Structure-guided design and immunological characterization of immunogens presenting the HIV-1 gp120 V3 loop on a CTB scaffold

Abstract

V3 loop is a major neutralizing determinant of the HIV-1 gp120. Using 3D structures of cholera toxin B subunit (CTB), complete V3 in the gp120 context, and V3 bound to a monoclonal antibody (mAb), we designed two V3-scaffold immunogen constructs (V3-CTB). The full-length V3-CTB presenting the complete V3 in a structural context mimicking gp120 was recognized by the large majority of our panel of 24 mAbs. The short V3-CTB presenting a V3 fragment in the conformation observed in the complex with the 447-52D Fab, exhibited high-affinity binding to this mAb. The immunogens were evaluated in rabbits using DNA-prime/protein-boost protocol. Boosting with the full-length V3-CTB induced high anti-V3 titers in sera that potently neutralize multiple HIV virus strains. The short V3-CTB was ineffective. The results suggest that very narrow antigenic profile of an immunogen is associated with poor Ab response. An immunogen with broader antigenic activity elicits robust Ab response.

Figure 1

The V3 peptide fragment (shown in magenta ribbon representation and sticks) bound to the broadly neutralizing mAb 447-52D (light and heavy chains are shown as green and yellow ribbons, respectively). Formation of a three-strand beta-sheet composed of two strands of the heavy chain CDR3 hairpin and one V3 strand, as well as tight binding of the conserved residue P313 in the GPGR motif at the tip of the loop can be observed. All molecular graphics images were prepared in Molsoft ICM-Pro.

Figure 2

Match of the CTB scaffold (blue) and V3 loop base (green) in the gp120 structure (grey). Close correspondence of the backbone traces of the two structures at the F42^CTB/N295^gp120 − A46^CTB/N332^gp120 junction can be observed.

Figure 3

A) Full-length V3-CTB amino-acid sequence (V3 loop insert is in bold); B) Short V3-CTB where only the V3 crown (shown in bold) is inserted. Additional mutations are introduced to enhance conformational stability of the insert (mutated positions are underlined).

Figure 4

Model of the short V3-CTB construct (ribbon and transparent surface) in complex with the Fab fragment of mAb 447-52D (magenta and blue ribbons for heavy and light chains, respectively).

Figure 5

Analysis of purity of recombinant CTB immunogens. Purified recombinant immunogens were analyzed by SDS/PAGE and stained with Coomassie. Lane 1: molecular weight markers with corresponding molecular weights (kDa); Lane 2: wide type CTB. Lane 3: full-length V3-CTB. Lane 4: short V3-CTB.

Figure 6

ELISA titration curves of immune sera vs. V3 fused to the Fc fragment of rabbit IgG. Immune sera were obtained two weeks after the second protein boost from rabbits designated as #6–10, boosted with the full length V3-CTB (red), or from rabbits #1–5, boosted with the short V3-CTB (blue).