The matured Abs bound HA with association rates that were approximately 40 times faster and dissociation rates that were slower than were found for UCA or the I-2 Ab. diverse antibodies elicited to a protein antigen bind to different epitopes on that protein (4), confounding efforts to convincingly demonstrate affinity maturation. There are at least five epitopes SMIP004 on the influenza virus hemagglutinin (HA) that binds the virions to host cells (5). By comparing the binding properties and structures of affinity matured Abs to one of the HA epitopes with those of their progenitor Abs, the elegant study by Schmidt et al. reported in PNAS (6) provides clear evidence for affinity maturation of Abs. The study is notable, moreover, for its focus on the human immune response to influenza virus vaccination with a conventional influenza seasonal vaccine (FLUZON flu shot). By bringing together diverse approaches (crystallography, molecular dynamics simulations, and kinetic studies), the authors shed intriguing light on how the affinity enhancement of affinity matured antibodies is achieved. Affinity maturation of Abs was originally established by measuring changes in a test animal of the serum Abs isolated from sequential bleedings taken over many weeks and months following antigen injection of a test animal (7, 8). In contrast to this longitudinal approach, the approach used by Schmidt et al. could be characterized as immunopaleontology. From a single bleeding drawn from a SMIP004 volunteer 1 wk after receiving a conventional seasonal flu shot injectionFluzone, a mixture of three inactivated strains of the virusmature B lymphocytes were isolated by cell sorting and individually screened for secretion of virus-neutralizing Ab. Then, using recently developed powerful methods for rapid cloning of Ig VH and VL genes from individual B cells (9C12) and determining their sequences, three Abs (called CH65, CH66, and CH67), each from a different B cell (i.e., plasmablast), were found to have similar sequences and to constitute a clonal lineage from which antecedent Abs could be inferred. These antecedents included an unmutated common ancestor (UCA) and an intermediate (I-2) SMIP004 of a clonal tree of which CH65, CH66, and CH67 constitute the terminal twigs. Expressed as recombinant mAbs, they all became available in essentially unlimited amounts for the remarkably comprehensive analyses by Schmidt et al. (6). The variable domain sequences of CH65 [previously described (13)] and CH66 were nearly identical, whereas the slightly different sequences of CH67 place it on another branch of the lineage. Crystal structures of the complexes formed by the HA antigen with the Abs antigen-binding domains (i.e., Fab) showed that all three Abs recognized the same epitope. This epitope was located in the HA global head at the site where HA binds sialic acids on host cells, a binding reaction essential for virus attachment to host cells and infection. Crystal structures of complexes of the HA global head with the Fab domain of each of these Abs showed the same protrusion of the Ab H chains complementarity determining region (CDR H3) loop into the HA epitope (Fig. 1). This mimics sialic acid and accounts for the ability of these Abs to block virion binding to cells, and thus broadly neutralize infectivity of many H1N1-type influenza virus strains (30 of 36 isolates obtained at various times over 30 y). Open in a separate window Fig. 1. CDR H3 (magenta) is conformationally diverse in the UCA antibody ( em A /em ) but more rigid in the affinity-matured antibody CH67, whether bound to influenza virus HA (red; em B /em ) or not bound ( em C /em ). With permission from S. C. Harrison and A. G. Schmidt. The CH65 and Thbd CH67 Abs had essentially the same intrinsic affinity for the HA epitope ( em K /em a = 2C2.8 106 M?1) whereas the UCA and the I-2 intermediate bound approximately 300 times more weakly ( em K /em a = 0.7C0.8 104 M?1). The matured Abs bound HA with association rates that were approximately 40 times faster and dissociation rates that were slower than were found for UCA or the I-2 Ab. That matured high-affinity Abs have faster on-rates than lower affinity Abs made earlier in response to the same epitope (a low molecular weight hapten) was seen previously by Foote and Milstein (8), who speculated that selection of high-affinity Ab-producing B cells might be driven by faster binding of antigen to cell-surface Ab. The mechanisms for obtaining faster on-rates by affinity maturation have, however, remained obscure. Schmidt et al. (6) now provide a clear example of a pathway by which this is achieved. The conformations adopted by the Ab heavy-chain third CDR (CDR H3) loops of the free matured Abs are very similar to that in the AbCantigen complexes, with their CDR H3 loop rigidly extended in a form able to insert into the sialic acid binding site of the HA (Fig. 1). In contrast, their UCA and I-2 predecessors exhibit far more CDR H3.