Analysis of antigenic determinant sites of influenza virus using epitope scanning method

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The purpose of this study is to synthesize peptides continuously which cover antigenic region of the hemagglutinin (HA) protein of influenza virus, and determine the antigenic determinant sites of the HA protein recognized by the human serum antibody. Furtheremore, we will analyze the degree of immunogenicity of each antigenic determinant site. We have obtained the following results until now.1. Multipin peptide synthesis was done according to the amino acid sequence of the HA polypeptide of influenza virus A/Kamata/14/91 (H3N2). The peptides consisting of eight or ten amino acids were continuously synthesized skipping two amino acids, covering the amino acid seuences between 100 to 300 of the HA polypeptide. Five paired sera of the patients who were infected with influenza A (H3N2) during the 1990/91 influenza season were used. The identification and the degree of immunogenicity of the ocntinuous epitopes were analyzed by ELISA assay.2. Five sera obtained after postinfection had 4 to 32-fold increased HI titers compared to those of the sera obtained at early stage of infection. However, the latter sera except one had HI titers of 20 to 40. When peptide antigens consisting of eight amino acids were used, about 1/3 of the peptides reacted with paired sera at defferent degrees. In one case who had a low HI titer at earky stage of infectin, postinfection serum reacted specifically with a few peptides. However, we could not specify antigenic determinant sites. We further synthesized peptides consisting of 10 amino acids. This time, antigenic determinant sites became more specifically identified compared to those of 8 amino acids, but further experiments were necessary to get clearcut conclusions.3. Further analyzes with the selection of patient's sera, determination of the number of amino acids consisting of the peptides to get more specificity, or severe control to see the degree of antigenicity, and so on, remained to be done.