Generation of monoclonal antibodies directed against the immunogenic glycoprotein K8.1 of human herpesvirus 8

Human Herpesvirus 8 (HHV-8) is clearly associated with Kaposi's sarcoma (KS ), body cavity-based lymphomas (BCBL), and certain forms of multifocal Cast leman's disease (MCD). It appears to be the sexually transmissible agent in volved in the development of AIDS-associated KS. HHV-8 genomes are invariab ly present in BCBL-derived cell lines where lytic replication of the virus can be induced by phorbol esters (PE), First-generation HHV-8 serological a ssays were based on these cell lines. More recently, several genes encoding HHV-8 antigens have been identified. One of the most reactive antigens is encoded by HHV-8 open reading frame K8.1, Although K8.1 does not exhibit ov ert sequence homology to any other known gene, it is likely to be analogous to gp220/350 of Epstein-Barr or gp150 of murine herpesvirus-68, virion-env elope glycoproteins involved in target cell recognition. Mice were immunize d with purified GST-K8.1 fusion protein expressed in E. coli, After fusion of murine plasma cells with the myeloma cell line P3-X63-Ag8, monoclonal an tibodies (MAbs) were generated, which are specifically directed against K8. 1 protein. The binding site for each MAb was identified by deletion mutant analysis using recombinant GST-K8.1 mutants and K8.1-specific peptides, Wit hout exception, the epitopes recognized by these MAbs were located within t he N-terminal part of the protein [amino acids (aa) 29 to 80], thus identif ying a highly immunogenic region. These antibodies will not only be useful tools for HHV-8 diagnostics, but will also facilitate the analysis of K8.1 function.