Project 2. Program CJirector/Principal Investigator (Last, First. Middle): G A R C I A - S A S T R E , A d o l f oPROJECT SU^/)^MRY (See instructions):Work in Project 2 will explore exciting recent developments in the study of cross-reactive immunity to influenzaviruses.The goal of this project is to determine the molecular and structural basis for broadly erbss-reactive human mAbresponses to influenza HAs; As the epitope sequences and structures reccjgnized by broadly cross-reactive Abs aredefined, we will be better able to rationally design broadly protective immuriogens. The work seeks to investigate therepertoire of human Abs recognizing three principal Gonserved regions of the HA molecule; a canonical stalk domainepitope, the long alpha-helix stalkdomain, and the globular head domain. The hypothesis is that, unexpectedly, thereare at least three immunogenic domains in influenza HA that retain highly conserved structural features and that mostadult healthy subjects do possess circulating cross-reactive B cell clones to these conserved sites on HA. Thesedomains are complex, however, and relatively inaccessible-for conventional Ab structures, w e have developed robusttechnologies for isolating human mAbs that reveal the relatively frequent nature of eross-reactive B ceils, and thegenetic'arid structural basis for broad cross-reactivity of the secreted Abs for influenza viruses of diverse subtypes. Wehypothesize that such cross-reactivity is driven by unusual features in the Ab repertoire^ especially an exceptionally highlevefof somatic point mutations and somatic insertions. This highly interactive project will engage in collaborative workwith Project 1 to determine mAb-HA co-crystal structures. Project 4 to study the biology of viral escape mutant viruses,;Project 5 to examine the effect of mutants on DCs, and will use the Glycan Array core to study glycosyiation in escapesites and the Animal Core to determine in vivQ potency of antibodies. Studies of such Abs and the sites recognized bythem will inform the rational design of universalinfluenza vaccines based on the underlying principles of heterosubtypjcimmunity; The stalk domain is of interest because highly eross-reactive Ab have been identified that recognized aconserved region in this area. However, the rare Abs identified to date do not possess both group 1 and 2 specificities, ,In preliminary datBi we show that it is possible to isolate such Abs. Using these unusual reagents, we will define thebasis for the group determinants or conserved elelmentS: by mapping the interaction of large panels of new Abs to the:stalk;region. We also will seek to isolate novel human Abs from human subjects that recognize the stalks of both majorHA antigenic groijps. The head domain is better studied, however there are only rare Abs:that exhibit cross-reactiyity forahtigenically distinct HAs. By large-scale sci-efening of B cells from subjectis immunized with seasonal and experimentalvaccines, we have identified cross-reactive Abs that i-ecdgnize the head domain of HAs of multiple subtypes ofinfluenza. By epitope mapping using biochemical arid biologic studies, coupled with studies to determine co-crystalstuctures, we will determine the structural basis for cross reactivity.RJELEVANGE (See instructions):Antibodies are the priricipal mediators of protection.induced against disease due to influenza followinginfection or vaccination, but influenza viruses exhibit a large amount of diversity, necessitating yearlyvaccination. Some unusual human antibodies, however, react against a wide variety of influenza viruses,suggesting that;a! universal vaccine might be possible. The proposed studies will identify potentialcomponents of a universal vaccine antigen.
