CHARACTERIZING IMMUNODOMINANT AND PROTECTIVE INFLUENZA HEMAGGLUTININ EPITOPES BY FUNCTIONAL-ACTIVITY AND RELATIVE BINDING TO MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-II SITES

In the present study the analysis of functional activity and major his tocompatibility complex (MHC) binding of two adjacent MHC class II-res tricted epitopes, located in the C-terminal 306-329 region of human in fluenza A virus hemagglutinin 1 subunit (HA1) conserved with subtype s equences and not affected by antigenic drift, was undertaken to explor e the hierarchy of local immnodominance. The functional activity of tw o T cell hybridomas of the memory/effector Th1 phenotype in combinatio n with in vivo immunization studies provided a good tool for investiga ting the functional characteristics of the T cell resonse. The in vitr o binding assays performed with a series of overlapping, N-terminal bi otinylated peptides covering the 306-341 sequence enabled us to compar e the relative binding efficiency of peptides, comprising two distinct epitopes of this region, to I-E-d expressed on living antigen-present ing cells. Our studies revealed that (i) immunization of BALB/c mice w ith the 306-329 H1 or H2 peptides resulted in the activation and proli feration of T cells recognizing both the 306-318 and the 317-329 epito pes, while the 306-329 H3 peptide elicits predomonantly 306-318-specif ic T cells, (ii) the 317-329 HA1 epitope of the H1 and H2 but not the H3 sequence is recognized by T cells and is available for recognition not only in the 317-329 peptide but also in the extended 306-329 or 30 6-341 peptides, (iii) the 306-318 and the 317-329 hemagglutinin peptid es encompassing the H1, H2 but not the H3 sequence bind with an appare ntly similar affinity to and therefore compete for I-E-d binding sites , and (iv) the 317-341, the 317-329 peptides and their truncated analo gs show subtype-dependent differences in MHC binding and those with lo wer binding capacity represent the H3 subtype sequences. These results demonstrate that differences in the binding capacity of peptides comp rising two non-overlapping epitopes located in the C-terminal 306-329 region of HA1 of all three subtype-specific sequences to MHC class II provide a rationale for the local and also for the previously observed in vivo immunodominance of the 306-318 region over the 317-329 epitop e in the H3 but not in the H1 or H2 sequences. In good correlation wit h the results of the binding and functional inhibition assays, these d ata demonstrate that in the H1 and H2 subtypes both regions are availa ble for T cell recognition, they compete for the same restriction elem ent with an appearently similar binding efficiency and, therefore, fun ction as co-dominant epitopes. Due to the stabilizing effect of the fu sion peptide, peptides comprising the 306-341 or 317-341 H1 sequences are highly immunogenic and elicit a protective immune response which i nvolves the production of antibodies and interleukin-2 and tumor necro sis factor producing effector Th1 cells both directed against the 317- 329 region. Based on the similarity of the I-E-d and HLA-DR1 peptide b inding grooves and motifs, these results suggest that amino acid subst itutions inserted to the H3 subtype sequence during viral evolution ca n modify the relative MHC binding capacity and invert the local hierar chy of immunodominance of two closely situated epitopes that are able to bind to the same MHC class II molecule.