Reactivity pattern of 92 monoclonal antibodies with 15 human papillomavirus types Raeda Z. Rizk, 1 Neil D. Christensen, 2 Kristina M. Michael, 1 Martin Mu ¨ ller, 1 Peter Sehr, 1 3 Tim Waterboer 1 and Michael Pawlita 1 Correspondence Michael Pawlita M.Pawlita@dkfz.de 1 Department of Genome Modifications and Cancer, Infection and Cancer Program, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 242, 69120 Heidelberg, Germany 2 Departments of Pathology and Microbiology and Immunology, The Jake Gittlen Cancer Research Foundation, College of Medicine, Pennsylvania State University, Hershey, PA 17033, USA Received 7 May 2007 Accepted 23 August 2007 Most anti-human papillomavirus (HPV) capsid antibody assays are based on virus-like particles (VLP). We evaluated glutathione S-transferase (GST)–L1 fusion proteins as ELISA antigens for determining type specificity and cross-reactivity of 92 VLP-specific monoclonal antibodies (mAb) generated against nine mucosal alpha papillomavirus types of species 7, 9 and 10. The antibody panel included 25 new mAb, and 24 previously published mAb are further characterized. We determined the cross-reactivity patterns with 15 different HPV types representing 6 species (alpha1, 2, 4, 7, 9 and 10) and neutralization and cross-neutralization properties with HPV types 6, 11, 16, 18 and 45. Eighty-nine (97 %) of the antibodies including 34, 71 and 14 recognizing neutralizing, conformational and linear epitopes, respectively, reacted with the GST–L1 protein of the HPV type used as immunogen, with log titres ranging from 2.0 to 7.3. Of these 89 antibodies, 52 % were monotypic, 20 % showed intra-species and 28 % inter-species cross-reactivity. Log neutralization titres to the immunogen HPV ranged from 1.7 to 5.6. A single cross-neutralizing mAb (H6.L12) was found. ELISA titres were always higher than neutralization titres. All neutralizing epitopes were conformational and mostly type-specific. Our data show that bacterially expressed, affinity-purified GST–L1 fusion proteins display a broad variety of epitopes and thus are well suited for detection of HPV antibodies. Cross-reactivity is associated with linear as well as conformational epitopes. Distantly related mucosal and skin alpha papillomaviruses share some conformational epitopes and the phylogenetic L1-based species definition may not define a serological unit since no species-specific epitope was found. INTRODUCTION Human papillomaviruses (HPV) are small (about 55 nm in diameter) non-enveloped DNA tumour viruses with a tropism for squamous epithelia. The icosahedral protein capsid is composed of 72 capsomeres (pentamers of the major capsid protein L1) and contains a single molecule of about 8 kbp closed circular double-stranded DNA. So far, more than 100 HPV types have been fully characterized by cloning and complete sequencing of their genomes. Classification of HPV is based on the major capsid protein L1 open reading frame nucleotide sequence: HPV of the same genus show at least 60 % sequence identity, those of the same species at least 70 %, those of the same type at least 90 % and those of the same subtype (variants) at least 98 % (de Villiers et al., 2004). HPV of the genus alpha (15 species) mostly infect anogenital and oral mucosa, some can additionally (species 2 and 8) or exclusively (species 4) infect the skin. Many alpha HPV can induce benign genital or common skin warts, while so called high-risk types (most frequently HPV types 16 and 18) (Munoz et al., 2003) belonging to species 5, 6, 7, 9 and 11, can induce intraepithelial neoplasia, the precursor of cervical cancer (Bosch & de Sanjose, 2003; Clifford et al., 2003; de Villiers et al., 2004). Upon infection with HPV, serum antibodies to L1 protein can develop (Dillner, 1999; van Doornum et al., 1998; Wang et al., 1996; Wideroff et al., 1999). This immune response is highly type-specific (Carter et al., 1996, 2000; Giroglou et al., 2001; Wang et al., 1997) and persists for years (Carter et al., 2000; Dillner, 1999). L1 antibodies are considered markers for current and past infection (Carter et al., 1996; Carter & Galloway, 1997; Kirnbauer, 1996) and are weakly associated with cervical cancer (van Doornum et al., 2003). In contrast, antibodies to the early oncoproteins E6 and E7 that are consistently expressed in HPV- transformed cells are strongly associated with cervical 3Present address: Joint DKFZ-EMBL Chemical Biology Core Facility, European Molecular Biology Laboratory (EMBL), Meyerhofstr. 1, 69117 Heidelberg, Germany. Journal of General Virology (2008), 89, 117–129 DOI 10.1099/vir.0.83145-0 0008-3145 G 2008 SGM Printed in Great Britain 117