title: Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab) and single chain variable fragment (ScFv) antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.
abstract: We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.
authors.affiliation: Laboratories of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: John M
authors.initials: JM
authors.lastname: Louis
authors.affiliation: Laboratories of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Annie
authors.initials: A
authors.lastname: Aniana
authors.affiliation: Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Katheryn
authors.initials: K
authors.lastname: Lohith
authors.affiliation: Laboratories of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Jane M
authors.initials: JM
authors.lastname: Sayer
authors.affiliation: Laboratories of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Julien
authors.initials: J
authors.lastname: Roche
authors.affiliation: Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Carole A
authors.initials: CA
authors.lastname: Bewley
authors.affiliation: Laboratories of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: G Marius
authors.initials: GM
authors.lastname: Clore
mesh_terms: D000595:Amino Acid Sequence; D057134:Antibodies, Neutralizing / Q000276:immunology*; D015483:HIV Antibodies / Q000276:immunology*; D015700:HIV Envelope Protein gp41 / Q000737:chemistry / Q000276:immunology*; D015658:HIV Infections / Q000276:immunology* / Q000821:virology; D015497:HIV-1 / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D057127:Single-Chain Antibodies / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D015483:HIV Antibodies; D015700:HIV Envelope Protein gp41; D007140:Immunoglobulin Fab Fragments; D057127:Single-Chain Antibodies
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