title: Enhanced exposure of the CD4-binding site to neutralizing antibodies by structural design of a membrane-anchored human immunodeficiency virus type 1 gp120 domain.
abstract: The broadly neutralizing antibody immunoglobulin G1 (IgG1) b12 binds to a conformationally conserved surface on the outer domain of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope (Env) glycoprotein. To develop outer domain proteins (ODs) that could be recognized selectively by CD4-binding-site (CD4-BS) antibodies, membrane-anchored ODs were generated from an HIV-1 clade B virus, TA1 R3A, which was highly sensitive to neutralization by the IgG1 b12 antibody. A 231-residue fragment of gp120 (residues 252 to 482) linked to transmembrane regions from CD4 showed b12 binding comparable to that of the native Env spike as measured by flow cytometry. Truncation of the beta 20-beta 21 hairpin (residues 422 to 436 to Gly-Gly) improved overall protein expression. Replacement of the immunodominant central 20 amino acids of the V3 loop (residues 302 to 323) with a basic hexapeptide (NTRGRR) increased b12 reactivity further. Surface calculations indicated that the ratio of b12 epitope to exposed immunogenic surface in the optimized OD increased to over 30%. This OD variant [OD(GSL)(Deltabeta 20-21)(hCD4-TM)] was recognized by b12 and another CD4-BS-reactive antibody, b13, but not by eight other CD4-BS antibodies with limited neutralization potency. Furthermore, optimized membrane-anchored OD selectively absorbed neutralizing activity from complex antisera and b12. Structurally designed membrane-anchored ODs represent candidate immunogens to elicit or to allow the detection of broadly neutralizing antibodies to the conserved site of CD4 binding on HIV-1 gp120.
authors.affiliation: Vaccine Research Center, NIAID, National Institutes of Health, Bldg. 40, 40 Convent Drive, Bethesda, MD 20892-3005, USA.
authors.forename: Lan
authors.initials: L
authors.lastname: Wu
authors.forename: Tongqing
authors.initials: T
authors.lastname: Zhou
authors.forename: Zhi-yong
authors.initials: ZY
authors.lastname: Yang
authors.forename: Krisha
authors.initials: K
authors.lastname: Svehla
authors.forename: Sijy
authors.initials: S
authors.lastname: O'Dell
authors.forename: Mark K
authors.initials: MK
authors.lastname: Louder
authors.forename: Ling
authors.initials: L
authors.lastname: Xu
authors.forename: John R
authors.initials: JR
authors.lastname: Mascola
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Burton
authors.forename: James A
authors.initials: JA
authors.lastname: Hoxie
authors.forename: Robert W
authors.initials: RW
authors.lastname: Doms
authors.forename: Peter D
authors.initials: PD
authors.lastname: Kwong
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Nabel
mesh_terms: D000595:Amino Acid Sequence; D001666:Binding Sites, Antibody; D015704:CD4 Antigens / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D015483:HIV Antibodies / Q000276:immunology / Q000378:metabolism*; D015699:HIV Envelope Protein gp120 / Q000276:immunology / Q000378:metabolism*; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D015704:CD4 Antigens; D000939:Epitopes; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D007074:Immunoglobulin G; C514793:gp120 protein, Human immunodeficiency virus 1