title: A native-like SOSIP.664 trimer based on an HIV-1 subtype B env gene.
abstract: Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike should expose as many epitopes as possible for broadly neutralizing antibodies (bNAbs) but few, if any, for nonneutralizing antibodies (non-NAbs). Soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A strain BG505 approach this ideal and are therefore plausible vaccine candidates. Here, we report on the production and in vitro properties of a new SOSIP.664 trimer derived from a subtype B env gene, B41, including how to make this protein in low-serum media without proteolytic damage (clipping) to the V3 region. We also show that nonclipped trimers can be purified successfully via a positive-selection affinity column using the bNAb PGT145, which recognizes a quaternary structure-dependent epitope at the trimer apex. Negative-stain electron microscopy imaging shows that the purified, nonclipped, native-like B41 SOSIP.664 trimers contain two subpopulations, which we propose represent an equilibrium between the fully closed and a more open conformation. The latter is different from the fully open, CD4 receptor-bound conformation and may represent an intermediate state of the trimer. This new subtype B trimer adds to the repertoire of native-like Env proteins that are suitable for immunogenicity and structural studies.
  
  
  OBJECTIVE
  The cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccine strategies are based on inducing neutralizing antibodies. For HIV-1, one approach involves using recombinant, soluble protein mimics of the native trimer. At present, the only reliable way to make native-like, soluble trimers in practical amounts is via the introduction of specific sequence changes that confer stability on the cleaved form of Env. The resulting proteins are known as SOSIP.664 gp140 trimers, and the current paradigm is based on the BG505 subtype A env gene. Here, we describe the production and characterization of a SOSIP.664 protein derived from a subtype B gene (B41), together with a simple, one-step method to purify native-like trimers by affinity chromatography with a trimer-specific bNAb, PGT145. The resulting trimers will be useful for structural and immunogenicity experiments aimed at devising ways to make an effective HIV-1 vaccine.
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: Pavel
authors.initials: P
authors.lastname: Pugach
authors.affiliation: 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.forename: Gabriel
authors.initials: G
authors.lastname: Ozorowski
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: Albert
authors.initials: A
authors.lastname: Cupo
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Ringe
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: Anila
authors.initials: A
authors.lastname: Yasmeen
authors.affiliation: 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.forename: Natalia
authors.initials: N
authors.lastname: de Val
authors.affiliation: Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Ronald
authors.initials: R
authors.lastname: Derking
authors.affiliation: 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.forename: Helen J
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: Jacob
authors.initials: J
authors.lastname: Korzun
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Golabek
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: de Los Reyes
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Ketas
authors.affiliation: 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.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Julien
authors.affiliation: Department of Immunology and Microbial Science, IAVI Neutralizing Antibody Center and Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA Ragon Institute of MGH, MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Burton
authors.affiliation: 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.forename: Ian A
authors.initials: IA
authors.lastname: Wilson
authors.affiliation: 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.forename: Rogier W
authors.initials: RW
authors.lastname: Sanders
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: P J
authors.initials: PJ
authors.lastname: Klasse
authors.affiliation: 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 abward@scripps.edu jpm2003@med.cornell.edu.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Ward
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA abward@scripps.edu jpm2003@med.cornell.edu.
authors.forename: John P
authors.initials: JP
authors.lastname: Moore
mesh_terms: D016915:AIDS Vaccines / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000276:immunology; D006801:Humans; D055503:Protein Multimerization; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D016915:AIDS Vaccines; D000939:Epitopes; D054299:env Gene Products, Human Immunodeficiency Virus