Surface Plasmon Resonance (SPR) binding analysis of cross-linked gp140-miniCD4 (gp140-S-S-M64U1) complex, in comparison to uncomplexed gp140, to ligands immobilized on CM5 sensor chip: (A) soluble CD4 (sCD4), (B) (CD4 binding site), (C) MAb 2G12 (glycans), and (D) MAb 17b (CD4i site).

Surface Plasmon Resonance (SPR) binding analysis of (A) gp140 and (B) cross-linked gp140-miniCD4 (gp140-S-S-M64U1) complex to two ligands immobilized on CM5 sensor chip: biotinylated, tyrosine-sulfated (at positions 10 and 14) 22-mer N-terminal CCR5 peptide [biotin-CCR5(SO3)] [30], [31] and a control, biotinylated non-sulfated 22-mer N-terminal CCR5 peptide [biotin-CCR5 (control)].

(A) Scheme of vaccination study in rabbits, showing time-point of administration of immunogens and collection of sera for evaluation. (B & C) SF162 gp140-specific binding antibody responses induced by various immunogens [gp140, M64U1 (miniCD4), gp140-S-S-M64U1 (cross-linked), or gp140+M64U1 (mixed)] in rabbit sera. Two weeks following the second (2wp2), third (2wp3) and fourth (2wp4) immunizations, individual sera were analyzed via ELISA and group geometric mean (geomean) titers calculated. Pre-immune sera were also evaluated to identify the level of non-specific reactivity. (B) Avidity of SF162 gp140-specific antibodies from the four groups of animals at 2wp2, 2wp3 and 2wp4 following immunization.

Analysis of breadth by neutralization of various tiered HIV-1 Env pseudoviruses by 2wp3 (p3) and 2wp4 (p4) sera from animals immunized with gp140 (open black circles, filled black circles), miniCD4 (M64U1) (open black triangles, filled black triangles) and two forms of the gp140-miniCD4 complexes: cross-linked (gp140-S-S-M64U1) (open blue circles, filled blue circles) and mixed (gp140+M64U1) (open red circles, filled red circles). ID50 neutralization titers are indicated against different viral isolates: SF162.LS, HxB2, MW965.26, BaL.26, SS1196.1, 6535.3, QH0692.42 and RHPA4259.7. The tiered categorization of the HIV-1 Env pseudoviruses is indicated. The subtypes are indicated within parenthesis. Neutralization of MLV is shown here as a control.

Determining epitope-specificity of sera using (A–C) a differential solid-phase adsorption method [38], and (D) V3-cyclic peptide competition assay. A representative profile of the antibody-specificity generated by sera from each of the groups is shown upon adsorption by the various antigen-coupled beads, as described in Material and Methods. Specificity of sera from group immunized with (A) gp140, (B) cross-linked gp140-S-S-M64U1 complex and (C) mixed gp140+M64U1 complex. The epitopes are highlighted atop each panel in grey. The beads (BSA-beads; black/grey curves) used as ‘control’ are shown on the first column. The second column shows specificity to ‘CD4BS’-eptiope (using gp120_D368R-beads; green curves) for gp140 immunized group and ‘CD4i’ epitope (using gp140-S-S-M64U1 complex; purple curves) for cross-linked or mixed gp140-miniCD4 complex immunized groups; the third column shows specificity for ‘V1V2’-loop (using gp120ΔV1V2-beads; orange curves) for gp140 immunized group and ‘CD4i’ epitope (using gp120_I420R-beads; cyan curves) for cross-linked or mixed gp140-miniCD4 complex immunized groups. The final column shows specificity to ‘V3’-loop (using gp120ΔV3-beads; blue curves). (D) Competition assay to inhibit binding of gp140 (left panel) and gp140-miniCD4 complex, cross-linked gp140-S-S-M64U1 (middle panel) or mixed gp140+M64U1 (right panel), immunized rabbit sera to gp140 by V3-cyclic peptide. As control, a V3-scrambled cyclic peptide was used (see Materials & Methods for details). The percent (%) inhibition, which is the extent of serum binding to gp140 inhibited by V3-peptide, is calculated as the ratio of serum binding in presence of V3-cyclic peptide to serum binding in presence of control peptide ×100.

Individual rabbit sera from the four groups (immunized with either gp140, miniCD4 M64U1, cross-linked gp140-S-S-M64U1 complex or mixed gp140+M64U1 complex) were tested for neutralizing activity against the isolate 7312A/V434A in the absence (open bars) or presence (filled bars) of sCD4. The ID50 neutralization titers are shown for the pre-immune (black), 2wp3 (red), and 2wp4 (blue) serum samples. The antigens used for immunization are indicated above each graph. Dashed lines indicate the lowest dilution tested.