title: Influences on the Design and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers.
abstract: We have investigated factors that influence the production of native-like soluble, recombinant trimers based on the env genes of two isolates of human immunodeficiency virus type 1 (HIV-1), specifically 92UG037.8 (clade A) and CZA97.012 (clade C). When the recombinant trimers based on the env genes of isolates 92UG037.8 and CZA97.012 were made according to the SOSIP.664 design and purified by affinity chromatography using broadly neutralizing antibodies (bNAbs) against quaternary epitopes (PGT145 and PGT151, respectively), the resulting trimers are highly stable and they are fully native-like when visualized by negative-stain electron microscopy. They also have a native-like (i.e., abundant) oligomannose glycan composition and display multiple bNAb epitopes while occluding those for nonneutralizing antibodies. In contrast, uncleaved, histidine-tagged Foldon (Fd) domain-containing gp140 proteins (gp140UNC-Fd-His), based on the same env genes, very rarely form native-like trimers, a finding that is consistent with their antigenic and biophysical properties and glycan composition. The addition of a 20-residue flexible linker (FL20) between the gp120 and gp41 ectodomain (gp41ECTO) subunits to make the uncleaved 92UG037.8 gp140-FL20 construct is not sufficient to create a native-like trimer, but a small percentage of native-like trimers were produced when an I559P substitution in gp41ECTO was also present. The further addition of a disulfide bond (SOS) to link the gp120 and gp41 subunits in the uncleaved gp140-FL20-SOSIP protein increases native-like trimer formation to ∼20 to 30%. Analysis of the disulfide bond content shows that misfolded gp120 subunits are abundant in uncleaved CZA97.012 gp140UNC-Fd-His proteins but very rare in native-like trimer populations. The design and stabilization method and the purification strategy are, therefore, all important influences on the quality of trimeric Env proteins and hence their suitability as vaccine components.
  
  
  OBJECTIVE
  Soluble, recombinant multimeric proteins based on the HIV-1 env gene are current candidate immunogens for vaccine trials in humans. These proteins are generally designed to mimic the native trimeric envelope glycoprotein (Env) that is the target of virus-neutralizing antibodies on the surfaces of virions. The underlying hypothesis is that an Env-mimetic protein may be able to induce antibodies that can neutralize the virus broadly and potently enough for a vaccine to be protective. Multiple different designs for Env-mimetic trimers have been put forth. Here, we used the CZA97.012 and 92UG037.8 env genes to compare some of these designs and determine which ones best mimic virus-associated Env trimers. We conclude that the most widely used versions of CZA97.012 and 92UG037.8 oligomeric Env proteins do not resemble the trimeric Env glycoprotein on HIV-1 viruses, which has implications for the design and interpretation of ongoing or proposed clinical trials of these proteins.
authors: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors: Rajesh P
authors: RP
authors: Ringe
authors: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors: Anila
authors: A
authors: Yasmeen
authors: Department of Integrative Structural and Computational Biology, IAVI Neutralizing Antibody Center, and Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA.
authors: Gabriel
authors: G
authors: Ozorowski
authors: Department of Chemistry, University of Kansas, Lawrence, Kansas, USA.
authors: Eden P
authors: EP
authors: Go
authors: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, United Kingdom.
authors: Laura K
authors: LK
authors: Pritchard
authors: Department of Medicinal Chemistry, University of Washington, Seattle, Washington, USA.
authors: Miklos
authors: M
authors: Guttman
authors: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors: Thomas A
authors: TA
authors: Ketas
authors: Department of Integrative Structural and Computational Biology, IAVI Neutralizing Antibody Center, and Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA.
authors: Christopher A
authors: CA
authors: Cottrell
authors: Department of Integrative Structural and Computational Biology, IAVI Neutralizing Antibody Center, and Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA.
authors: Ian A
authors: IA
authors: Wilson
authors: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors: Rogier W
authors: RW
authors: Sanders
authors: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors: Albert
authors: A
authors: Cupo
authors: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, United Kingdom.
authors: Max
authors: M
authors: Crispin
authors: Department of Medicinal Chemistry, University of Washington, Seattle, Washington, USA.
authors: Kelly K
authors: KK
authors: Lee
authors: Department of Chemistry, University of Kansas, Lawrence, Kansas, USA.
authors: Heather
authors: H
authors: Desaire
authors: Department of Integrative Structural and Computational Biology, IAVI Neutralizing Antibody Center, and Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA.
authors: Andrew B
authors: AB
authors: Ward
authors: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors: P J
authors: PJ
authors: Klasse
authors: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA jpm2003@med.cornell.edu.
authors: John P
authors: JP
authors: Moore
mesh_terms: D000911:Antibodies, Monoclonal; D002152:Calorimetry, Differential Scanning; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D015497:HIV-1 / Q000235:genetics*; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D009500:Neutralization Tests; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D020349:Surface Plasmon Resonance; D054299:env Gene Products, Human Immunodeficiency Virus / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D054299:env Gene Products, Human Immunodeficiency Virus