Immunogenic Display of Purified Chemically Cross-Linked HIV-1 Spikes

J Virol. 2015 Jul;89(13):6725-45. doi: 10.1128/JVI.03738-14. Epub 2015 Apr 15.


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.

Importance: 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • AIDS Vaccines / chemistry
  • AIDS Vaccines / immunology
  • Animals
  • Antibodies, Monoclonal / immunology
  • Antibodies, Neutralizing / immunology
  • Cross-Linking Reagents
  • Drug Carriers
  • Epitopes / chemistry
  • Epitopes / immunology
  • HIV Antibodies / immunology
  • HIV-1 / chemistry
  • HIV-1 / immunology*
  • Macromolecular Substances / immunology
  • Macromolecular Substances / ultrastructure
  • Microscopy, Electron, Transmission
  • Nanoparticles / ultrastructure
  • Protein Binding
  • Protein Stability
  • Rabbits
  • env Gene Products, Human Immunodeficiency Virus / chemistry
  • env Gene Products, Human Immunodeficiency Virus / immunology*


  • AIDS Vaccines
  • Antibodies, Monoclonal
  • Antibodies, Neutralizing
  • Cross-Linking Reagents
  • Drug Carriers
  • Epitopes
  • HIV Antibodies
  • Macromolecular Substances
  • env Gene Products, Human Immunodeficiency Virus