title: Chemical Cross-Linking Stabilizes Native-Like HIV-1 Envelope Glycoprotein Trimer Antigens.
abstract: Major neutralizing antibody immune evasion strategies of the HIV-1 envelope glycoprotein (Env) trimer include conformational and structural instability. Stabilized soluble trimers such as BG505 SOSIP.664 mimic the structure of virion-associated Env but nevertheless sample different conformational states. Here we demonstrate that treating BG505 SOSIP.664 trimers with glutaraldehyde or a heterobifunctional cross-linker introduces additional stability with relatively modest effects on antigenicity. Thus, most broadly neutralizing antibody (bNAb) epitopes were preserved after cross-linking, whereas the binding of most weakly or nonneutralizing antibodies (non-NAb) was reduced. Cross-linking stabilized all Env conformers present within a mixed population, and individual conformers could be isolated by bNAb affinity chromatography. Both positive selection of cross-linked conformers using the quaternary epitope-specific bNAbs PGT145, PGT151, and 3BC315 and negative selection with non-NAbs against the V3 region enriched for trimer populations with improved antigenicity for bNAbs. Similar results were obtained using the clade B B41 SOSIP.664 trimer. The cross-linking method may, therefore, be useful for countering the natural conformational heterogeneity of some HIV-1 Env proteins and, by extrapolation, also vaccine immunogens from other pathogens.
  
  
  OBJECTIVE
  The development of a vaccine to induce protective antibodies against HIV-1 is of primary public health importance. Recent advances in immunogen design have provided soluble recombinant envelope glycoprotein trimers with near-native morphology and antigenicity. However, these trimers are conformationally flexible, potentially reducing B-cell recognition of neutralizing antibody epitopes. Here we show that chemical cross-linking increases trimer stability, reducing binding of nonneutralizing antibodies while largely maintaining neutralizing antibody binding. Cross-linking followed by positive or negative antibody affinity selection of individual stable conformational variants further improved the antigenic and morphological characteristics of the trimers. This approach may be generally applicable to HIV-1 Env and also to other conformationally flexible pathogen antigens.
authors.affiliation: The Sir William Dunn School of Pathology, The University of Oxford, Oxford, United Kingdom.
authors.forename: Torben
authors.initials: T
authors.lastname: Schiffner
authors.affiliation: Department of Integrative Structural and Computational Biology, IAVI Neutralizing Antibody Center, Collaboration for AIDS Vaccine Discovery, 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: The Sir William Dunn School of Pathology, The University of Oxford, Oxford, United Kingdom.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Russell
authors.affiliation: Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Steven W
authors.initials: SW
authors.lastname: de Taeye
authors.affiliation: Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Alba Torrents
authors.initials: AT
authors.lastname: de la Peña
authors.affiliation: Department of Integrative Structural and Computational Biology, IAVI Neutralizing Antibody Center, Collaboration for AIDS Vaccine Discovery, 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 Integrative Structural and Computational Biology, IAVI Neutralizing Antibody Center, Collaboration for AIDS Vaccine Discovery, 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 Integrative Structural and Computational Biology, IAVI Neutralizing Antibody Center, Collaboration for AIDS Vaccine Discovery, and Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California, USA.
authors.forename: Travis
authors.initials: T
authors.lastname: Nieusma
authors.affiliation: The Sir William Dunn School of Pathology, The University of Oxford, Oxford, United Kingdom.
authors.forename: Florian
authors.initials: F
authors.lastname: Brod
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 Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA.
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: John P
authors.initials: JP
authors.lastname: Moore
authors.affiliation: Department of Integrative Structural and Computational Biology, IAVI Neutralizing Antibody Center, Collaboration for AIDS Vaccine Discovery, and 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: The Sir William Dunn School of Pathology, The University of Oxford, Oxford, United Kingdom quentin.sattentau@path.ox.ac.uk.
authors.forename: Quentin J
authors.initials: QJ
authors.lastname: Sattentau
mesh_terms: D057134:Antibodies, Neutralizing / Q000276:immunology; D003432:Cross-Linking Reagents / Q000378:metabolism; D015483:HIV Antibodies / Q000276:immunology; D015488:HIV Antigens / Q000276:immunology* / Q000378:metabolism*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D054299:env Gene Products, Human Immunodeficiency Virus / Q000276:immunology* / Q000378:metabolism*
chemical_list: D057134:Antibodies, Neutralizing; D003432:Cross-Linking Reagents; D015483:HIV Antibodies; D015488:HIV Antigens; D054299:env Gene Products, Human Immunodeficiency Virus