CHARACTERIZATION OF EVENTS DURING THE LATE STAGES OF HPV16 INFECTION IN-VIVO USING HIGH-AFFINITY SYNTHETIC FABS TO E4

HPV late gene expression is initiated as an infected basal cell migrat es through the differentiating layers of the epidermis, resulting in t he onset of vegetative viral DNA replication and the expression of vir al late proteins. We have used a large synthetic immunoglobulin librar y displayed on phage (diversity 6.5 x 10(10) phage) to isolate three F abs (TVG405, 406, and 407) which recognize distinct epitopes on the E4 late protein of HPV16. A C-terminal monoclonal (TVG404) was generated by hybridoma technology, and N-terminal polyclonal antiserum was prep ared by peptide immunization (alpha N-term). The most potent antibody (TVG405) had an affinity for E4 of approximately 1.0 nM. All antibodie s recognized the protein in paraffin-embedded archival material, allow ing us to map events in the late stages of virus infection. Expression of E4 in vivo does not coincide with synthesis of the major virus coa t protein L1, but precedes it by 1 or 2 cell layers in premalignant le sions caused by HPV16 and by up to 20 cell layers in HPV63-induced war ts. In higher grade lesions associated with HPV16, E4 is produced in t he absence of L1. By contrast, vegetative viral DNA replication and E4 expression correlate exactly and in some lesions begin as the infecte d epithelial cell leaves the basal layer. Differentiation markers such as filaggrin, loricrin, and certain keratins are not detectable in E4 -positive cells, and nuclear degeneration is delayed. HPV16 E4 has a f ilamentous distribution in the lower epithelial layers, but associates with solitary perinuclear structures in more differentiated cells. An tibodies to the N-terminus of the protein stained these structures poo rly. Our findings are compatible with a role for the HPV16 E4 protein in vegetative DNA replication or in modifying the phenotype oi the inf ected cell to favor virus synthesis or virus release. The Fabs will be of value in the evaluation of model systems for mimicking HPV infecti on in vitro. (C) 1997 Academic Press.