title: Improved design of an antigen with enhanced specificity for the broadly HIV-neutralizing antibody b12.
abstract: In an attempt to design immunogens that elicit broadly HIV-neutralizing antibodies, we recently engineered monomeric HIV-1 gp120 to bind preferentially b12, a broadly neutralizing antibody to the CD4-binding site (CD4bs) on gp120, by mutating four central residues in the CD4bs to alanine and introducing extra N-glycosylation sites potentially to mask unwanted B-cell epitopes. Despite the favorable antigenicity of this mutant, it harbors two potential caveats that may limit its effectiveness to elicit b12-like antibodies: (i) b12-binding affinity is reduced relative to wild-type gp120 and (ii) binding of some non-neutralizing antibodies to the N-terminal C1 region of gp120 is still observed. Here, we sought to correct these potential limitations. By reverting one of the added N-glycosylation sites on the gp120 core, b12 binding was improved without affecting the epitope-masking properties of the original mutant. Furthermore, truncation of the gp120 N-terminus eliminated binding of the anti-C1 antibodies. Finally, based on the binding profiles of additional non-neutralizing antibodies tested here, further N-glycosylation sites were incorporated to mask their corresponding epitopes. The resulting hyperglycosylated gp120 variants bind b12 and another broadly neutralizing antibody, 2G12, with apparent affinities approaching that of wild-type gp120, but do not bind 21 non- or weakly neutralizing antibodies to seven different epitopes on gp120. These hyperglycosylated variants expand our panel of glycoengineered gp120s that are currently being evaluated for their ability to elicit broadly neutralizing antibodies.
authors.affiliation: Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA. rpanto@scripps.edu
authors.forename: R
authors.initials: R
authors.lastname: Pantophlet
authors.forename: I A
authors.initials: IA
authors.lastname: Wilson
authors.forename: D R
authors.initials: DR
authors.lastname: Burton
mesh_terms: D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015704:CD4 Antigens / Q000378:metabolism; D000939:Epitopes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D015483:HIV Antibodies / Q000378:metabolism; D015488:HIV Antigens / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D000939:Epitopes; D015483:HIV Antibodies; D015488:HIV Antigens; D015699:HIV Envelope Protein gp120