title: Stabilizing exposure of conserved epitopes by structure guided insertion of disulfide bond in HIV-1 envelope glycoprotein.
abstract: Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will 'snap' Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to 'lock' gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules.
authors.affiliation: Vaccines Research, Novartis Vaccines & Diagnostics Inc., Cambridge, Massachusetts, United States of America.
authors.forename: Aemro
authors.initials: A
authors.lastname: Kassa
authors.forename: Antu K
authors.initials: AK
authors.lastname: Dey
authors.forename: Pampi
authors.initials: P
authors.lastname: Sarkar
authors.forename: Celia
authors.initials: C
authors.lastname: Labranche
authors.forename: Eden P
authors.initials: EP
authors.lastname: Go
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Clark
authors.forename: Yide
authors.initials: Y
authors.lastname: Sun
authors.forename: Avishek
authors.initials: A
authors.lastname: Nandi
authors.forename: Karin
authors.initials: K
authors.lastname: Hartog
authors.forename: Heather
authors.initials: H
authors.lastname: Desaire
authors.forename: David
authors.initials: D
authors.lastname: Montefiori
authors.forename: Andrea
authors.initials: A
authors.lastname: Carfi
authors.forename: Indresh K
authors.initials: IK
authors.lastname: Srivastava
authors.forename: Susan W
authors.initials: SW
authors.lastname: Barnett
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001665:Binding Sites; D015704:CD4 Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D004220:Disulfides / Q000737:chemistry*; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D005260:Female; D015699:HIV Envelope Protein gp120 / Q000008:administration & dosage / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006801:Humans; D007114:Immunization; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011817:Rabbits; D020349:Surface Plasmon Resonance; D054299:env Gene Products, Human Immunodeficiency Virus / Q000008:administration & dosage / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: D000906:Antibodies; D015704:CD4 Antigens; D004220:Disulfides; D000939:Epitopes; D015699:HIV Envelope Protein gp120; D008024:Ligands; D054299:env Gene Products, Human Immunodeficiency Virus; C097729:gp140 envelope protein, Human immunodeficiency virus 1