title: Llama antibody fragments recognizing various epitopes of the CD4bs neutralize a broad range of HIV-1 subtypes A, B and C.
abstract: Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120(Ds2)), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B'/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides.
authors.affiliation: Biomolecular Imaging, Department Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Nika
authors.initials: N
authors.lastname: Strokappe
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Szynol
authors.forename: Marlèn
authors.initials: M
authors.lastname: Aasa-Chapman
authors.forename: Andrea
authors.initials: A
authors.lastname: Gorlani
authors.forename: Anna
authors.initials: A
authors.lastname: Forsman Quigley
authors.forename: David Lutje
authors.initials: DL
authors.lastname: Hulsik
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.forename: Robin
authors.initials: R
authors.lastname: Weiss
authors.forename: Hans
authors.initials: H
authors.lastname: de Haard
authors.forename: Theo
authors.initials: T
authors.lastname: Verrips
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D057134:Antibodies, Neutralizing / Q000235:genetics; D001667:Binding, Competitive; D015704:CD4 Antigens; D002161:Camelids, New World / Q000235:genetics / Q000276:immunology*; D022801:Complementarity Determining Regions / Q000235:genetics; D003429:Cross Reactions; D000939:Epitopes / Q000235:genetics; D015483:HIV Antibodies / Q000235:genetics; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000276:immunology; D015497:HIV-1 / Q000145:classification* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007114:Immunization; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D017386:Sequence Homology, Amino Acid; D054299:env Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000276:immunology
chemical_list: D057134:Antibodies, Neutralizing; D015704:CD4 Antigens; D022801:Complementarity Determining Regions; D000939:Epitopes; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D054299:env Gene Products, Human Immunodeficiency Virus; C514793:gp120 protein, Human immunodeficiency virus 1; C097729:gp140 envelope protein, Human immunodeficiency virus 1