National Institute for Medical Research, London, United Kingdom.
PLoS One. 2012;7(12):e49974. doi: 10.1371/journal.pone.0049974. Epub 2012 Dec 3.
High-risk human papillomavirus (HPV) infections are the cause of nearly all cases of cervical cancer. Although the detection of HPV DNA has proved useful in cervical diagnosis, it does not necessarily predict disease presence or severity, and cannot conclusively identify the causative type when multiple HPVs are present. Such limitations may be addressed using complementary approaches such as cytology, laser capture microscopy, and/or the use of infection biomarkers. One such infection biomarker is the HPV E4 protein, which is expressed at high level in cells that are supporting (or have supported) viral genome amplification. Its distribution in lesions has suggested a role in disease staging. Here we have examined whether type-specific E4 antibodies may also allow the identification and/or confirmation of causal HPV-type. To do this, type-specific polyclonal and monoclonal antibodies against three E4 proteins (HPV-16, -18, and -58) were generated and validated by ELISA and western blotting, and by immunohistochemistry (IHC) staining of epithelial rafts containing these individual HPV types. Type-specific detection of HPV and its associated disease was subsequently examined using formalin-fixed paraffin-embedded cervical intra-epithelial neoplasias (CIN, (n = 247)) and normal controls (n = 28). All koilocytotic CIN1 lesions showed type-specific E4 expression of their respective HPV types. Differences were noted amongst E4 expression patterns in CIN3. HPV-18 E4 was not detected in any of the 6 HPV-18 DNA-positive CIN3 lesions examined, whereas in HPV-16 and -58 CIN3, 28/37 (76%) and 5/9 (55.6%) expressed E4 respectively, usually in regions of epithelial differentiation. Our results demonstrate that type-specific E4 antibodies can be used to help establish causality, as may be required when multiple HPV types are detected. The unique characteristics of the E4 biomarker suggest a role in diagnosis and patient management particularly when used in combination.
高危型人乳头瘤病毒（HPV）感染是导致几乎所有宫颈癌病例的原因。尽管 HPV DNA 的检测已被证明在宫颈诊断中有用，但它并不能必然预测疾病的存在或严重程度，并且当存在多种 HPV 时，不能明确确定致病类型。这些局限性可以通过细胞学、激光捕获显微镜和/或使用感染生物标志物等互补方法来解决。一种这样的感染生物标志物是 HPV E4 蛋白，它在支持（或曾经支持）病毒基因组扩增的细胞中高水平表达。其在病变中的分布表明它在疾病分期中具有作用。在这里，我们研究了针对 HPV 型特异性 E4 抗体是否也可以用于鉴定和/或确认因果 HPV 型。为此，我们针对三种 E4 蛋白（HPV-16、-18 和 -58）生成了型特异性多克隆和单克隆抗体，并通过 ELISA 和 Western blot 以及含有这些 HPV 型的上皮筏的免疫组织化学（IHC）染色进行了验证。随后使用福尔马林固定石蜡包埋的宫颈上皮内瘤变（CIN，（n=247））和正常对照（n=28）检查了针对 HPV 及其相关疾病的型特异性检测。所有 koilocytotic CIN1 病变均显示出各自 HPV 型的型特异性 E4 表达。在 CIN3 中，E4 表达模式存在差异。在 6 例 HPV-18 DNA 阳性的 CIN3 病变中，均未检测到 HPV-18 E4，而在 HPV-16 和 -58 CIN3 中，分别有 28/37（76%）和 5/9（55.6%）表达 E4，通常在上皮分化区域。我们的结果表明，型特异性 E4 抗体可用于帮助确定因果关系，当检测到多种 HPV 型时可能需要这样做。E4 生物标志物的独特特征表明它在诊断和患者管理中具有作用，特别是当与其他方法联合使用时。
