title: Structure-Based Design of a Soluble Prefusion-Closed HIV-1 Env Trimer with Reduced CD4 Affinity and Improved Immunogenicity.
abstract: The HIV-1 envelope (Env) trimer is a target for vaccine design as well as a conformational machine that facilitates virus entry by transitioning between prefusion-closed, CD4-bound, and coreceptor-bound conformations by transitioning into a postfusion state. Vaccine designers have sought to restrict the conformation of the HIV-1 Env trimer to its prefusion-closed state as this state is recognized by most broadly neutralizing, but not nonneutralizing, antibodies. We previously identified a disulfide bond, I201C-A433C (DS), which stabilizes Env in the vaccine-desired prefusion-closed state. When placed into the context of BG505 SOSIP.664, a soluble Env trimer mimic developed by Sanders, Moore, and colleagues, the engineered DS-SOSIP trimer showed reduced conformational triggering by CD4. Here, we further stabilize DS-SOSIP through a combination of structure-based design and 96-well-based expression and antigenic assessment. From 103 designs, we identified one, named DS-SOSIP.4mut, with four additional mutations at the interface of potentially mobile domains of the prefusion-closed structure. We also determined the crystal structures of DS-SOSIP.4mut at 4.1-Å resolution and of an additional DS-SOSIP.6mut variant at 4.3-Å resolution, and these confirmed the formation of engineered disulfide bonds. Notably, DS-SOSIP.4mut elicited a higher ratio of tier 2 autologous titers versus tier 1 V3-sensitive titers than BG505 SOSIP.664. DS-SOSIP.4mut also showed reduced recognition of CD4 and increased thermostability. The improved antigenicity, thermostability, and immunogenicity of DS-SOSIP.4mut suggest utility as an immunogen or a serologic probe; moreover, the specific four alterations identified here, M154, M300, M302, and L320 (4mut), can also be transferred to other HIV-1 Env trimers of interest to improve their properties.IMPORTANCEOne approach to elicit broadly neutralizing antibodies against HIV-1 is to stabilize the structurally flexible HIV-1 envelope (Env) trimer in a conformation that displays predominantly broadly neutralizing epitopes and few to no nonneutralizing epitopes. The prefusion-closed conformation of HIV-1 Env has been identified as one such preferred conformation, and a current leading vaccine candidate is the BG505 DS-SOSIP variant, comprising two disulfides and an Ile-to-Pro mutation of Env from strain BG505. Here, we introduced additional mutations to further stabilize BG505 DS-SOSIP in the vaccine-preferred prefusion-closed conformation. In guinea pigs, our best mutant, DS-SOSIP.4mut, elicited a significantly higher ratio of autologous versus V3-directed neutralizing antibody responses than the SOSIP-stabilized form. We also observed an improvement in thermostability and a reduction in CD4 affinity. With improved antigenicity, stability, and immunogenicity, DS-SOSIP.4mut-stabilized trimers may have utility as HIV-1 immunogens or in other antigen-specific contexts, such as with B-cell probes.
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Gwo-Yu
authors.initials: GY
authors.lastname: Chuang
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Geng
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Marie
authors.initials: M
authors.lastname: Pancera
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Xu
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Cheng
authors.initials: C
authors.lastname: Cheng
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Priyamvada
authors.initials: P
authors.lastname: Acharya
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Chambers
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Aliaksandr
authors.initials: A
authors.lastname: Druz
authors.affiliation: Electron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.
authors.forename: Yaroslav
authors.initials: Y
authors.lastname: Tsybovsky
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Timothy G
authors.initials: TG
authors.lastname: Wanninger
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Yongping
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Doria-Rose
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Ivelin S
authors.initials: IS
authors.lastname: Georgiev
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Gorman
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: M Gordon
authors.initials: MG
authors.lastname: Joyce
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Sijy
authors.initials: S
authors.lastname: O'Dell
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Tongqing
authors.identifier: http://orcid.org/0000-0002-3935-4637
authors.initials: T
authors.lastname: Zhou
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Adrian B
authors.initials: AB
authors.lastname: McDermott
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA jmascola@nih.gov pkwong@mail.nih.gov.
authors.forename: John R
authors.initials: JR
authors.lastname: Mascola
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA jmascola@nih.gov pkwong@mail.nih.gov.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Kwong
mesh_terms: D016915:AIDS Vaccines / Q000737:chemistry / Q000276:immunology; D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood / Q000276:immunology; D015704:CD4 Antigens / Q000276:immunology* / Q000378:metabolism*; D006168:Guinea Pigs; D015483:HIV Antibodies / Q000097:blood / Q000276:immunology; D015488:HIV Antigens / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000276:immunology*; D006801:Humans; D055503:Protein Multimerization; D055550:Protein Stability; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism
chemical_list: D016915:AIDS Vaccines; D057134:Antibodies, Neutralizing; D015704:CD4 Antigens; D015483:HIV Antibodies; D015488:HIV Antigens; D054299:env Gene Products, Human Immunodeficiency Virus
keywords: HIV-1; immunogen design; protein stabilization