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

Abstract

A major goal for HIV-1 vaccine development is an ability to elicit strong and durable broadly neutralizing antibody (bNAb) responses. The trimeric envelope glycoprotein (Env) spikes on HIV-1 are known to contain multiple epitopes that are susceptible to bNAbs isolated from infected individuals. Nonetheless, all trimeric and monomeric Env immunogens designed to date have failed to elicit such antibodies. We report the structure-guided design of HIV-1 cyclically permuted gp120 that forms homogeneous, stable trimers, and displays enhanced binding to multiple bNAbs, including VRC01, VRC03, VRC-PG04, PGT128, and the quaternary epitope-specific bNAbs PGT145 and PGDM1400. Constructs that were cyclically permuted in the V1 loop region and contained an N-terminal trimerization domain to stabilize V1V2-mediated quaternary interactions, showed the highest homogeneity and the best antigenic characteristics. In guinea pigs, a DNA prime-protein boost regimen with these new gp120 trimer immunogens elicited potent neutralizing antibody responses against highly sensitive Tier 1A isolates and weaker neutralizing antibody responses with an average titer of about 115 against a panel of heterologous Tier 2 isolates. A modest fraction of the Tier 2 virus neutralizing activity appeared to target the CD4 binding site on gp120. These results suggest that cyclically permuted HIV-1 gp120 trimers represent a viable platform in which further modifications may be made to eventually achieve protective bNAb responses.

Keywords: CD4 binding site; Env trimers; cyclic permutation; human immunodeficiency virus (HIV); immunogen design; immunogenicity; protein conformation; protein engineering; protein stability; protein stabilization, dynamics.

FIGURE 1.

Engineered cyclically permuted gp120 immunogens form homogeneous, stable trimers. A, schematic representation of the four different immunogens characterized in this study. Cyclically permuted gp120 trimers (JRFL-hCMP-V1cyc and JRCSF-hCMP-V1cyc) were designed by joining native N and C termini of gp120 with a flexible, soluble 20-residue linker and new N and C termini were created by opening the V1 loop followed by fusion of an hCMP trimerization domain to the new N terminus. A more conventional gp120 trimer (JRFLgp120-L6-hCMP) was made by connecting the hCMP trimerization domain to the C terminus of JRFL gp120 with a 6-residue flexible linker. L6, 6-residue linker; L20, 20-residue linker. All constructs were secreted from HEK 293T cells under control of the CMV promoter with a tpa leader. B, purified proteins were subjected to BN-PAGE followed by Western blotting with rabbit anti-gp120 polyclonal sera to characterize the oligomeric state of the proteins. Cyclically permuted gp120 trimers appeared as trimers, whereas JRFL gp120 and JRFLgp120-L6-hCMP appeared as a mixture of oligomers. C, analytical gel-filtration chromatography of cyclically permuted constructs on a Superdex-200 column in PBS buffer at room temperature. The absorbance at 220 nm is shown as a function of the elution volume. Cyclically permuted gp120 trimers showed a single peak corresponding to a trimer, whereas JRFL gp120 and JRFLgp120-L6-hCMP showed two major peaks corresponding to a dimer and a monomer. D, the mass fractions and corresponding molecular weights of each peak for the proteins analyzed by SEC-MALS. E, the functional stability of all proteins was tested by monitoring binding to bNAbs, VRC01 (CD4bs specific), and PGT128 (V3 loop glycan specific) before and after heating. Proteins were incubated at 60 or 100 °C for 1 h and cooled down to room temperature to monitor binding to bNAbs VRC01 (left panel) and PGT128 (right panel) immobilized on the surface of a CM5 chip. Biacore response units were plotted after incubation at different temperatures to compare stabilities. Cyclically permuted gp120 trimers are stable at 60 °C showing similar binding before and after heating to VRC01 and PGT128 antibodies. They retained detectable binding to these antibodies even after incubation at 100 °C for 1 h. F, temperature-induced equilibrium unfolding transitions of monomeric JRFL gp120 (black open circles, T_m = 337.1 K (64.0 °C)), and trimeric JRFL-hCMP-V1cycgp120 (red open circles, T_m = 341.8 K (68.7 °C)). The lines through the symbols represent fits using a two-state model.

FIGURE 2.

Raw EM images of negative stained gp120 and gp120 complexed with mAb PGDM1400. A, raw EM image of cyclically permuted gp120 particles embedded in negative stain. Panel on the right shows projections of excised gp120 particles. B, raw EM image of particles of gp120 complexed with PGDM1400 embedded in negative stain. Panel on the right shows projections of excised particles. C, representative class averages of gp120 and gp120 complexed with mAb PGDM1400. PGDM1400 antibodies are marked with white arrows.

FIGURE 3.

Guinea pig immunizations. A, immunization parameters and time lines of DNA prime and protein boost in guinea pigs. Immunization and bleed time points are indicated in weeks. B, gp120-specific ELISA end point titers of sera collected from immunized animals. Horizontal lines represent mean titers. Preimmune sera obtained before immunization had no detectable anti-gp120 titer. A Mann-Whitney test was performed to compare end point titers, between different time points. *, p < 0.05; ns, no significant difference (p > 0.05) between two compared groups.

FIGURE 4.

Antisera from cyclically permuted gp120 trimers elicit broad cross-reactive neutralizing responses. TZM-bl ID₅₀ values (neutralizing titers) for week 20 antisera from guinea pigs in TZM-bl cell-based neutralization assays. The neutralizing activity of antisera from each group of guinea pigs was tested against Tier 1 and Tier 2 HIV-1 isolates from a global panel and additional Tier-2 isolates, which included viruses from clades A, B, C, and CRFs. The neutralization titers are corrected for their respective preimmune backgrounds. The color code indicates the following: ID₅₀ ≤ 60 or ≤ 2-fold of MuLV neutralizing titers are in white, ID₅₀ from 60 to 90 are in yellow, ID₅₀ from 90 to 500 are in orange, and ID₅₀ >500 are highlighted in red. Group number and corresponding immunogen used for vaccination are indicated in the first column.

FIGURE 5.

Relative to the other two immunogens, cyclically permuted gp120 trimers elicited significantly higher neutralizing titers against Tier 2 HIV-1 isolates. Scatter plot of neutralization ID₅₀ values of week 20 antisera from JRFL gp120 (orange), JRFL-hCMP-V1cyc (red), JRCSF-hCMP-V1cyc (green), and JRFLgp120-L6-hCMP (blue). Error bars in the diagram represents mean ± S.E. Neutralization ID₅₀ values of: A, all Tier 2; B, clade B and C viruses from Tier 2; and C, non-clade B and C Tier 2 viruses are represented after subtracting preimmune titers. Neutralizing ID₅₀ titers elicited by JRFL gp120 were compared with corresponding ID₅₀ titers elicited by other immunogens by a Mann-Whitney test.

FIGURE 6.

Mapping of CD4 binding site specificities in antisera from cyclically permuted gp120 trimers. A, neutralization activity of IgG purified from pooled week 20 sera against JRFL and JRFL-N279A pseudoviruses. Introduction of the N279A mutation in Env makes JRFL pseudovirus insensitive to neutralization by the CD4 binding site specific, VRC01-like antibodies. The percent neutralization as a function of IgG concentration is shown. Error bars represent the S.D. from two independent experiments. Only IgG from antisera of cyclically permuted gp120 trimers showed decreased neutralization against JRFL-N279A pseudovirus indicating the presence of CD4 binding site specific neutralizing antibodies. B, binding of pooled antisera to CD4 binding site focused outer domain fragments (b122a and ODEC) and JRFL gp120. Antigens were coated directly onto ELISA plate wells and probed with pooled antisera from week 20. The absorbance at 410 nm is proportional to the amount of bound antiserum. Antisera from cyclically permuted gp120 trimers show higher binding to the CD4 binding site focused outer domain immunogens than antisera from JRFL gp20 and JRFLgp120-L6-hCMP.

FIGURE 7.

A, approximate positions of new N termini of gp120 in V1cyc and V2cyc, respectively, mapped onto the structure of the BG505 SOSIP.664 HIV-1 Env trimer (Protein Data Bank code 4TVP). The color scheme of the structures is as follows: gp120 (orange), gp41 (cyan), V1V2 region (dark red), residue 141 of V1 region (blue spheres), residue 187 of V2 region (yellow spheres), and region 136–154 of V1 loop (black). The trimer axis is represented as a purple rod. The hCMP trimerization domain was fused to the above N-terminal positions to trimerize gp120. The chain direction at the N termini of V1cyc and V2cyc are indicated by black arrowheads. Residue 144 (the actual N terminus of V1cyc) in JRFL is absent in BG505 because of a deletion of residues 142–149. Hence residue 141 is shown as the N terminus. B, superimposition of core gp120 bound to CD4bs non-neutralizing F105 Fab (PDB code 3HI1) onto gp140 from the BG505 Env trimer (PDB code 4TVP). The color scheme of the structure is as follows: F105 bound gp120 (green), V1V2 region (dark red), and the remaining gp120 subunit from BG505 Env trimer (orange), gp41 from BG505 Env trimer (cyan), F105 Fab light chain (gray), and heavy chain (black) of F105 antigen binding fragment Fab. Inability of F105 to bind gp120 in the presence of the V1V2 region is indicated with a dotted circle demonstrating steric overlap between the F105 light chain and V1V2 in the superimposed structure. C, superimposition of core gp120 bound to CD4bs neutralizing VRC01 Fab (PDB code 3NGB), onto gp140 from the BG505 Env trimer (PDB code 4TVP) shows the absence of any steric clash between the Fab and native trimer. The color scheme of the structure is as follows: VRC01 bound gp120 (green), V1V2 region (dark red), and the gp120 subunit (orange) from BG505 Env trimer, gp41 from BG505 Env trimer (cyan), VRC01 Fab light chain (gray), and heavy chain (black).