title: A dimerized single-chain variable fragment system for the assessment of neutralizing activity of phage display-selected antibody fragments specific for cytomegalovirus.
abstract: Cytomegalovirus (CMV) causes severe sequelae in congenitally infected newborns and may cause life-threatening disease in immuno-deficient patients. Recent findings demonstrate the possibility to alleviate the disease by infusing intravenous immunoglobulin G (IgG) preparations, indicating that antibodies are an effective therapeutic option. Modern molecular methodologies, like phage display, allow for the development of specific antibodies targeting virtually any antigen, including those of CMV. However, such methodologies do not in general result in products that by themselves mediate biological activity. To facilitate a semi-high-throughput approach for functional screening in future efforts to develop efficacious antibodies against CMV, we have integrated two different approaches to circumvent potential bottlenecks in such efforts. Firstly, we explored an approach that permits easy transfer of antibody fragment encoding genes from commonly used phage display vectors into vectors for the production of divalent immunoglobulins. Secondly, we demonstrate that such proteins can be applied in a novel reporter-based neutralization assay to establish a proof-of-concept workflow for the generation of neutralizing antibodies against CMV. We validated our approach by showing that divalent antibodies raised against the antigenic domain (AD)-2 region of gB effectively neutralized three different CMV strains (AD169, Towne and TB40/E), whereas two antibodies against the AD-1 region of gB displayed minor neutralizing capabilities. In conclusion, the methods investigated in this proof-of-concept study enables for a semi-high-throughput workflow in the screening and investigation of biological active antibodies.
authors.affiliation: Department of Immunotechnology, Lund University, BMC D13, SE-221 84 Lund, Sweden. Fredrika.Carlsson@immun.lth.se
authors.forename: Fredrika
authors.initials: F
authors.lastname: Carlsson
authors.forename: Mirko
authors.initials: M
authors.lastname: Trilling
authors.forename: Franck
authors.initials: F
authors.lastname: Perez
authors.forename: Mats
authors.initials: M
authors.lastname: Ohlin
mesh_terms: D057134:Antibodies, Neutralizing / Q000276:immunology* / Q000302:isolation & purification; D000914:Antibodies, Viral / Q000276:immunology* / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods; D003587:Cytomegalovirus / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000276:immunology* / Q000517:prevention & control; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D006801:Humans; D009500:Neutralization Tests; D019151:Peptide Library; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D004279:DNA, Viral; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies