title: Immunogenic Display of Purified Chemically Cross-Linked HIV-1 Spikes.
abstract: HIV-1 envelope glycoprotein (Env) spikes are prime vaccine candidates, at least in principle, but suffer from instability, molecular heterogeneity and a low copy number on virions. We anticipated that chemical cross-linking of HIV-1 would allow purification and molecular characterization of trimeric Env spikes, as well as high copy number immunization. Broadly neutralizing antibodies bound tightly to all major quaternary epitopes on cross-linked spikes. Covalent cross-linking of the trimer also stabilized broadly neutralizing epitopes, although surprisingly some individual epitopes were still somewhat sensitive to heat or reducing agent. Immunodepletion using non-neutralizing antibodies to gp120 and gp41 was an effective method for removing non-native-like Env. Cross-linked spikes, purified via an engineered C-terminal tag, were shown by negative stain EM to have well-ordered, trilobed structure. An immunization was performed comparing a boost with Env spikes on virions to spikes cross-linked and captured onto nanoparticles, each following a gp160 DNA prime. Although differences in neutralization did not reach statistical significance, cross-linked Env spikes elicited a more diverse and sporadically neutralizing antibody response against Tier 1b and 2 isolates when displayed on nanoparticles, despite attenuated binding titers to gp120 and V3 crown peptides. Our study demonstrates display of cross-linked trimeric Env spikes on nanoparticles, while showing a level of control over antigenicity, purity and density of virion-associated Env, which may have relevance for Env based vaccine strategies for HIV-1.
  
  
  OBJECTIVE
  The envelope spike (Env) is the target of HIV-1 neutralizing antibodies, which a successful vaccine will need to elicit. However, native Env on virions is innately labile, as well as heterogeneously and sparsely displayed. We therefore stabilized Env spikes using a chemical cross-linker and removed non-native Env by immunodepletion with non-neutralizing antibodies. Fixed native spikes were recognized by all classes of known broadly neutralizing antibodies but not by non-neutralizing antibodies and displayed on nanoparticles in high copy number. An immunization experiment in rabbits revealed that cross-linking Env reduced its overall immunogenicity; however, high-copy display on nanoparticles enabled boosting of antibodies that sporadically neutralized some relatively resistant HIV-1 isolates, albeit at a low titer. This study describes the purification of stable and antigenically correct Env spikes from virions that can be used as immunogens.
authors: Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California, USA.
authors: Daniel P
authors: DP
authors: Leaman
authors: Department of Integrative Structural and Computational Biology, Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, IAVI Neutralizing Antibody Center and Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, California, USA.
authors: Jeong Hyun
authors: JH
authors: Lee
authors: Department of Integrative Structural and Computational Biology, Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, IAVI Neutralizing Antibody Center and Collaboration for AIDS Vaccine Discovery, The Scripps Research Institute, La Jolla, California, USA.
authors: Andrew B
authors: AB
authors: Ward
authors: Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California, USA zwick@scripps.edu.
authors: Michael B
authors: MB
authors: Zwick
mesh_terms: D016915:AIDS Vaccines / Q000737:chemistry / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D057134:Antibodies, Neutralizing / Q000276:immunology; D003432:Cross-Linking Reagents; D004337:Drug Carriers; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D015483:HIV Antibodies / Q000276:immunology; D015497:HIV-1 / Q000737:chemistry / Q000276:immunology*; D046911:Macromolecular Substances / Q000276:immunology / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles / Q000648:ultrastructure; D011485:Protein Binding; D055550:Protein Stability; D011817:Rabbits; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000276:immunology*
chemical_list: D016915:AIDS Vaccines; D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D003432:Cross-Linking Reagents; D004337:Drug Carriers; D000939:Epitopes; D015483:HIV Antibodies; D046911:Macromolecular Substances; D054299:env Gene Products, Human Immunodeficiency Virus