title: Baculovirus expression of a human G2-specific, neutralizing IgG monoclonal antibody to Puumala virus.
abstract: We amplified by polymerase chain reaction the heavy- and light-chain antibody genes of a human hybridoma secreting a neutralizing, IgG monoclonal antibody to the G2 protein of Puumala virus. The heavy- and light-chain genes were cloned and sequenced and the deduced amino acids were aligned with those of other human antibodies to identify the constant and variable regions. The genes were cloned into the baculovirus plasmid transfer vector pACUW51 such that the heavy-chain and light-chain genes were under control of the baculovirus polyhedrin or p10 promoters, respectively. The transfer vector plasmid was cotransfected into cultured Spodoptera frugiperda (Sf9) cells with linearized DNA of the baculovirus Autographa californica nuclear polyhedrosis virus, and recombinant baculoviruses were selected by plaque formation on monolayers of Sf9 cells. Expression and secretion of an IgG monoclonal antibody was confirmed by assay of recombinant-infected Sf9 cell supernatants for the presence of the heavy and light chains. Specificity of the expressed human antibody was determined by immune-precipitation of radiolabeled Puumala virus proteins and by ELISA with Puumala virus-infected cell lysates. Similar quantities of the expressed IgG and the authentic monoclonal antibody neutralized Puumala virus in plaque-reduction neutralization assays. Neither the authentic nor the recombinant antibody could passively protect hamsters from challenge with Puumala virus; however, our results demonstrate the potential of this methodology for production of biologically active neutralizing antibodies.
authors.affiliation: Virology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702-5011, USA.
authors.forename: M
authors.initials: M
authors.lastname: Liang
authors.forename: M
authors.initials: M
authors.lastname: Guttieri
authors.forename: A
authors.initials: A
authors.lastname: Lundkvist
authors.forename: C
authors.initials: C
authors.lastname: Schmaljohn
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D000914:Antibodies, Viral / Q000235:genetics* / Q000276:immunology; D000918:Antibody Specificity; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D006224:Cricetinae; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005803:Genes, Immunoglobulin; D006476:Orthohantavirus / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D016133:Polymerase Chain Reaction; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D017931:DNA Primers; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains