Fleury D, et al. A complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site. Nat Struct Biol. 1999;6:530. [PubMed] 11. Fleury D, Wharton SA, Skehel JJ, Knossow M, Bizebard T. Antigen distortion allows influenza virus to escape neutralization. Nat Struct Biol. 1998;5:119. [PubMed] 12. Xu R, et al. Structural basis of preexisting immunity to the 2009 H1N1 pandemic influenza virus. Science. 2010;328:357. [PMC free article] [PubMed] 13. Ekiert DC, et al. Antibody recognition of a highly conserved influenza virus epitope. Science. 2009;324:246. [PMC free article] [PubMed] 14. Throsby M, et al. Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells. PLoS One. 2008;3:e3942. [PMC free article] [PubMed]
15. Hemagglutinins cluster into two distinct groups on the basis of their primary sequence. Group 1 HAs include 10 of the 16 subtypes: H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16. Group 2 HAs account for the remaining 6 subtypes: H3, H4, H7, H10, H14, and H15.
16. Kashyap AK, et al. Combinatorial antibody libraries from survivors of the Turkish H5N1 avian influenza outbreak reveal virus neutralization strategies. Proc Natl Acad Sci U S A. 2008;105:5986. [PubMed] 17. Sui J, et al. Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol. 2009;16:265. [PMC free article] [PubMed] 18. Chen GL, Subbarao K. Attacking the flu: neutralizing antibodies may lead to ‘universal’ vaccine. Nat Med. 2009;15:1251. [PMC free article] [PubMed] 19. Wang TT, Palese P. Universal epitopes of influenza virus hemagglutinins? Nat Struct Mol Biol. 2009;16:233. [PubMed] 20. Bommakanti G, et al. Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge. Proc Natl Acad Sci U S A. 2010;107:13701. [PubMed] 21. Corti D, et al. Heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine. J Clin Invest. 2010;120:1663. [PMC free article] [PubMed] 22. Steel J, et al. Influenza virus vaccine based on the conserved hemagglutinin stalk domain. MBio. 2010;1 [PMC free article] [PubMed] 23. Wang TT, et al. Vaccination with a synthetic peptide from the influenza virus hemagglutinin provides protection against distinct viral subtypes. Proc Natl Acad Sci U S A. 2010;107:18979. [PubMed] 24. Wang TT, et al. Broadly protective monoclonal antibodies against H3 influenza viruses following sequential immunization with different hemagglutinins. PLoS Pathog. 2010;6:e1000796. [PMC free article] [PubMed] 25. Wei CJ, et al. Induction of broadly neutralizing H1N1 influenza antibodies by vaccination. Science. 2010;329:1060. [PubMed] 26. Wrammert J, et al. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection. J Exp Med. 2011;208:181. [PMC free article] [PubMed]
28. Although CR8020 binds to H4 and H14, the affinity is probably too low to result in in vitro neutralization, at least at the concentration tested here. Neutralization of H14 and H15 viruses was not tested.
29. All increases in body weight were statistically significant (p 0.018), except for the H7N7 challenge group treated with 3mg/kg CR8020 (p=0.168).
30. Wilson IA, Skehel JJ, Wiley DC. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981;289:366. [PubMed]
31. The final model includes HA1 residues 11–328, HA2 residues 1–175, CR8020 heavy chain residues 2–222 and light chain residues 1–212. The asymmetric unit contains one HA protomer bound to on CR8020 Fab, with the additional protomers in the trimer generated by crystallographic symmetry operations.
32. In this regard, CR8020 can be thought of as being analogous to the anti-HIV envelope antibodies 2F5, 4E10, and Z13. These antibodies recognize the membrane-proximal external region (MPER), a short helical peptide from the gp41 subunit that is closely associated with the viral envelope.
33. CR8020 also contacts: 1) HA1 residue 325, near the C-terminus of the chain, 2) HA2 residue 25, in the fourth strand out in the small -sheet, 3) HA2 residue 38, at the bottom of helix A, and 4) HA2 residues 146 and 150, in a short helix adjacent to the sheet. It is worth noting that, in addition to protein-protein interactions, the Fab also makes contacts with the core fucose of a universally conserved glycan linked to Asn154 in HA2, although the extent of the contribution of this interaction to the overall binding energy is unclear.
34. Bao Y, et al. The influenza virus resource at the National Center for Biotechnology Information. J Virol. 2008;82:596. [PMC free article] [PubMed]
36. In this analysis, residues are considered conserved if they fall within one of the following groups: 1) Asp/Asn/Glu/Gln; 2) Phe/Tyr; 3) Ile/Leu/Val/Met; 4) Lys/Arg; or 5) Ser/Thr.
37. Lorieau JL, Louis JM, Bax A. The complete influenza hemagglutinin fusion domain adopts a tight helical hairpin arrangement at the lipid:water interface. Proc Natl Acad Sci U S A. 2010;107:11341. [PubMed] 38. Twenty of the first 23 positions in HA2 are well-conserved across all subtypes (See Fig. S3). Two of the remaining positions (HA2 positions 12 and 15) have differing, group-specific residues. The final position, HA2 residue 19 is also conserved as D or N across most subtypes from both groups. However, we regard this substitution as non-conservative in the context of CR8020, since D19N mutations escape virus neutralization. 39. Chen J, Skehel JJ, Wiley DC. N- and C-terminal residues combine in the fusion-pH influenza hemagglutinin HA(2) subunit to form an N cap that terminates the triple-stranded coiled coil. Proc Natl Acad Sci U S A. 1999;96:8967. [PubMed]
40. While there is clear density for the VL:Arg53 side chain placing the guanidinium moiety in close proximity to HA2:Asp19, the preferred rotamer for the Arg side chain cannot be assigned unambiguously. Various rotamers consistent with the observed electron density result in charged atom contact distances of ~4–4.5Å. Although somewhat longer than expected for a salt bridge that would make a major contribution to antibody binding, this discrepancy may be due in part to shielding by a nearby sulfate from the crystallization solution, which is sandwiched between the VL:Arg53–Arg54 side chains.
41. Most of the group 2 HAs with Asp19Asn are restricted to a single lineage of the H7 subtype, and disproportionate sampling in birds in this region may exaggerate the prevalence of Asp19Asn substitutions in the H7 population. See discussion in the SOM on Science Online.
42. Neutralization of H15 has not been tested, but the KD for CR8020 binding is comparable to that of H7, which is neutralized.
43. Neutralization of H14 viruses has not been tested, but comparable binding of CR8020 to H4 and H14 suggests that H14 will not be neutralized.
44. H14 also has a Glu325G mutation in HA1, which has a negligible effect on CR8020 in a HK68 background.
45. This scenario is reminiscent of the group 1 restriction of CR6261, which cannot interact with group 2 viruses, such as H3 and H7, at least in part due to a conserved glycan at HA1:Asn38, and the more general and well-documented use of glycans to mask and unmask surfaces to evade immune recognition, such as vividly illustrated in the evolution of human H1N1 viruses (12, 56)
46. Bullough PA, Hughson FM, Skehel JJ, Wiley DC. Structure of influenza haemagglutinin at the pH of membrane fusion. Nature. 1994;371:37. [PubMed]
47. In contrast, a control mAb against the HA1 head bound to A/Wisconsin/67/2005 HA in all three conformations and binding was only lost after DTT treatment, which dissociates HA1 from HA2 in the post-fusion state.
48. As evidenced by the fact that in this case treatment with DTT did not diminish CR8057 binding to A/Wisconsin/67/2005 HA. In line with its narrow spectrum of neutralization (Table S1), CR8057 did not bind to any conformation of the HAs of A/Hong Kong/1/1968 or A/Hong Kong/24/1985.
49. Since the initial crystals only appeared between 3 and 7 days after the start of the experiment, CR8020 must be capable of retaining HA in the pre-fusion state for several days at low pH.
50. Xu R, Wilson IA. Structural characterization of an early fusion intermediate of influenza virus hemagglutinin. J Virol. 2011;85:5172. [PMC free article] [PubMed] 51. Russ G, Polakova K, Kostolansky F, Styk B, Vancikova M. Monoclonal antibodies to glycopolypeptides HA1 and HA2 of influenza virus haemagglutinin. Acta Virol. 1987;31:374. [PubMed] 52. Vareckova E, Mucha V, Wharton SA, Kostolansky F. Inhibition of fusion activity of influenza A haemagglutinin mediated by HA2-specific monoclonal antibodies. Arch Virol. 2003;148:469. [PubMed] 53. Yoshida R, et al. Cross-protective potential of a novel monoclonal antibody directed against antigenic site B of the hemagglutinin of influenza A viruses. PLoS Pathog. 2009;5:e1000350. [PMC free article] [PubMed] 54. Hashem AM, et al. Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus. Biochem Biophys Res Commun. 2010;403:247. [PubMed] 55. Stropkovska A, et al. Broadly cross-reactive monoclonal antibodies against HA2 glycopeptide of Influenza A virus hemagglutinin of H3 subtype reduce replication of influenza A viruses of human and avian origin. Acta Virol. 2009;53:15. [PubMed] 56. Wei CJ, et al. Cross-neutralization of 1918 and 2009 influenza viruses: role of glycans in viral evolution and vaccine design. Sci Transl Med. 2010;2:24ra21 . [PMC free article] [PubMed] 57. Skehel JJ, Wiley DC. Influenza haemagglutinin. Vaccine. 2002;20(Suppl 2):S51. [PubMed]