E4 antibodies facilitate detection and type-assignment of active HPV infection in cervical disease
Heather Griffin, Zhonglin Wu, Rebecca Marnane, Vincent Dewar, Anco Molijn, Wim Quint, Christine Van Hoof, Frank Struyf, Brigitte Colau, David Jenkins, John Doorbar, Heather Griffin, Zhonglin Wu, Rebecca Marnane, Vincent Dewar, Anco Molijn, Wim Quint, Christine Van Hoof, Frank Struyf, Brigitte Colau, David Jenkins, John Doorbar
Abstract
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.
Conflict of interest statement
Competing Interests: VD, FS, BC and DJ are or were, at the time of the study, employees of GlaxoSmithKline Biologicals, Rixensart. CVH is an employee of XpePharma and Science, Wavre. AM, WQ and DJ are employees of DDL Diagnostic Laboratory. BC and FS own shares and options to shares in GSK. JD is designated inventor on United Kingdom patent PCT/GB97/03321 (filed in December 1997) and PCT/GB01/01176 (filed in April 2001) owned by MRC. There are no other products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
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