The long-term goal of this research is to understand the immunochemical basis of human immune responsiveness to inhaled pollen allergens. We will study two homologous groups of ragweed allergenic proteins, the Amb Vs and the Amb VIs. Responsiveness to the Amb V homologues is known to be associated with HLA-DR2/Dw2 (DR2.2), and DR2.12 in Orientals; responsiveness to Amb VI is associated with DR5 (DRw11) plus DR2.21 (and probably also DR2.22). Interestingly, responsiveness to Amb a VI is negatively associated with DR2.2. Within a homologous group, we postulate that the portions of these molecules (agretopes) that bind to the Class II MHC (Ia) molecules are similar (accounting for the same Ia restriction), but that the T-cell and B-cell epitopes are usually different for the individual homologues. Analysis of these epitopes will be facilitated by use of NMR analysis and computer modeling. We will sequence Amb V cDNAs of several ragweed species. Protein structures of Amb a V and Amb t V will be determined by NMR spectroscopy, and the structures of further Amb Vs by NMR or computer modeling. Functional, recombinant Amb a V, Amb t V and Amb p V proteins will be produced by cloning the respective cDNAs into the pGEX-2T expression vector in E. coli. Mutations that are potentially informative in defining Ia/T- cell and B-cell epitopes will be identified from the NMR structures and by computer modeling. We will then produce the Amb V variants by site- directed mutagenesis and expression of the cDNAs. The Ia-binding and T- cell-binding portions of the Ia/Tcell epitopes, as well as the B-cell epitopes of the Amb V molecules will be defined by examining these mutants, as well as using synthetic peptides (native and variant sequences) of Amb Vs. Mutant Amb Vs that have altered immunological properties will be analyzed by NMR to examine the effects of altering selected amino acids. Since the Amb Vs have a complex array of disulfide bonds that are implicated in T-cell epitopes, these studies will provide a valuable model for the molecular analysis of Ia/T-cell epitopes containing disulfide(s). We will transfect HLA-DRA and DRB1*1501 (encoding DRbetaI2.2), and mutants of the betaI gene, into a human B-cell line that does not express Ia or into mouse L cells. Using these constructs, we will determine which Ia residues are critical for binding to Amb V peptides. Similar studies, including cDNA cloning and sequencing, will be performed in the case of the Amb VIs; in this case, it will also be interesting to examine the structural basis for why the immune responsiveness is positively associated with DR5/DR2.21, but negatively associated with DR2.2. A unique asset will be the availability of two well-characterized populations: ca 400 clinic patients and ca 500 subjects from an epidemiologic-genetic study of human immune response to allergens. Subjects from different racial and ethnic origins, included in an international HLA and Allergy project, will also be studied.
