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. 2003 May;77(10):5889-901.
doi: 10.1128/jvi.77.10.5889-5901.2003.

Hyperglycosylated mutants of human immunodeficiency virus (HIV) type 1 monomeric gp120 as novel antigens for HIV vaccine design

Ralph Pantophlet  1 , Ian A Wilson, Dennis R Burton
Affiliations

Hyperglycosylated mutants of human immunodeficiency virus (HIV) type 1 monomeric gp120 as novel antigens for HIV vaccine design

Ralph Pantophlet et al. J Virol. 2003 May.

Abstract

The ability to induce broadly neutralizing antibodies should be a key component of any forthcoming vaccine against human immunodeficiency virus type 1. One potential vaccine candidate, monomeric gp120, has generally failed to elicit such antibodies. We postulated that gp120 might be a better immunogen if it could be engineered to preferentially bind known broadly neutralizing antibodies. In a first study, we found that four alanine substitutions on the perimeter of the so-called Phe-43 cavity of gp120 could reduce binding of weakly neutralizing CD4-binding site antibodies (R. Pantophlet, E. O. Saphire, P. Poignard, P. W. H. I. Parren, I. A. Wilson, and D. R. Burton, J. Virol. 77:642-658, 2003), while slightly enhancing binding of the potent, broadly neutralizing antibody b12. In the present study, we sought to reduce or abolish the binding of a wider range of nonneutralizing antibodies, by incorporating extra N-glycosylation motifs at select positions into the hypervariable loops and the gp120 core. A hyperglycosylated mutant containing seven extra glycosylation sequons (consensus sequences) and the four alanine substitutions described above did not bind an extensive panel of nonneutralizing and weakly neutralizing antibodies, including a polyclonal immunoglobulin preparation (HIVIG) of low neutralizing potency. Binding of b12, at lowered affinity, and of four antibodies to the C1 and C5 regions was maintained. Removal of N- and C-terminal residues in the C1 and C5 regions, respectively, reduced or abolished binding of the four antibodies, but this also adversely affected b12 binding. The hyperglycosylated mutant and its analogues described here are novel antigens that may provide a new approach to eliciting antibodies with b12-like neutralizing properties.

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Figures

FIG. 1.
FIG. 1.
Binding of CD4bs MAbs (A and C) and non-CD4bs MAbs (B and D) to gp120JR-FL and mutant GDMR in ELISA. (A and B) Antibody binding to wild-type gp120. (C and D) Antibody binding to gp120 of mutant GDMR. Supernatants containing monomeric gp120 were captured onto ELISA plate wells and probed with various concentrations of MAb, starting at 10 μg/ml. Bound antibody was detected with alkaline phosphatase-conjugated secondary antibody. Absorbance was measured at 405 nm.
FIG. 2.
FIG. 2.
Binding of V3 loop MAbs to wild-type gp120JR-FL and glycoprotein of mutant P313N in Western blot and in ELISA. (A) Wild-type gp120 (lane 1) and mutant glycoprotein (lane 2) were reacted with MAb b12 (1 μg/ml). (B) Glycoproteins were reacted with MAbs 19b, 447-52D, and loop 2 (pooled mixture; 1 μg of each per ml). Molecular mass indicators (bars) and the average molecular masses of wild-type and mutant glycoproteins (arrows) are shown on the left in kilodaltons. (C and D) MAb binding to wild-type gp120 (C) and mutant P313N (D) was determined by ELISA.
FIG. 3.
FIG. 3.
Binding of MAb b12 to gp120 with added N-glycosylation sequons. Wild-type gp120 and mutant glycoproteins (labeled I to VII) were captured onto ELISA wells and probed with MAb b12 (10 ng/ml). HIVIG (1 μg/ml) was used to ensure that equivalent amounts of protein were captured. Bound antibody was detected with peroxidase-conjugated secondary antibody, and the absorbance was measured at 450 nm.
FIG. 4.
FIG. 4.
Location of introduced N-glycan attachment sites and alanine substitutions, mapped onto the gp120 core. N-linked oligosaccharides (yellow) were modeled onto the core structure (48) according to the most likely glycoforms, as inferred from a previous study (122). The putative locations of the V1 and V2 loops are also indicated in each panel. The 20-Å bar represents the average width of a typical antibody-combining site. (A) Depiction of the gp120 core is shown from the perspective of CD4. The attachment sites of the extra glycans are labeled and colored dark blue; dark blue spheres indicate those in the V1 and V2 loops. The alanine substitutions at positions 473 to 476 (GDMR) are labeled and colored red. The perspectives shown in panels B and C are also indicated by arrows. (B) View of the gp120 outer domain (48, 116). The spatial location of the V3 and V4 loops, which are proposed to extend from the protein surface, are also indicated. (C) View of the proposed gp41-gp120 interface (48, 116). Figures were made with RasMol (93) and modified by using Adobe Photoshop.
FIG. 5.
FIG. 5.
Determination of the molecular mass of mutant mCHO*-GDMR by Western blot. Wild-type gp120JR-FL and mutant glycoprotein were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with a polyclonal antibody prearation generated against the C5 region of gp120 (5 μg/ml). Lane 1, wild type; lane 2, mutant mCHO*-GDMR. Molecular mass indicators are shown on the left, and the average molecular mass of each glycoprotein is denoted on the right in kilodaltons.
FIG. 6.
FIG. 6.
Binding of anti-gp120 MAbs to wild-type (A to D, left) and mutant mCHO*-GDMR (E to H, right) glycoproteins. (A and E) Binding of MAbs to the C1, C5, C1-C4, and C1-C5 domains; (B and F) binding MAbs to the CD4bs; (C and G) binding of MAbs to the coreceptor-binding site; (D and H) binding of MAbs to the V2 and V3 loops. Captured glycoproteins were probed with MAb concentrations indicated on the x axis. The absorbance was measured at 405 nm.
FIG. 7.
FIG. 7.
Glycoproteins of mutants mCHO*-GDMR and mCHO*-GDMR ΔN/C (N and C termini deleted), captured onto ELISA plate wells with indicated antibodies (10 μg/ml) and detected with biotinylated MAb 2G12 (1μg/ml). Gray bars represent mutant mCHO*-GDMR; dark bars represent mutant mCHO*-GDMR ΔN/C. The absorbance was measured at 450 nm.
FIG. 8.
FIG. 8.
Reactivity of HIVIG (open symbols) and MAb b12 (solid symbols) in ELISA with wild-type gp120 and mutant glycoproteins. wt, wild-type gp120; GDMR, mutant GDMR in which residues at positions 473 to 476 in gp120 have been replaced with alanine; GDMR-P313N, similar to mutant GDMR with the addition of an N-glycosylation site in the V3 loop; mCHO*-GDMR, similar to mutant GDMR-P313N, with the addition of six glycosylation motifs (two in the V1 loop, one in the V2 loop, and three on the gp120 core).
FIG. 9.
FIG. 9.
Binding of CD4bs MAbs and MAb A32 to glycoprotein of mutant mCHO*. This mutant contains all of the glycosylation sequons that are also present in mutant mCHO*-GDMR but lacks the alanine substitutions on the edge of the Phe-43 cavity.
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