HIV-1 gp120-CD4-Induced Antibody Complex Elicits CD4 Binding Site-Specific Antibody Response in Mice

Abstract

Elicitation of broadly neutralizing Ab (bNAb) responses toward the conserved HIV-1 envelope (Env) CD4 binding site (CD4bs) by vaccination is an important goal for vaccine development and yet to be achieved. The outcome of previous immunogenicity studies suggests that the limited accessibility of the CD4bs and the presence of predominant nonneutralizing determinants (nND) on Env may impede the elicitation of bNAbs and their precursors by vaccination. In this study, we designed a panel of novel immunogens that 1) preferentially expose the CD4bs by selective elimination of glycosylation sites flanking the CD4bs, and 2) minimize the nND immune response by engineering fusion proteins consisting of gp120 Core and one or two CD4-induced (CD4i) mAbs for masking nND epitopes, referred to as gp120-CD4i fusion proteins. As expected, the fusion proteins possess improved antigenicity with retained affinity for VRC01-class, CD4bs-directed bNAbs and dampened affinity for nonneutralizing Abs. We immunized C57BL/6 mice with these fusion proteins and found that overall the fusion proteins elicit more focused CD4bs Ab response than prototypical gp120 Core by serological analysis. Consistently, we found that mice immunized with selected gp120-CD4i fusion proteins have higher frequencies of germinal center-activated B cells and CD4bs-directed memory B cells than those inoculated with parental immunogens. We isolated three mAbs from mice immunized with selected gp120-CD4i fusion proteins and found that their footprints on Env are similar to VRC01-class bNAbs. Thus, using gp120-CD4i fusion proteins with selective glycan deletion as immunogens could focus Ab response toward CD4bs epitope.

FIGURE 1.. Design of the gp120-CD4i fusion proteins.

(A) Model of YU2 gp120 core (PDB: 3TGQ) in complex with VRC01 germline Ab (VRC01GL, PDB: 4jpk). Of note two N-glycans, N276 (modeled from PDB: 5FYK) and N463 (modeled as oligomanose from http://www.glycosciences.de) surrounding the gp120 CD4bs epitope clash with VRC01 GL. (B) CoreD with two mutations (N276D/N463D) that remove the N276 and N463 N-glycans respectively. Connectivity of gp120-CD4i fusion proteins: (C) CoreD-A32, CoreD (V44-E492) gp120 shown in green, A32 in red; (D) CoreD-17b with circular permutation (PM): (S199-E492) gp120 shown in green, (V44-T123) gp120 in gold, 17b in blue; (E) A32-CoreD-17b with circular permutation: A32 in red, the other elements are depicted as in (D). 5× ((G4S)₄) linker is depicted as red dash line. (F) Linear schematic presentation of the YU2gp120 core-based gp120-CD4i fusion protein immunogens. CoreD-PM stands for the CoreD with circular permutation shown in (D) and (E). 4× and 5× depicts (G4S)₄ and (G4S)₅ flexible peptide linker, respectively.

FIGURE 2.. gp120-CD4i fusion proteins display retained or enhanced affinity for CD4bs bNAbs with minimized non-bNAb binding.

(A) ELISA binding of gp120-CD4i fusion proteins to CD4bs bNAbs (VRC01 & PGV04) and non-bNAbs (F105, GE136 & GE148), in comparison to Core & CoreD. (B) gp120-CD4i fusion protein binding affinity (dissociation constant, K_D) for CD4bs ligands (VRC01, PGV04 and CD4Ig) assessed by biolayer light interferometry (BLI). (C) Detailed binding kinetic analysis of gp120-CD4i fusion proteins binding to CD4bs ligands by BLI. k_on, association rate; k_off, dissociation rate; K_D, affinity or dissociation constant, is calculated as k_off/k_on. Overall, most of the CoreD-CD4i fusion proteins have enhanced or retained affinity, K_D, for CD4bs bNAbs, compared with the parental gp120 core protein.

FIGURE 3.. Selected gp120-CD4i fusion proteins display elevated binding affinity for VRC01 germline precursor, VRC01 GL, assessed by biolayer light interferometry (BLI).

(A) BLI response of gp120-CD4i fusion proteins in comparison with Core, CoreD, and trimeric Env immunogens (BG505 SOSIP variants & 16055 NFL TD CC ΔGly4). (B) Detailed binding kinetic analysis of gp120-CD4i fusion proteins binding to VRC01 GL by BLI, in comparison with Core, CoreD, and trimeric Env immunogens. k_on, k_off, and K_D are defined as in Figure 2C.

FIGURE 4.. gp120-CD4i fusion protein immunogenicity study design.

(A) C57BL/6 mice (N=4 mice/group) were divided into 10 groups to be immunized with various immunogens formulated with adjuvant (adjuplex) including Core, CoreD, single and double CD4i fusion proteins, with Env trimer BG505 SOSIP.664 as reference immunogen and PBS as negative control. (B) Immunization and sampling schedule. Mice were intramuscularly (I.M.) immunized with 10 μg of Env immunogens in adjuplex on weeks 0, 4, and 8 (indicated by blue arrow) respectively for a total 3 times. Blood sampling was performed on weeks 0 (pre-immune serum), 2, 6 and 11 (2 or 3 weeks post each immunization) (marked by Xs). Upon termination on week 11, the lymph nodes and spleens of the immune mice were harvested for analysis.

FIGURE 5.. Antigen probes used to investigate Ab response in mice.

(A) The design of CoreD-n5i5-17b fusion protein as antigen probe. CoreD (green) is connected to n5i5 ScFv (gold) and 17b scFv (blue), with 3× and 4× linkers (red) denoting 3 and 4 tandem (G4S) flexible peptide linker modules, respectively. (B) ELISA binding profile of CoreD-n5i5-17b: left, it binds strongly to CD4bs bNAb VRC01 and poorly to non-bNAbs F105 and GE136, as well as CD4i mAbs, A32 & 17b; right, the CD4bs knock out mutant variant (D368R/D474A) shows attenuated binding affinity for CD4bs bNAb VRC01 compared to the wildtype (WT, black) antigen probe. (C) Schematic presentation of the surface structural models of the Env antigen probes used for profiling immune mouse serum Ab response specificity. CD4bs epitope is denoted in yellow color. RSC3 resurfaced regions are shown in red. CD4i scFvs are depicted in blue.

FIGURE 6.. Antigen binding specificity of the antibody responses in immune mouse sera assessed by ELISA.

(A) Immune mouse sera (week 11) display various level of binding activity to antigen probes presented as the areas under the curve (AUC), shown as mean ± standard deviation. Antigen probes include CoreD, RSC3, and BG505 SOSIP trimer. Animal sera were classified into 4 clusters depending on inoculated immunogens as shown in Figure 4 for comparison. Asterisk denotes statistically significant difference (* P<0.05, ** P<0.01, *** P<0.001, one-way ANOVA). (B) CD4bs Ab response of the week 11 immune mouse sera presented as the ELISA binding AUC difference (ΔAUC) between the wildtype (WT) antigen probe and the CD4bs knockout (KO) mutant, CoreD-n5i5-17b WT vs D368R/D474A, RSC3 WT vs ΔRSC3 (Δ371I) (11), and BG505 SOSIP WT vs D368R, respectively, shown as mean ± standard deviation. Statistical analysis result is indicated as in (A). (C) The Spearman correlation between CD4bs response (mean ΔAUC in (B)) of week 11 immune sera from mice immunized with individual immunogen and difference of respective immunogen affinity for CD4bs bNAb vs non-bNAb (Log K_D (VRC01/F105)), with CoreD-A32 & BG505 SOSIP trimer (denoted in dash-lined boxes) excluded from the analysis as outliners.

FIGURE 7.. Germinal center (GC) and CD4bs-directed B cell responses in immune mice.

Lymphocytes from draining (inguinal) lymph nodes of immune mice on week 11 were stained with antigen probe and mAbs for surface markers followed by FACS analysis. (A) Frequency of CD4bs+ B cells in immune mice, which are initially gated as CD19⁺B220⁺CD3⁻Gr1⁻F4/80⁻IgD⁻, followed by binding phenotype to CD4bs-specific antigen probe pair (CoreD-n5i5-17b WT⁺ CD4bs KO⁻). Shown are representative plots. (B) Frequency of B cells in GC in immune mice. GC B cells are initially gated as CD19⁺B220⁺CD3⁻Gr1⁻F4/80⁻IgD⁻, followed by GC-specific markers GL7⁺Fas⁺. (C) Correlation of GC and CD4bs+ B cell frequencies in naïve and six immunized animal groups, using the nonparametric Spearman test. (D) Frequency of CD4bs-directed memory B cells in immune mice. Memory B cells are gated as CD19⁺B220⁺CD3⁻Gr1⁻F4/80⁻IgD⁻CD38⁺. CD4bs-directed memory B cells are subsequently gates by CD4bs-specific antigen probe pair (CoreD-n5i5-17b WT⁺ CD4bs KO⁻) binding phenotype as in (A). * P<0.05, ** P<0.01, one-way ANOVA.

FIGURE 8.. Isolation of mAbs from mice immunized with double CD4i-fusion protein A32-CoreD-17b.

(A) Gating strategy of single cell sorting for CD4bs-directed class-switched B cells in A32-CoreD-17b-immunized mice. Class-switched B cells were gated as CD19⁺B220⁺CD3⁻Gr1⁻F4/80⁻Aqua blue (death)⁻IgM⁻IgD⁻, of which CD4bs-specificity was indicated by binding phenotype of CoreD-n5i5-17b WT⁺ CD4bs KO⁻ using CoreD-n5i5-17b antigen probes. (B) The Ig H and L chain gene family usage and genetic features of three mice mAbs, 1A10, 1A11 and 1D3. SHM, somatic hypermutation level, presented at both nucleotide (nt) and amino acid sequence (aa) levels. CDRs are delineated according to the IMGT definition.

FIGURE 9.. Antigen binding specificities of mAbs from mice immunized with double CD4i-fusion protein A32-CoreD-17b.

(A) Mouse mAbs (1A10, 1A11 and 1D3) display similar binding specificity with CD4bs bNAb VRC01 to sorting probes, CoreD-n5i5-17b WT and CD4bs KO mutant, D368R/D474A by ELISA. (B) Competition of biotin-labeled prototypical CD4bs ligands, VRC01 (left) and CD4Ig (right), respectively, with mouse mAbs as competitor for binding to CoreD-n5i5-17b. +++: 75–100% competition, ++: 50–75% competition. (C) ELISA binding profiles of mouse mAbs to various antigens, with intact 276 & 463 glycans (glycan 276/463^on) or removed (glycan 276/463^off). Binding was categorized based on OD450 values at the highest concentration of mAb and the 50% effective concentration (EC₅₀, μg/mL) values: ++++: OD450 > 3.0, EC₅₀ < 0.1; +++: OD450 > 3.0, EC₅₀ > 0.1; ++: 1.0 < OD450 < 3.0; +: 0.2 < OD450 < 1.0; -: OD450 < 0.2. (D) Kinetic parameters of mouse mAbs binding to gp120-CD4i fusion proteins assessed by BLI. k_on, k_off, and K_D are defined as in Figure 2C.

FIGURE 10.. The footprints of mouse CD4bs mAbs on Env surface determined by differential binding to selected CD4bs mutants of antigen CoreD-n5i5-17b.

(A) A mini panel of CoreD-n5i5-17b mutants for fine mapping the critical contact residues in the Env CD4bs region for mouse mAb recognition. Left, a list of the mutations in the context of CoreD-n5i5-17b sequence; Right, the residues with mutations used for CD4bs mapping are highlighted in orange on the YU2 gp120 core molecular surface (PDB: 3tgq). Four Env major regions involved in CD4bs bNAb binding are marked in blue: Loop D, V5, CD4 BLP (CD4 binding loop), and C5. (B) Binding affinity of CoreD-n5i5-17b mutant antigens with various CD4bs bNAbs and mouse mAbs relative to the wild-type (WT) CoreD-n5i5-17b, is presented in percentage (%) and color-coded. The relative binding affinity was assessed by biolayer light interferometry. (C) Summary of mouse CD4bs mAb footprints on the surface of CoreD-n5i5-17b, in comparison with mature CD4bs bNAbs, VRC01 & PGV04, and naïve precursor, VRC01 GL, as determined in (B). The effect of mutations on antibody binding is color-coded as in (B).

FIGURE 11.. Env binding mode similarity and divergence between mouse CD4bs mAb 1D3 and bNAb VRC01.

(A) Fits of SAXS experimental data (blue) to all-atom models (red) of VRC01 Fab (left) or 1D3 Fab (right) complexed with CoreD-n5i5-17b. VRC01 Fab position was derived from experimental X-ray structure of VRC01 Fab: gp120 core complex (PDB: 3ngb). 1D3 Fab binding mode to CoreD-n5i5-17b was predicted with Rosetta docking and fit into the experimental SAXS scattering curve. (B) Molecular shapes (shown as transparent surfaces) of VRC01 or 1D3 Fab: CoreD-n5i5-17b complexes determined by SAXS and aligned with representative all-atom structures. VRC01 complex is shown in light grey; 1D3 complex is in light magenta. Envelopes were drawn over the filtered averaged DAMMIN structures. All-atom structural models of the complexes were overlaid and fit into the SAXS density with Chimera for comparison. The CoreD-n5i5-17b model is shown in cyan spheres, and VRC01 (grey) and 1D3 (magenta) Fabs are shown as cartoons. (C) Binding mode of mouse and human CD4bs mAbs in the context of Env trimer, shown as a light blue semitransparent surface. Angle of approach of CD4bs non-neutralizing Ab F105 (defined as the angle between the antibody and trimer axis) is different from that of bNAb VRC01 or mouse 1D3 Abs. Models of the complexes with VRC01 (grey), 1D3 (light magenta), and F105 (red) were superposed using common gp120 core unit with Chimera (46). In the right side insert, the steric clash of the F105 light chain with the trimer apex loop cluster is denoted in a yellow circle.