title: Development of functional human monoclonal single-chain variable fragment antibody against HIV-1 from human cervical B cells.
abstract: A panel of novel recombinant single-chain variable fragment (scFv) antibody against human immunodeficiency virus type-1 (HIV-1) was isolated and characterized. We generated human scFvs using RNA harvested from cervical B lymphocytes of Kenyan prostitutes who are highly exposed to HIV-1, but remain persistently seronegative. The variable regions of the heavy (VH) and light (VL) chain antibody genes were selected as hybrids using guided-selection with the VL and VH, respectively, of a derivative of IgGb(12) using the phagemid vector pComb3X. IgGb(12) is a previously well-characterized HIV-1 neutralizing human monoclonal antibody (MAb). One of the hybrid scFv, IgA6/4L, neutralizes HIV-1 infectivity in in vitro cell culture assay. The cervical VH and VL chain antibody genes were connected by a DNA linker and subcloned in pComb3X. The cervical scFv clones were functional in recognizing HIV-1 gp120 by enzyme-linked immunosorbant assay (ELISA) and on cells in flow cytometry. Whole IgGb(12) does not inhibit binding of clones IgA6/5k nor IgA6/30lambda to gp120, which suggests that they bind different epitopes. Nucleotide sequence analysis of the cervical scFv show the clones are unique and reveal interesting characteristics of human cervical V gene pools. This work demonstrates, for the first time, cloning of a functional scFv MAb to a sexually transmitted disease pathogen from local cervical B-cell pools in exposed humans.
authors.affiliation: Monoclonal Antibody Section, National Centre for Foreign Animal Disease, 1015 Arlington St., Winnipeg, Manitoba, Canada. berryjd@inspection.gc.ca
authors.forename: Jody D
authors.initials: JD
authors.lastname: Berry
authors.forename: John
authors.initials: J
authors.lastname: Rutherford
authors.forename: Gregg J
authors.initials: GJ
authors.lastname: Silverman
authors.forename: Rupert
authors.initials: R
authors.lastname: Kaul
authors.forename: Marikka
authors.initials: M
authors.lastname: Elia
authors.forename: Sarah
authors.initials: S
authors.lastname: Gobuty
authors.forename: Roberta
authors.initials: R
authors.lastname: Fuller
authors.forename: Francis A
authors.initials: FA
authors.lastname: Plummer
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Barbas
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D001483:Base Sequence; D002584:Cervix Uteri / Q000166:cytology / Q000276:immunology*; D005260:Female; D015483:HIV Antibodies / Q000235:genetics* / Q000276:immunology; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D007630:Kenya; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D015483:HIV Antibodies; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins