title: Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes.
abstract: Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1envgenes do not yield fully native-like trimers efficiently. One suchenvgene is CZA97.012 from a neutralization-resistant (tier 2) clade C virus. As appropriately purified, native-like CZA97.012 SOSIP.664 trimers induce autologous neutralizing antibodies (NAbs) efficiently in immunized rabbits, we sought to improve the efficiency with which they can be produced and to better understand the limitations to the original design. By using structure- and antigenicity-guided mutagenesis strategies focused on the V2 and V3 regions and the gp120-gp41 interface, we developed the CZA97 SOSIP.v4.2-M6.IT construct. Fully native-like, stable trimers that display multiple bNAb epitopes could be expressed from this construct in a stable CHO cell line and purified at an acceptable yield using either a PGT145 or a 2G12 bNAb affinity column. We also show that similar mutagenesis strategies can be used to improve the yields and properties of SOSIP.664 trimers of the DU422, 426c, and 92UG037 genotypes.IMPORTANCERecombinant trimeric proteins based on HIV-1envgenes are being developed for future vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein (Env) structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. The vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. Because HIV-1 is extremely variable, a practical vaccine may need to incorporate Env trimers derived from multiple different virus sequences. Accordingly, we need to understand how to make recombinant trimers from many differentenvgenes. Here, we show how to produce trimers from a clade C virus, CZA97.012, by using an array of protein engineering techniques to improve a prototypic construct. We also show that the methods may have wider utility for otherenvgenes, thereby further guiding immunogen design.
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
authors.forename: Rajesh P
authors.initials: RP
authors.lastname: Ringe
authors.affiliation: Department of Integrative Structural and Computational Biology, International AIDS Vaccine Initiative Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, California, USA.|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: Anila
authors.initials: A
authors.lastname: Yasmeen
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: Victor M
authors.initials: VM
authors.lastname: Cruz Portillo
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 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 Integrative Structural and Computational Biology, International AIDS Vaccine Initiative Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, California, USA.|Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Kimmo
authors.initials: K
authors.lastname: Rantalainen
authors.affiliation: Department of Integrative Structural and Computational Biology, International AIDS Vaccine Initiative Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, California, USA.|Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Lauren G
authors.initials: LG
authors.lastname: Holden
authors.affiliation: Department of Integrative Structural and Computational Biology, International AIDS Vaccine Initiative Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, California, USA.|Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Cottrell
authors.affiliation: Department of Integrative Structural and Computational Biology, International AIDS Vaccine Initiative Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, California, USA.|Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA.|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 Integrative Structural and Computational Biology, International AIDS Vaccine Initiative Neutralizing Antibody Center and the Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, California, USA.|Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA.
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.
authors.forename: P J
authors.initials: PJ
authors.lastname: Klasse
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA jpm2003@med.cornell.edu.
authors.forename: John P
authors.initials: JP
authors.lastname: Moore
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000096:biosynthesis / Q000276:immunology; D016466:CHO Cells; D003412:Cricetulus; D000939:Epitopes / Q000276:immunology; D005838:Genotype; D015483:HIV Antibodies / Q000096:biosynthesis / Q000276:immunology; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D007114:Immunization; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods; D055503:Protein Multimerization; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D012995:Solubility; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D057134:Antibodies, Neutralizing; D000939:Epitopes; D015483:HIV Antibodies; D011994:Recombinant Proteins; D054299:env Gene Products, Human Immunodeficiency Virus
keywords: Env trimers; HIV-1 vaccine