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)蛋白抗原区的多肽，并确定人血清抗体识别的HA蛋白的抗原决定簇位置。此外，我们还将分析每个抗原决定簇的免疫原性程度。到目前为止，我们已经取得了以下结果。根据甲型流感病毒A/Kamata/14/91(H3N2)株HA多肽的氨基酸序列进行多肽合成。由8个或10个氨基酸组成的多肽被连续合成，跳过两个氨基酸，覆盖了HA多肽的100-300个氨基酸序列。使用1990/91年流感季节感染甲型H3N1流感患者的5份配对血清。采用酶联免疫吸附试验对连续表位的鉴定和免疫原性程度进行分析。与感染早期的血清相比，感染后获得的5份血清的HI效价增加了4~32倍。然而，后一批血清的HI效价除一份外，其余均为20~40。当使用由8个氨基酸组成的多肽抗原时，约有1/3的多肽与配对血清发生不同程度的反应。1例感染早期HI滴度低的病例，感染后血清与少数多肽发生特异性反应。然而，我们不能指定抗原决定簇的位置。我们进一步合成了由10个氨基酸组成的多肽。这一次，与8个氨基酸相比，抗原决定簇的识别变得更加特异，但需要进一步的实验才能得到明确的结论。进一步的分析随着患者血清的选择，氨基酸组成的多肽数量的测定以获得更多的特异性，或严格控制以查看抗原性的程度，等等，仍有待于做。

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.