title: Covalent trimers of the internal N-terminal trimeric coiled-coil of gp41 and antibodies directed against them are potent inhibitors of HIV envelope-mediated cell fusion.
abstract: We have engineered two soluble, covalently linked, trimeric polypeptides, N35CCG-N13 and N34CCG comprising only the internal trimeric coiled-coil of the ectodomain of HIV-1 gp41. Both trimers inhibit human immunodeficiency virus, type 1 (HIV-1) envelope (Env)-mediated cell fusion at nanomolar concentrations by targeting the exposed C-terminal region of the gp41 ectodomain in the prehairpin intermediate state. The IC50 values for N35CCG-N13 and N34CCG are approximately 15 and approximately 95 nM, respectively, in a quantitative vaccinia virus-based reporter gene assay for HIV-1 Env-mediated cell fusion using Env from the T cell tropic strain LAV. Polyclonal antibodies were raised against N35CCG-N13 and a tightly binding fraction of anti-N35CCG-N13 inhibits T cell and macrophage tropic HIV-1 Env-mediated cell fusion with respective IC50 values of approximately 0.5 and approximately 1.5 microg/ml at 37 degrees C. The tightly binding anti-N35CCG-N13 antibody fraction targets the exposed internal trimeric coiled-coil in the prehairpin intermediate state of gp41 in a manner analogous to peptides derived from the C region of the gp41 ectodomain. The potency of the tightly binding anti-N35CCG-N13 antibody fraction in the fusion assay is comparable with that of the broadly neutralizing monoclonal antibody 2G12. These results indicate that N35CCG-N13 is a potential anti-HIV therapeutic agent and represents a suitable immunogen for the generation of neutralizing monoclonal antibodies targeted to the internal trimeric coiled-coil of gp41. The data on the tightly binding anti-N35CCG-N13 antibody fraction demonstrate that the internal trimeric coiled-coil of gp41 in the prehairpin intermediate state is accessible to antibodies and that access is not restricted by either antibody size or the presence of a kinetic barrier.
authors.affiliation: Laboratories of Chemical Physics and Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Louis
authors.forename: Issa
authors.initials: I
authors.lastname: Nesheiwat
authors.forename: LengChee
authors.initials: L
authors.lastname: Chang
authors.forename: G Marius
authors.initials: GM
authors.lastname: Clore
authors.forename: Carole A
authors.initials: CA
authors.lastname: Bewley
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology*; D001483:Base Sequence; D001704:Biopolymers; D002459:Cell Fusion; D017931:DNA Primers; D015700:HIV Envelope Protein gp41 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data
chemical_list: D000906:Antibodies; D001704:Biopolymers; D017931:DNA Primers; D015700:HIV Envelope Protein gp41