The V1/V2 domain of the
HIV-1 envelope protein gp120 possesses two important
epitopes: a
glycan-dependent
epitope recognized by the prototypic broadly neutralizing
monoclonal antibody (bN-mAb), PG9, as well as an
epitope recognized by non-
neutralizing antibodies that has been associated with protection from
HIV infection in the RV144
HIV vaccine trial. Because both of these
epitopes are poorly immunogenic in the context of full length envelope
proteins, immunization with properly folded and glycosylated fragments (scaffolds) represents a potential way to enhance the immune response to these specific
epitopes. Previous studies showed that V1/V2 domain scaffolds could be produced from a few selected isolates, but not from many of the isolates that would be advantageous in a
multivalent vaccine. In this paper, we used a
protein engineering approach to improve the conformational stability and antibody binding activity of V1/V2 domain scaffolds from multiple diverse isolates, including several that were initially unable to bind the prototypic PG9 bN-mAb. Significantly, this effort required replicating both the correct
glycan structure as well as the β-sheet structure required for PG9 binding. Although scaffolds incorporating the
glycans required for PG9 binding (e.g., mannose-5) can be produced using glycosylation inhibitors (e.g.,
swainsonine), or mutant cell lines (e.g. GnTI(-) 293 HEK), these are not practical for
biopharmaceutical production of
proteins intended for clinical trials. In this report, we describe engineered
glycopeptide scaffolds from three different clades of HIV-1 that bind PG9 with high affinity when expressed in a wildtype cell line suitable for
biopharmaceutical production. The mutations that improved PG9 binding to scaffolds produced in normal cells included
amino acid positions outside of the antibody contact region designed to stabilize the β-sheet and turn structures. The scaffolds produced address three major problems in
HIV vaccine development: (1) improving antibody responses to poorly immunogenic
epitopes in the V1/V2 domain; (2) eliminating antibody responses to highly immunogenic (decoy)
epitopes outside the V1/V2 domain; and (3) enabling the production of V1/V2 scaffolds in a cell line suitable for
biopharmaceutical production.