Targeted N-glycan deletion at the receptor-binding site retains HIV Env NFL trimer integrity and accelerates the elicited antibody response

PLoS Pathog. 2017 Sep 13;13(9):e1006614. doi: 10.1371/journal.ppat.1006614. eCollection 2017 Sep.

Abstract

Extensive shielding by N-glycans on the surface of the HIV envelope glycoproteins (Env) restricts B cell recognition of conserved neutralizing determinants. Elicitation of broadly neutralizing antibodies (bNAbs) in selected HIV-infected individuals reveals that Abs capable of penetrating the glycan shield can be generated by the B cell repertoire. Accordingly, we sought to determine if targeted N-glycan deletion might alter antibody responses to Env. We focused on the conserved CD4 binding site (CD4bs) since this is a known neutralizing determinant that is devoid of glycosylation to allow CD4 receptor engagement, but is ringed by surrounding N-glycans. We selectively deleted potential N-glycan sites (PNGS) proximal to the CD4bs on well-ordered clade C 16055 native flexibly linked (NFL) trimers to potentially increase recognition by naïve B cells in vivo. We generated glycan-deleted trimer variants that maintained native-like conformation and stability. Using a panel of CD4bs-directed bNAbs, we demonstrated improved accessibility of the CD4bs on the N-glycan-deleted trimer variants. We showed that pseudoviruses lacking these Env PNGSs were more sensitive to neutralization by CD4bs-specific bNAbs but remained resistant to non-neutralizing mAbs. We performed rabbit immunogenicity experiments using two approaches comparing glycan-deleted to fully glycosylated NFL trimers. The first was to delete 4 PNGS sites and then boost with fully glycosylated Env; the second was to delete 4 sites and gradually re-introduce these N-glycans in subsequent boosts. We demonstrated that the 16055 PNGS-deleted trimers more rapidly elicited serum antibodies that more potently neutralized the CD4bs-proximal-PNGS-deleted viruses in a statistically significant manner and strongly trended towards increased neutralization of fully glycosylated autologous virus. This approach elicited serum IgG capable of cross-neutralizing selected tier 2 viruses lacking N-glycans at residue N276 (natural or engineered), indicating that PNGS deletion of well-ordered trimers is a promising strategy to prime B cell responses to this conserved neutralizing determinant.

MeSH terms

  • Animals
  • Antibodies, Neutralizing / immunology*
  • B-Lymphocytes / immunology
  • CD4 Antigens / immunology
  • Enzyme-Linked Immunosorbent Assay
  • Epitopes, B-Lymphocyte / immunology
  • Female
  • HIV Antibodies / immunology*
  • HIV-1 / immunology
  • Imaging, Three-Dimensional
  • Lymphocyte Activation / immunology
  • Microscopy, Electron
  • Mutagenesis, Site-Directed
  • Polysaccharides / immunology*
  • Rabbits
  • env Gene Products, Human Immunodeficiency Virus / immunology*

Substances

  • Antibodies, Neutralizing
  • CD4 Antigens
  • Epitopes, B-Lymphocyte
  • HIV Antibodies
  • Polysaccharides
  • env Gene Products, Human Immunodeficiency Virus