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

本研究的目的是连续合成覆盖流感病毒血凝素（HA）蛋白抗原区域的多肽，并确定人血清抗体识别的HA蛋白的抗原决定位点。此外，我们将分析每个抗原决定位点的免疫原性程度。到目前为止，我们已经获得了以下结果。根据流感病毒A/Kamata/14/91 (H3N2) HA多肽的氨基酸序列合成多针肽。跳过两个氨基酸，连续合成由8个或10个氨基酸组成的肽，覆盖HA多肽的100到300个氨基酸序列。使用1990/91年流感季节感染甲型流感（H3N2）患者的5对血清。用ELISA法分析连续表位的鉴定和免疫原性程度。与感染早期获得的血清相比，感染后获得的5种血清HI滴度增加了4至32倍。然而，除1例外，其余血清的HI滴度均为20 ~ 40。当使用由8种氨基酸组成的肽抗原时，约有1/3的肽与配对的血清发生不同程度的反应。在感染早期低HI滴度的一个病例中，感染后血清与一些肽特异性反应。然而，我们无法确定抗原决定位点。我们进一步合成了由10个氨基酸组成的肽。这一次，与8种氨基酸相比，抗原决定位点得到了更明确的鉴定，但需要进一步的实验来得到明确的结论。进一步的分析与患者血清的选择，确定组成肽的氨基酸的数量以获得更多的特异性，或严格控制看抗原性的程度，等等，还有待于做。

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.