Expression and immunoreactivity of high-risk human papillomavirus E4 prtein

某些被称为高危型的人乳头瘤病毒类型被认为会导致宫颈癌和宫颈上皮内瘤变（CIN）。最近已经知道这些类型的E4蛋白在CIN上皮中大量表达。然而，这种表达尚未在临床样本中广泛研究。本研究旨在检测E4抗体在特定HPV类型中的表达模式。我们获得了7种不同的针对HPV 11、16、18、31、33、56型E4蛋白的多克隆抗体，这些抗体来自免疫兔并纯化。在开始这个实验时，我们尝试在细胞学样本中使用三种不同的HPV基因分型方法对细胞学异常的女性进行HPV基因分型。这些方法现在已经在日本商业化了。我们发现不同的化验结果在一些被检查的样品中显示不一致。在感染不同HPV类型的病例中，更经常观察到不一致的结果。正如我们先前预期的那样，这种类型的多重HPV型感染在细胞学异常的女性中更为常见。现在我们将在这些显示不一致结果的ease中分析更多细节。这是E4进一步实验研究的关键一步。在大多数确定HPV型别的CIN1和cin2,3病变中，从中层到浅层上皮均有E4表达。部分病例表达丰富。然而，一些免疫细胞也被这些抗体染色。我们必须弄清楚对免疫细胞的阳性反应仅仅是由于交叉反应还是由于这些免疫细胞中存在E4。此外，我们必须在免疫染色中测试这些抗体的类型特异性。E4蛋白免疫染色法可用于高危HPV感染的诊断，并且E4可能是治疗性HPV疫苗的良好靶点。

Certain human papillomavirus types named high-risk types are thought to cause cervical cancer and cervical intraepithelial neoplasia (CIN). It has been recently known that E4 proteins of these types are abundantly expressed in CIN epithelium. However, such expression has not been investigated extensively in clinical samples. This study is to examine the expression pattern using E4 antibodies for specific HPV types.We obtained seven different polyclonal antibodies against E4 proteins of HPV types 11, 16, 18, 31, 33, 56, which were produced from immunized rabbits and purified. lb start this experiment, we tried to do HPV genotyping in cytological samples using three different HPV genotyping methods in women who were abnormal in cytology. These methods are now commercially available in Japan. We found that different assays have shown discordant results in some of the samples examined. Discordant results were more frequently observed in the cases infected with different HPV types. This type of multiple HPV type infection were more common in women having abnormal cytology as we have previously expected. Now we are going to analyze more detail in these eases showing discordant results. This is a crucial step for further experiment for E4 study.E4 expression has been confirmed on epithelium from middle to superficial layers in most of CIN1 and CIN2, 3 lesions in which HPV types were determined. Abundant expression was seen in some cases. However, some immune cells were also stained by these antibodies. We have to figure out the positive reaction to immune cell is merely due to cross-reaction or due to presence of E4 in these immune cells. Furthermore, we have to test type-specificity of these antibodies in immune-staining. E4 protein immunostaining method may be useful for diagnostic purpose of high-risk HPV infection, and furthermore E4 might be a good target for therapeutic HPV vaccine.