Biochemically defined HIV-1 envelope glycoprotein variant immunogens display differential binding and neutralizing specificities to the CD4-binding site

Abstract

HIV-1 gp120 binds the primary receptor CD4. Recently, a plethora of broadly neutralizing antibodies to the gp120 CD4-binding site (CD4bs) validated this region as a target for immunogen design. Here, we asked if modified HIV-1 envelope glycoproteins (Env) designed to increase CD4 recognition might improve recognition by CD4bs neutralizing antibodies and more efficiently elicit such reactivities. We also asked if CD4bs stabilization, coupled with altering the Env format (monomer to trimer or cross-clade), might better elicit neutralizing antibodies by focusing the immune response on the functionally conserved CD4bs. We produced monomeric and trimeric Envs stabilized by mutations within the gp120 CD4bs cavity (pocket-filling; PF2) or by appending heterologous trimerization motifs to soluble Env ectodomains (gp120/gp140). Recombinant glycoproteins were purified to relative homogeneity, and ligand binding properties were analyzed by ELISA, surface plasmon resonance, and isothermal titration microcalorimetry. In some formats, the PF2 substitutions increased CD4 affinity, and importantly, PF2-containing proteins were better recognized by the broadly neutralizing CD4bs mAbs, VRC01 and VRC-PG04. Based on this analysis, we immunized selected Env variants into rabbits using heterologous or homologous regimens. Analysis of the sera revealed that homologous inoculation of the PF2-containing, variable region-deleted YU2 gp120 trimers (ΔV123/PF2-GCN4) more rapidly elicited CD4bs-directed neutralizing antibodies compared with other regimens, whereas homologous trimers elicited increased neutralization potency, mapping predominantly to the gp120 third major variable region (V3). These results suggest that some engineered Env proteins may more efficiently direct responses toward the conserved CD4bs and be valuable to elicit antibodies of greater neutralizing capacity.

FIGURE 1.

Schematic to illustrate the different Env formats and inoculation rationale. A, schematic representation of the recombinant gp120 core/PF2, the gp120ΔV123/PF2 trimer, and gp140 trimer illustrating the different Env formats. The recombinant uncleaved gp140 trimer is composed of gp120 (gray), the gp41 ectodomain (brown), the heterologous trimerization motif (gold or green), and His₆ tag. The HXBc2 core protein and the YU2 gp120ΔV123 trimers are color-coded similarly to the gp140 trimer schematic. B, schematics of the experimental rationale for the prime/boosting of core-to-trimer or trimer-to-core which increases or decreases the elements of Env contained in each immunogen, as illustrated Env format alteration. The goal of the experiment is to focus the B cell immune response on the conserved CD4-binding site shared by all immunogens (red). Several of the constructs contained substitutions in the Phe-43 cavity (pocket-filling) that enhances CD4 affinity, with the goal to more efficiently engage B cells with enhanced B cell receptor affinity for the CD4-binding site. Dashed lines indicate the Env loop and N- and C-terminal truncations. The cross-clade gp140-F trimers for boosting used constructs derived from the clade B YU2 and CAAN strains or the clade C ZA012 strain.

FIGURE 2.

Gel filtration, SDS-PAGE, and blue native gel analysis of purified Env. A, reducing SDS-PAGE of affinity-purified Env used for rabbit inoculations is labeled as shown. B, gel filtration profiles of affinity-purified gp140 trimers; the fractions that were combined to isolate homogenous trimers are boxed in gray. C, gel filtration profiles of selected gp140-F trimers that were further purified by size exclusion chromatography. D, blue native gel. Lane 1, HXBc2 gp120 core/PF2; lane 2, YU2 gp120ΔV123/PF2-GCN4; lanes 3–7: ZA012, CAAN/PF2, CAAN, YU2/PF2 and YU2 gp140-F trimers.

FIGURE 3.

Antigenicity of recombinant Env variants. A, recognition by the CD4bs neutralizing ligands assessed by ELISA of the following proteins: HXBc2 gp120 core/PF2, YU2 gp120ΔV123/PF2-GCN4, YU2 gp140-F (−/+PF2), CAAN gp140-F (−/+PF2), and ZA012 gp140-F. HIV IG was used as an internal control for the relative concentrations of the gp140 trimers. B, representative SPR curves (left panel) and ITC figure (right panel) of soluble CD4 binding with CAAN gp140-F trimer. RU, response units.

FIGURE 4.

Neutralization profile of rabbit immune sera tested against a panel of HIV-1 isolates. A, schematic diagram of the heterologous prime/boost immunization regimen and homologous regimen. Rabbits were inoculated at 4-week intervals with the different Env formats, two times for each format. One week after each inoculation, antisera were collected for analysis. B, all sera tested were collected after six inoculations of Env/adjuvant. Neutralization by preimmune sera was used to confirm that there was negligible nonspecific effect in the sera prior to inoculation. Neutralization values of greater than 1000 are boxed in the darkest maroon; values between 40 and 1000 are in red, and those between 10 and 40 are in yellow; the values that are still over control group (SIVmac) are bold.

FIGURE 5.

Characterization of probes for differential neutralization inhibition assay and mapping of nonpotent CD4bs neutralizing antibodies in antisera. Based on HXBc2 coreV3S, we designed two probes, TriMut with triple mutations (I423M, N425K, and G431E) and TriMut368/70 with two additional mutations (D368R and E370F), to map the CD4bs neutralizing antibodies present in sera by differential neutralization adsorption. The probes were preincubated, respectively, with serially diluted antisera in the neutralization assay, and differences between TriMut compared with the CD4bs “knock-out” mutations at 368/70 revealed the extent of neutralization directed to the CD4bs. Sera from rabbits of four groups were selected; one group of animals had been inoculated with heterologous prime/boost regimens (two inoculations with HXBc2 core/PF2, two inoculations with YU2 gp120ΔV123/PF2-GCN4, and two final inoculations with CAAN gp140-F), and three groups of animals were inoculated with a homologous regimen (six inoculations with YU2 gp120ΔV123/PF2-GCN4, or YU2 gp140-F, or CAAN gp140-F). A, binding profile of the probes with a panel of CD4bs antibodies and CD4-Ig. Binding of antibodies to TriMut and TriMut368/70 was determined by ELISA and SPR. B, validation of the differential neutralization absorption assay. Neutralization absorption of the CD4bs mAb, VRC01, the non-CD4bs, and co-receptor binding site mAb 17b was measured. C, differential adsorption neutralization assay against the HXBc2 virus by selected sera. Differential neutralization absorption curves of post-2 and post-6 sera from four rabbits are shown as representative examples. D, comparison of CD4bs neutralizing antibodies elicited with the different immunization regimens. Difference of neutralization absorbed by the two probes at 1:12 serum dilution was plotted by data derived from each respective group of animals to determine relative content of HXBc2 neutralizing CD4bs-directed antibodies.