title: Design, expression, and immunogenicity of a soluble HIV trimeric envelope fragment adopting a prefusion gp41 configuration.
abstract: The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) is comprised of non-covalently associated gp120/gp41 subunits that form trimeric spikes on the virion surface. Upon binding to host cells, Env undergoes a series of structural transitions, leading to gp41 rearrangement necessary for fusion of viral and host membranes. Until now, the prefusion state of gp41 ectodomain (e-gp41) has eluded molecular and structural analysis, and thus assessment of the potential of such an e-gp41 conformer to elicit neutralizing antibodies has not been possible. Considering the importance of gp120 amino (C1) and carboxyl (C5) segments in the association with e-gp41, we hypothesize that these regions are sufficient to maintain e-gp41 in a prefusion state. Based on the available gp120 atomic structure, we designed several truncated gp140 variants by including the C1 and C5 regions of gp120 in a gp41 ectodomain fragment. After iterative cycles of protein design, expression and characterization, we obtained a variant truncated at Lys(665) that stably folds as an elongated trimer under physiologic conditions. Several independent biochemical/biophysical analyses strongly suggest that this mini-Env adopts a prefusion e-gp41 configuration that is strikingly distinct from the postfusion trimer-of-hairpin structure. Interestingly, this prefusion mini-Env, lacking the fragment containing the 2F5/4E10 neutralizing monoclonal antibody binding sites, displays no detectable HIV-neutralizing epitopes when employed as an immunogen in rabbits. The result of this immunogenicity study has important implications for HIV-1 vaccine design efforts. Moreover, this engineered mini-Env protein should facilitate three-dimensional structural studies of the prefusion e-gp41 and serve to guide future attempts at pharmacologic and immunologic intervention of HIV-1.
authors.affiliation: Laboratory of Immunobiology and Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Zhi-Song
authors.initials: ZS
authors.lastname: Qiao
authors.forename: Mikyung
authors.initials: M
authors.lastname: Kim
authors.forename: Bruce
authors.initials: B
authors.lastname: Reinhold
authors.forename: David
authors.initials: D
authors.lastname: Montefiori
authors.forename: Jia-huai
authors.initials: JH
authors.lastname: Wang
authors.forename: Ellis L
authors.initials: EL
authors.lastname: Reinherz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D016367:Baculoviridae / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry; D015686:Gene Products, env / Q000737:chemistry* / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000737:chemistry / Q000378:metabolism; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D007313:Insecta; D008027:Light; D008156:Luciferases / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008854:Microscopy, Electron; D008954:Models, Biological; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012542:Scattering, Radiation; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D054299:env Gene Products, Human Immunodeficiency Virus
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D000939:Epitopes; D015686:Gene Products, env; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; D011134:Polysaccharides; D054299:env Gene Products, Human Immunodeficiency Virus; C097729:gp140 envelope protein, Human immunodeficiency virus 1; D049452:Green Fluorescent Proteins; D008156:Luciferases; D010447:Peptide Hydrolases; D014357:Trypsin; D008239:Lysine