Variable loop glycan dependency of the broad and potent HIV-1-neutralizing antibodies PG9 and PG16

Abstract

The HIV-1-specific antibodies PG9 and PG16 show marked cross-isolate neutralization breadth and potency. Antibody neutralization has been shown to be dependent on the presence of N-linked glycosylation at position 160 in gp120. We show here that (i) the loss of several key glycosylation sites in the V1, V2, and V3 loops; (ii) the generation of pseudoviruses in the presence of various glycosidase inhibitors; and (iii) the growth of pseudoviruses in a mutant cell line (GnT1(-/-)) that alters envelope glycosylation patterns all have significant effects on the sensitivity of virus to neutralization by PG9 and PG16. However, the interaction of antibody is not inhibited by sugar monosaccharides corresponding to those found in glycans on the HIV surface. We show that some of the glycosylation effects described are isolate dependent and others are universal and can be used as diagnostic for the presence of PG9 and PG16-like antibodies in the sera of HIV-1-infected patients. The results suggest that PG9 and PG16 recognize a conformational epitope that is dependent on glycosylation at specific variable loop N-linked sites. This information may be valuable for the design of immunogens to elicit PG9 and PG16-like antibodies, as well as constructs for cocrystallization studies.

FIG. 1.

Monosaccharide competition for MAb binding to Env. (A) Monosaccharide competition of PG9 binding to gp120_DU422 in ELISA. (B) Monosaccharide competition of 2G12 binding to gp120_JR-FL in ELISA. (C) Mannose competition of MAbs to fixed JR-CSF Env-expressing 293T cells by flow cytometry.

FIG. 2.

Sensitivity of PG9 and PG16 neutralization to substitutions eliminating N-linked glycosylation sites. (A) JR-CSF PG9; (B) JR-CSF PG16; (C) JR-FL E168K PG9; (D) JR-FL E168K PG16.

FIG. 3.

N-linked glycosylation pathway to show the formation of high-mannose, complex, and hybrid glycans. Glycosidase inhibitors (red) are shown underneath the enzyme they inhibit.

FIG. 4.

Neutralization activity of MAbs against JR-CSF pseudoviruses made in the presence of glycosidase inhibitors. (A) b12; (B) 2G12; (C) PG9; (D) PG16.

FIG. 5.

Neutralization activity of MAbs against JR-FL E168K pseudoviruses made in the presence of glycosidase inhibitors. (A) b12; (B) 2G12; (C) PG9; (D) PG16.

FIG. 6.

Neutralization activity of MAbs against 92RW020 and SF162 K160N pseudoviruses when made in the presence of glycosidase inhibitors. (A) 92RW020 PG9; (B) 92RW020 PG16; (C) SF162 K160N PG9; (D) SF162 K160N PG16.

FIG. 7.

Cell surface binding of MAbs to swainsonine-treated HIV JR-FL E168K Env-transfected cells compared to untreated HIV JR-FL E168K Env cells. (A) PG9; (B) PG16; (C) b12.

FIG. 8.

Neutralization of glycan mixed trimer viruses by PG9, PG16, and b12. Cells were transfected with various proportions of JR-FL E168K and JR-FL E168K N189A. The percentage refers to the amount of JR-FL E168K N189A plasmid used. (A) PG9; (B) PG16; (C) 2G12. (D) The percent neutralization at an antibody concentration of 10 μg/ml was plotted against the ratio of “suboptimal” envelope (i.e., ratio of JR-FL E186K). The data fit best to the equation N = N_plateau + [(1 − X)³ + 3X(1 − X)² + 3(1 − X)X²](N_max − N_plateau), where N is the neutralization and X is the ratio of suboptimal substitution (i.e., only one “optimal” monomer is required per trimer to reach maximum neutralization) (53, 59).