title: Site-directed primary in vitro immunization: production of HIV-1 neutralizing human monoclonal antibodies from lymphocytes obtained from seronegative donors.
abstract: The design of an in vitro immunization system based on a synthetic heterotope immunogen, which was a peptide containing both T- and B-cell epitopes, that elicited a neutralizing, primary human humoral immune response against the human immunodeficiency virus (HIV-1) is reported here. This heterotope construct contained the major neutralizing B-cell epitope, within the V3 region of glycoprotein 120 (gp120), linked to a promiscuous helper T-cell epitope of tetanus toxin. The peptide was used to induce a human humoral in vitro immune response against the V3 region, using lymphocytes obtained from healthy, sero-negative blood donors. The in vitro immunized peripheral blood lymphocytes were Epstein-Barr virus infected and the antibody response to the synthetic peptide was evaluated using a solid-phase ELISA with the recombinant C-terminal fragment of gp120 (pB1, amino acid residues 287-467, derived from the HIV-1 LAI isolate). The heterotope construct yielded a significantly frequency of specifically immunized B cells, in contrast to the control immunizations with individual T and B epitopes, mixtures of these epitopes or no immunogen at all. This approach allowed us to generate human monoclonal antibodies, using lymphocytes derived from sero-negative donors, that cross-neutralized several HIV-1 strains, inhibited syncytia formation as well as prevented spreading of the viral infection from cell to cell. Thus, site-directed in vitro immunization using synthetic heterotopes might prove valuable in the dissection and induction of a protective humoral immune response.
authors.affiliation: Department of Immunotechnology, Lund University, Sweden.
authors.forename: L T
authors.initials: LT
authors.lastname: Chin
authors.forename: J
authors.initials: J
authors.lastname: Hinkula
authors.forename: M
authors.initials: M
authors.lastname: Levi
authors.forename: M
authors.initials: M
authors.lastname: Ohlin
authors.forename: B
authors.initials: B
authors.lastname: Wahren
authors.forename: C A
authors.initials: CA
authors.lastname: Borrebaeck
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D002460:Cell Line; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D015483:HIV Antibodies / Q000096:biosynthesis*; D015658:HIV Infections / Q000276:immunology; D015497:HIV-1 / Q000276:immunology*; D004854:Herpesvirus 4, Human; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007114:Immunization; D008214:Lymphocytes / Q000276:immunology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D015483:HIV Antibodies; D010455:Peptides