Designed Cyclic Permutants of HIV-1 gp120: Implications for Envelope Trimer Structure and Immunogen Design

Abstract

Most HIV-1 broadly neutralizing antibodies are directed against the gp120 subunit of the env surface protein. Native env consists of a trimer of gp120-gp41 heterodimers, and in contrast to monomeric gp120, preferentially binds CD4 binding site (CD4bs)-directed neutralizing antibodies over non-neutralizing ones. Some cryo-electron tomography studies have suggested that the V1V2 loop regions of gp120 are located close to the trimer interface. We have therefore designed cyclically permuted variants of gp120 with and without the h-CMP and SUMO2a trimerization domains inserted into the V1V2 loop. h-CMP-V1cyc is one such variant in which residues 153 and 142 are the N- and C-terminal residues, respectively, of cyclically permuted gp120 and h-CMP is fused to the N-terminus. This molecule forms a trimer under native conditions and binds CD4 and the neutralizing CD4bs antibodies b12 with significantly higher affinity than wild-type gp120. It binds non-neutralizing CD4bs antibody F105 with lower affinity than gp120. A similar derivative, h-CMP-V1cyc1, bound the V1V2 loop-directed broadly neutralizing antibodies PG9 and PG16 with ∼20-fold higher affinity than wild-type JRCSF gp120. These cyclic permutants of gp120 are properly folded and are potential immunogens. The data also support env models in which the V1V2 loops are proximal to the trimer interface.

Design of an Escherichia coli Expressed HIV-1 gp120 Fragment Immunogen That Binds to b12 and Induces Broad and Potent Neutralizing Antibodies

“…In order to enhance the binding signal to PGT128, we used a cyclically permuted trimeric gp120, which binds to PGT128 with 10-fold higher affinity than monomeric gp120. This molecule (ccmp-gp120 trimer) has been described previously (35). All three sera showed similar binding profiles (Fig.…”

Structure-based Design of Cyclically Permuted HIV-1 gp120 Trimers That Elicit Neutralizing Antibodies

“…The trimers also bound preferentially to the trimer-specific antibodies PGDM1400 (Table 1 and supplemental Fig. S5E) as well as PG9 and PG16 (37). This data indicates that trimerization of cyclically permuted gp120 at the V1 loop retains native-like quaternary epitopes at the V1V2 region.…”

“…To overcome this problem, we designed gp120 derivatives that are cyclically permuted at the V1 loop region of gp120 at sites where mutations and insertions are tolerated (supplemental Fig. S1) (37). To this end, the native N and C termini of the gp120 molecule were connected with a 20-amino acid linker and new N and C termini were created within the V1 loop region at specific positions in the gp120 sequence.…”

“…S5B). We have previously reported that cyclically permuted gp120 trimers show improved affinity to neutralizing antibody b12 and lower affinity for non-neutralizing CD4bs antibody F105 relative to monomeric gp120 (37). The F105 binding studies were repeated (Table 1, supplemental Fig.…”

“…To minimize conformational dynamics in these loops, based on available structural information, we inserted a heterologous trimerization domain into the V1 loop region to trimerize the gp120 molecule at the apex instead of at the C terminus of gp120 as had been done previously. We have previously designed cyclically permuted gp120 by making new N and C termini at the V1 loop and by connecting the native N and C termini with an appropriate flexible linker (37). Here we describe the antigenicity and immunogenicity of a new generation of cyclically permuted gp120s that exhibit improved homogeneity and trimer stability.…”