F10 competition assays performed with H1CA0409, H2SIN57 or H5VN04 coated on MSD plates further indicated that all anti-H5VN04 phage Ab are stem directed and 10 cross-reacted with H1CA0409; 2 cross-reacted with H2 A/Singapore/1/1957 (H2SIN57); and 9 cross-reacted with both H1CA0409 and H2SIN57 (Figure S4A in Text S1)

F10 competition assays performed with H1CA0409, H2SIN57 or H5VN04 coated on MSD plates further indicated that all anti-H5VN04 phage Ab are stem directed and 10 cross-reacted with H1CA0409; 2 cross-reacted with H2 A/Singapore/1/1957 (H2SIN57); and 9 cross-reacted with both H1CA0409 and H2SIN57 (Figure S4A in Text S1). show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met) and Phe54), and CDR-H3-Tyr at positions 981; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase- recognition motifs. A semi-synthetic phage-display library screen designed to investigate AID/pol restrictions resulted in PF-04217903 methanesulfonate the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a) facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of germline genes that alone make contact with HA and prevent virus entry and emergence of escape mutants. Our study was undertaken to gain an understanding of what structural requirements enable a rearranged Ab to become a potent cross-neutralizing antibody. We found that in addition to a critical amino acid triad consisting of a pair of anchor residues in CDR-H2 and a properly positioned CDR-H3 Tyr, distinctive V-segment substitutions that arise in positions that are distinct from phase I AID somatic hypermutation (SHM) hotspot motifs are often required. As few as two V-segment SHM can fulfill this role which appears to facilitate the optimal binding of CDR-H2 Phe54 and CHR-H3-Tyr into adjacent hydrophobic pockets in the HA stem. These studies provide new information on the SHM requirements for germline gene in broadly neutralizing antibodies against the stem domain of group 1 influenza A viruses (HV1-69 sBnAbs). PF-04217903 methanesulfonate While germline gene is highly utilized in the population Rabbit Polyclonal to p47 phox [17], the regulation of this germline gene usage during development and adaptive immune responses has only recently been reexplored [18] following some initial investigations [19]C[21]. In addition, details of the molecular events that are involved in the elicitation of HV1-69-sBnAbs by vaccination or seasonal influenza infection remains unknown. The highly immunogenic globular head [1], [10], [11] is thought to be a main impediment for sBnAb elicitation as the stem epitopes have been shown to be readily accessible to sBnAbs [22]. In this study we sought to better define the V-segment amino acid substitutions and CDR-H3 amino acids within rearranged germline genes that are preferentially used to allow an germline based Ab to become a potent HV1-69-sBnAb. Analysis of 38 HV1-69-sBnAbs recovered from 8 laboratories (Table S1 in Text S1) indicates that broad-spectrum binding and neutralization is conveyed by a triad of critical anchor residues composed of two CDR-H2 residues including a hydrophobic residue at position 53 and Phe54, and properly positioned CDR-H3 tyrosines. In addition, we define distinctive V-segment mutations within the CDR H1/H2/H4 loops. Moreover, these V-segment mutations occur in positions that are sparse in activation-induced cytidine deaminase (AID) and polymerase eta (pol ) consensus hot-spot motifs. Together with panning of a semi-synthetic Ab library against H5/H1 HAs, mutagenesis studies, and structural modeling PF-04217903 methanesulfonate we demonstrate that HV1-69-sBnAbs can be evolved from a germline gene with as few as two V-segment substitutions with one occurring at CDR-H2 Ile52Ser, and by properly positioned Tyr in the CDR-H3 domain. The CDR-H2 substitutions at positions Ile52Ser and Pro52aGly/Ala are predicted to function not by making new contacts with the epitope themselves, but rather by enabling conformational changes within the CDR loops that facilitate optimal insertion of two major anchor residues CDR-H2 Phe54 and CDR-H3-Tyr98 into adjacent pockets in the stem. Our immunogenetic and structural studies demonstrate that the generation of critical SHM for HV1-69-sBnAbs does not occur through the classical phase I AID repair mechanism that takes place directly under WRCY/RGYW motifs, instead by phase 2 long-patch error-prone repair or random non-AID SHM events. Further, these results suggest that the secondary AID repair mechanisms as described here may not require B cell recycling/reentry in the germinal center [23], rather by alternative routes such as short term entry into the germinal center or in a specialized extra-follicular location such as the marginal zone [24]. Results HV1-69-sBnAb CDR-H3 Tyrs together with CDR-H2 Phe54 mediate high affinity binding to adjacent pockets in the HA stem The co-crystal structures of the HV1-69-sBnAbs, F10 [1], PF-04217903 methanesulfonate CR6261 [4], and CR9114 [7] with H5VN04 established that binding is mediated exclusively by the heavy chains. To further explore binding similarities in the co-crystal structures, binding free energy contributions for the heavy chain CDR residues were estimated by using the ANCHOR server [25].