Detection of neutralizing antibodies against human papillomaviruses (HPV) by inhibition of gene transfer mediated by HPV pseudovirions.

The goal of this study was to develop a human papillomavirus (HPV) neutralization assay using HPV pseudovirions generated in vitro. For this purpose, gene transfer efficiency of HPV virus-like particles (VLPs) was improved by using direct interaction between a reporter plasmid and the VLPs. Electron microscopic observation of the interaction between DNA molecules and VLPs revealed that VLPs always interact with a single DNA molecule and that VLPs bind to the end of linearized DNA molecules. An 100-fold improvement in the gene transfer was obtained by simple interaction between a linearized DNA molecule and VLPs. Moreover, direct interaction methods offer the possibility of transferring plasmids a size higher than that of the papillomavirus genome. The approach that we developed to generate HPV-16 and HPV-31 pseudovirions proved to be suitable for testing neutralizing antibodies in human sera both after immunization and after natural infection.

Marek's disease virus (MDV) homologues of herpes simplex virus type 1 UL49 (VP22) and UL48 (VP16) genes: high-level expression and characterization of MDV-1 VP22 and VP16.

Genes UL49 and UL48 of Marek's disease virus 1 (MDV-1) strain RB1B, encoding the respective homologues of herpes simplex virus type 1 (HSV-1) genes VP22 and VP16, were cloned into a baculovirus vector. Seven anti-VP22 MAbs and one anti-VP16 MAb were generated and used to identify the tegument proteins in cells infected lytically with MDV-1. The two genes are known to be transcribed as a single bicistronic transcript, and the detection of only one of the two proteins (VP22) in MSB-1 lymphoma and in chicken embryo skin cells infected with MDV-1 prompted the study of the transcription/translation of the UL49-48 sequence in an in vivo and in vitro expression system. VP16 was expressed in vitro at detectable levels, whereas it could only be detected at a lower level in a more controlled environment. It was demonstrated that VP22 is phosphorylated in insect cells and possesses the remarkable property of being imported into all cells in a monolayer. VP22 localized rapidly and efficiently to nuclei, like its HSV-1 counterpart. The DNA-binding property of VP22 is also reported and a part of the region responsible for this activity was identified between aa 16 and 37 in the N-terminal region of the protein.