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Sci. attachment and to identify multiple epitopes on VZV-specific proteins. INTRODUCTION Varicella-zoster computer virus (VZV), the etiologic agent of varicella (chickenpox) and zoster (shingles), is an exclusively human pathogen and a member of the family of enveloped DNA viruses. Herpesviruses cause both lytic and latent infections. Lytic infection requires membrane fusion, an event governed by a core complex consisting of conserved glycoproteins gB, gH, SSR128129E and gL. Along with membrane fusion, VZV gH and gL are also involved in computer virus egress and are essential for computer virus replication (1C4). In addition to mediating Rabbit Polyclonal to CSTF2T computer virus access, VZV glycoproteins can traffic from infected cells to uninfected cells (5). VZV glycoproteins induce strong humoral immune responses both in naturally infected individuals and in varicella or zoster vaccine recipients (6C10). While VZV gE is the most immunogenic and predominant glycoprotein in VZV-infected cell membranes, antibodies to VZV gH are the major neutralizing antibodies (11C16). It is likely that neutralizing activity by the SSR128129E gH/gL complex effectively prevents cell-to-cell computer virus spread (5, 17C20). Analysis of VZV attachment and membrane fusion requires highly specific neutralizing antibodies. Hybridoma cell lines and phage display libraries produce human anti-VZV gE and gH monoclonal antibodies (MAbs) that neutralize computer virus contamination (21C24). Monoclonal antibodies that identify the VZV gH/gL protein complex hold promise in therapies including passive transfer of neutralizing VZV antibodies (15, 16, 25, 26). Here we present a new method for building a recombinant human monoclonal anti-VZV antibody and show that this antibody detects a conformational epitope around the gH/gL complex and neutralizes computer virus. MATERIALS AND METHODS Cells and computer virus. Human lung fibroblast (HFL) and human embryonic kidney (HEK-EBNA 293) cells (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s altered Eagle’s medium supplemented with 4 mM l-glutamine (DMEM; Sigma-Aldrich, St. Louis, MO) and 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA). VZV was propagated by cocultivating infected cells with uninfected cells as explained previously (27). Infected HFL cultures were harvested at the height of virus-induced cytopathic effect, usually at 3 days postinfection (dpi). Construction of recombinant antibody. Blood was collected 7 days after immunization with zoster vaccine (Zostavax; Merck, Whitehouse Station, NJ) from a consenting healthy 59-year-old man with a history of varicella at age 7 and who was not taking any immunosuppressive drugs. White blood cells were isolated using Vacutainer CPT tubes (Becton-Dickinson, Franklin Lakes, NJ). B cell subsets and circulating CD19+ CD20? CD38++ CD27++ SSR128129E plasma blasts were sorted using specific fluorescent antibodies against CD3 (fluorescein isothiocyanate [FITC]), CD20 (FITC), CD14 (FITC), CD19 (Pac-blue), CD27 (R-phycoerythrin [R-PE]), and CD38 (allophycocyanin [APC]-Cy5.5), as described previously (28) and as diagrammed in Fig. 1. Plasma blasts were deposited at 1 cell per well in 96-well SSR128129E plates and lysed, and cDNA was synthesized immediately as explained previously (29). Heavy and light chain variable (V) region sequences expressed by single plasma blasts were amplified using a set of family-based leader and framework primers (29). V region sequences were analyzed using DNAsis software (San Francisco, CA), and sequences were aligned to immunoglobulin (Ig) databases (V base [http://www2.mrc-lmb.cam.ac.uk/vbase]) to identify the closest human VH and VL germ collection sequences. Flag-tagged bivalent human IgG1 antibodies were produced in HEK293 cells from a single plasma blast clone, using established vectors for expression (30), affinity purified on protein A-Sepharose beads, concentrated to about 500 l using Centricon YM 30 centrifugal filter devices (Millipore, Bedford, MA), and dialyzed overnight at 4C in phosphate-buffered saline (PBS). The antibody, named rec-RC IgG, was quantified (bicinchoninic acid [BCA] protein assay; Pierce Chemical, Rockford, IL), supplemented with 0.1% protease-free and IgG-free bovine serum albuminC0.002% NaN3, and stored at 4C (30). Open in a separate windows Fig 1 Cloning of VZV-specific monoclonal antibodies from your circulating plasma blast pool after Zostavax immunization. Seven days after Zostavax immunization, circulating plasma blasts (CD19+ CD27+ CD38+ CD3? CD20?) were sorted (1 cell/well), and heavy and light chain variable regions from each cell were amplified. One pair of identical heavy and light chain variable regions was expressed in HEK293 cells, generating the monoclonal recombinant antibody rec-RC IgG. Dot plots show the expression of CD27 and CD38 among CD19-gated cells that were CD3 and CD20 unfavorable. FITC, fluorescein isothiocyanate;.