In the remaining four copies, only the variable domains are well defined, as observed in other Fab and Ig-domain containing structures [1], [22], [23]. a D190E variant, the D225G variant (NY1918), or the D190E/D225G double mutant (AV1918).(PDF) ppat.1003067.s006.pdf (531K) GUID:?6C07ED66-48BA-40AC-8AEA-C4E709C5F9B6 Table S4: 1F1-HA contacts. Summary of interacting residue pairs from chains A, B, M, and N in the TNRC23 BRAF inhibitor 1F1-Sc1918 crystal structure, generated using CONTACSYM [42].(PDF) ppat.1003067.s007.pdf (350K) GUID:?9B787F70-C346-4B2B-A184-7320DFDACBF0 Abstract Most monoclonal antibodies (mAbs) to the influenza A virus hemagglutinin (HA) head domain exhibit very limited breadth of inhibitory activity due to antigenic drift in field strains. However, mAb 1F1, isolated from a 1918 influenza pandemic survivor, inhibits select human H1 viruses (1918, 1943, 1947, and 1977 isolates). The crystal structure of 1F1 in complex with the 1918 HA shows that 1F1 contacts residues that are classically defined as belonging to three distinct antigenic sites, Sa, Sb and Ca2. The 1F1 heavy chain also reaches into BRAF inhibitor the receptor binding site (RBS) and interacts with residues that contact sialoglycan receptors and determine HA receptor specificity. The 1F1 epitope is remarkably similar to the previously described murine HC63 H3 epitope, despite significant sequence differences between H1 and H3 HAs. Both antibodies potently inhibit receptor binding, but only HC63 can block the pH-induced conformational changes in HA that drive membrane fusion. Contacts within the RBS suggested that 1F1 may be sensitive to changes that alter HA receptor binding activity. Affinity assays confirmed that sequence changes that switch the HA to avian receptor specificity affect binding of 1F1 and a mAb possessing a closely related heavy chain, 1I20. To characterize 1F1 cross-reactivity, additional escape mutant selection and site-directed mutagenesis were performed. Residues 190 and 227 in the 1F1 epitope were found to be critical for 1F1 reactivity towards 1918, 1943 and 1977 HAs, as well as for 1I20 reactivity towards the 1918 HA. Therefore, 1F1 heavy-chain interactions with conserved RBS residues likely contribute BRAF inhibitor to its ability to inhibit divergent HAs. Author Summary Influenza infection kills thousands of people every year and causes major pandemics every few decades. The most lethal outbreak of influenza known was the 1918 H1N1 influenza pandemic that killed an estimated 20 to 100 million people. The 1918 virus was likely introduced into the human population from birds. We previously described five human neutralizing antibodies from survivors of the 1918 pandemic that bind the hemagglutinin (HA) surface antigen. Here, we define the binding sites of antibodies 1F1 and 1I20 on the 1918 HA and demonstrate that these overlap with the glycan receptor binding site. The glycan specificity differs between human and avian viruses for the linkages of the sialylated sugar receptors [human (2C6) or avian (2C3)]. 1F1 and 1I20 binds viruses that contain HA residues that mediate preference for 2C6 sialylated sugars. Three other control antibodies were not affected by preferences for the linkages of the sialylated sugar receptors because they bind elsewhere. Since the receptor-binding site is relatively conserved, this may explain the cross-reactivity of 1F1 and the enhanced binding of 1F1 and 1I20 to HAs with human receptor specificity. Introduction The hemagglutinin (HA) protein of influenza viruses binds to sialic acid receptors on host cells and is the major target of neutralizing antibodies. Amino-acid changes in the immunodominant HA antigenic sites that arise in response to immune selective pressure (antigenic drift) enable seasonal influenza A viruses to cause repeated epidemics and necessitate continuous reevaluation of BRAF inhibitor the composition of influenza vaccines. Characterization of antibodies that display the ability to cross-neutralize divergent viruses may suggest strategies to elicit more BRAF inhibitor broadly protective immunity. The broadest cross-reactive influenza mAbs described to date recognize conserved regions of the HA stem [1], [2], [3], [4], [5], [6] as compared to the HA head.