Bacterially expressed HIV-1 gp120 outer-domain fragment immunogens with improved stability and affinity for CD4-binding site neutralizing antibodies

J Biol Chem. 2018 Sep 28;293(39):15002-15020. doi: 10.1074/jbc.RA118.005006. Epub 2018 Aug 9.


Protein minimization is an attractive approach for designing vaccines against rapidly evolving pathogens such as human immunodeficiency virus, type 1 (HIV-1), because it can help in focusing the immune response toward conserved conformational epitopes present on complex targets. The outer domain (OD) of HIV-1 gp120 contains epitopes for a large number of neutralizing antibodies and therefore is a primary target for structure-based vaccine design. We have previously designed a bacterially expressed outer-domain immunogen (ODEC) that bound CD4-binding site (CD4bs) ligands with 3-12 μm affinity and elicited a modest neutralizing antibody response in rabbits. In this study, we have optimized ODEC using consensus sequence design, cyclic permutation, and structure-guided mutations to generate a number of variants with improved yields, biophysical properties, stabilities, and affinities (KD of 10-50 nm) for various CD4bs targeting broadly neutralizing antibodies, including the germline-reverted version of the broadly neutralizing antibody VRC01. In contrast to ODEC, the optimized immunogens elicited high anti-gp120 titers in rabbits as early as 6 weeks post-immunization, before any gp120 boost was given. Following two gp120 boosts, sera collected at week 22 showed cross-clade neutralization of tier 1 HIV-1 viruses. Using a number of different prime/boost combinations, we have identified a cyclically permuted OD fragment as the best priming immunogen, and a trimeric, cyclically permuted gp120 as the most suitable boosting molecule among the tested immunogens. This study also provides insights into some of the biophysical correlates of improved immunogenicity.

Keywords: glycosylation; hydrogen–deuterium exchange; mutagenesis; protein design; protein refolding; vaccine development; yeast surface display.

Publication types

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

MeSH terms

  • AIDS Vaccines / chemistry
  • AIDS Vaccines / immunology*
  • AIDS Vaccines / therapeutic use
  • Animals
  • Antibodies, Monoclonal / chemistry
  • Antibodies, Monoclonal / immunology
  • Antibodies, Neutralizing / immunology
  • Binding Sites
  • Broadly Neutralizing Antibodies
  • CD4 Antigens / chemistry
  • CD4 Antigens / immunology*
  • Crystallography, X-Ray
  • Epitopes / chemistry
  • Epitopes / immunology
  • HIV Antibodies / chemistry
  • HIV Antibodies / immunology
  • HIV Envelope Protein gp120 / chemistry*
  • HIV Envelope Protein gp120 / genetics
  • HIV Envelope Protein gp120 / immunology
  • HIV Infections / genetics
  • HIV Infections / immunology*
  • HIV-1 / chemistry*
  • HIV-1 / immunology
  • HIV-1 / pathogenicity
  • Humans
  • Ligands
  • Protein Binding
  • Rabbits


  • AIDS Vaccines
  • Antibodies, Monoclonal
  • Antibodies, Neutralizing
  • Broadly Neutralizing Antibodies
  • CD4 Antigens
  • Epitopes
  • HIV Antibodies
  • HIV Envelope Protein gp120
  • Ligands
  • VRC01 monoclonal antibody
  • gp120 protein, Human immunodeficiency virus 1

Associated data

  • PDB/1G9M
  • PDB/3NGB