The determination in this laboratory of the complete antigenic structures of several
proteins initially relied on a multi-approach complex chemical strategy and revealed that antigenic sites are surface locations which could be either 'continuous' or 'discontinuous' in architecture. More recently, we introduced a simplified comprehensive synthetic approach for the localization of the continuous antigenic sites of a
protein. The approach depends on the synthesis of consecutive overlapping
peptides, of uniform size and overlaps, and encompass the entire
protein chain, from the beginning to the end. The latter approach is rather costly and labor-intensive, especially when applied to large
protein molecules. All these studies showed, however, that
protein antigenic sites occupy surface areas on a
protein molecule. In order to render the determination of
protein antigenic sites more feasible within a reasonable period of time, we considered that only the
protein surface needs to be examined. Thus, for a
protein of known three-dimensional structure, the
protein surface can be readily screened for the continuous antigenic sites by the systematic synthesis and examination of immunochemical activity of all exposed segments of the
protein. We have applied this approach here to influenza A virus
hemagglutinin. Twelve
peptides (11 reported for the first time here, and one reported previously), representing continuous surface segments of the molecule, have so far been synthesized, purified, characterized and their immunochemical activity studied. The
peptides were found to bind anti-
viral antibodies raised in outbred mice and
antibodies in human sera from individuals that had suffered a recent
influenza A
infection. In one mouse strain (Balb/c; H-2d) so far examined, several of the
peptides stimulated an in vitro proliferative response of T cells from virus (X-31)- primed mice. Finally,
antisera to the
peptides were raised in mice and, as expected, were found to bind to intact virus. In most cases, anti-
peptide antibodies, did not bind disrupted virus. These studies indicate that
protein 'continuous' antigenic sites can be localized by systematic synthetic scanning of the surface. It is emphasized that the approach is useful only for the localization of 'continuous' sites. The results also reveal that the antigenic structure of influenza virus
hemagglutinin is more complex than has hitherto been suspected.