Characterization of B-Cells Epitopes on HPV Capsid Proteins

Abstract

Human Papillomavirus (HPV) are considered as the major etiologic cause for a variety of benign and malignant epithelial lesions. The so-called high risk-HPV types are related to cervical cancer, while low-risk types are associated with benign mucosal lesions only. HPV types 16 and 18 are the most frequently found types in cervical cancers. Antibodies to HPV major capsid protein, L1, can develop in response to natural HPV infection as well as after vaccination. In contrast, antibodies to early, non-structural proteins, mainly E6 and E7 which are consistently expressed in HPVtransformed cells, have been found to be strongly associated with invasive carcinoma. Virus-like particles (VLP) which mimic morphologically and immunologically the native virions have been used so far as standard serological antigens. This PhD thesis had two main objectives, first to characterize the bacterially expressed, affinity-purified glutathione S-transferase fusion proteins (GST) as alternative antigens for serology in terms of displayed epitope repertoire, using a panel of 92 VLP-specific monoclonal antibodies (mAb) generated against 9 mucosal alpha papillomavirus types. The second objective was to analyze in details the natural as well as HPV vaccine-induced immune response in patients with cervical intraepithelial neoplasias (CIN II/III). Additionally, an antibody blocking assay based on Maltose binding protein (MBP)-fusions was established to analyze the typespecificity of HPV antibodies. It is shown here that GST-L1 fusion proteins display a broad variety of epitopes and thus are well suited for detection of human HPV antibodies. Cross-reactivity is associated with linear as well as conformational epitopes and can be intra- and/or interspecies specific and follows the phylogenetic relationships only loosely. Neutralizing epitopes are always conformational and mostly, but not always typespecific. The established MBP-16L1 allowed blocking the binding of mono-specific as well as cross-reactive antibodies to GST-16L1 but not that of specific antibodies to other HPV types. Vaccination of patients with cervical intraepithelial neoplasias with HPV16L1/E7 chimeric VLP induced strong 16L1 specific and weaker 16E7 specific responses as well as L1 neutralizing antibodies. The response was positively correlated with vaccine dose the immune status before vaccination. Vaccination-induced crossreactivity to both L1 and E7 was across species and the cross-reactive sera were non cross-neutralizing for distantly related HPV. Chimeric VLP vaccination also induced antibody responses to other HPV16 early proteins suggesting that these proteins were released from the lesions in response to vaccination. In conclusion, bacterially expressed GST fusion proteins are good candidates to be used as antigens in HPV serology and are also useful tools to define and characterize the complex patterns of conformational and linear cross-reactive epitopes.