Epitope mapping of a monoclonal antibody specific for human herpesvirus 6 variant A immediate-early 2 protein

Background: Human herpesvirus 6 (HHV-6) variants A and B are distinct viruses that differ in their biological properties and association to disease. Diagnostic tools able to discriminate between these two variants and between active or latent HHV-6 infection are much needed. In our effort to develop variant-specific antibodies against HHV-6 immediate-early (IE) proteins, we had previously generated P6H8, a monoclonal antibody (mAb) reacting specifically with the immediate-early 2 (IE2) proteins from HHV-6A. Objectives: To characterize the P6H8 HHV-6 variant specific mAb and evaluate its potential as part of a variant-specific diagnostic tool for HHV-6A infection. Consequently, our objective was to map the epitope recognized by P6H8. Study design: In order to map P6H8 reactivity by Western blotting, we generated deletion mutants of IE2 protein as well as various GST-IE2 fusion proteins. HHV-6A infected cells were used to demonstrate P6H8 reactivity against native IE2. A synthetic peptide corresponding to the P6H8 epitope was used to confirm our results and block P6H8 reactivity. Results: We mapped the P6H8 epitope to amino acids 1078-1089 of HHV-6A IE2. A peptide (FTPFYYQSSRTR) recreating this epitope was effective in blocking the recognition of both native and recombinant IE2 by P6H8. Conclusions: Our work provides a precise characterization of the P6H8 mAb and its specificity toward the IE2 protein of HHV-6 variant A which could prove useful for the differential diagnostic of active infection by HHV-6. (c) 2007 Elsevier B.V. All rights reserved.