Antibodies against Manalpha1,2-Manalpha1,2-Man oligosaccharide structures recognize envelope glycoproteins from HIV-1 and SIV strains

Abstract

Design of an envelope glycoprotein (Env)-based vaccine against human immunodeficiency virus type-1 (HIV-1) is complicated by the large number of N-linked glycans that coat the protein and serve as a barrier to antibody-mediated neutralization. Compared to normal mammalian glycoproteins, high-mannose-type glycans are disproportionately represented on the gp120 subunit of Env. These N-glycans serve as a target for a number of anti-HIV molecules that bind terminal alpha1,2-linked mannose residues, including lectins and the monoclonal antibody 2G12. We created a Saccharomyces cerevisiae glycosylation mutant, Deltamnn1Deltamnn4, to expose numerous terminal Manalpha1,2-Man residues on endogenous hypermannosylated glycoproteins in the yeast cell wall. Immunization of rabbits with whole cells from this mutant induced antibodies that bound to a broad range of Env proteins, including clade A, B, and C of HIV and simian immunodeficiency virus (SIV). The gp120 binding activity of these immune sera was due to mannose-specific immunoglobulin, as removal of high-mannose glycans and alpha1,2-linked mannoses from gp120 abrogated serum binding. Glycan array analysis with purified IgG demonstrated binding mainly to glycans with Manalpha1,2-Manalpha1,2-Man trisaccharides. Altogether, these data demonstrate the immunogenicity of exposed polyvalent Manalpha1,2-Manalpha1,2-Man structures on the yeast cell wall mannan and their ability to induce antibodies that bind to the HIV Env protein. The yeast strain and sera from this study will be useful tools for determining the type of mannose-specific response that is needed to develop neutralizing antibodies to the glycan shield of HIV.

Fig. 1

N-Glycans produced in S. cerevisiae strains. (A) Depiction of expected structures of polymannose-type N-glycans produced in wild-type and Δmnn1Δmnn4 (DM) yeast strains, with the indicated type and linkage of each monosaccharide. The shaded boxes represent repeated oligosaccharide units, with an average of ten units per N-glycan. Expected structures of N-glycans from WT and DM yeast are based on previous studies of WT yeast and the Δmnn1 mutant (Ballou ; Dean 1999). Man₈GlcNAc₂, an important glycan in the 2G12 epitope, is depicted with the D1 and D3 arms. (B) Whole yeast cell ELISA testing anti-α1,3-Man (left) and 2G12 (right) binding to wild type, DM, and TM yeast coated at 2.5 × 10⁶ cells/well. (C) Western blots testing anti-Gp38, Ecm33, α1,3-Man, and 2G12 binding to 4.8 μg of total cell lysate from WT, DM, and TM yeast.

Fig. 2

Immune sera elicited with S. cerevisiae DM yeast crossreacted with gp120 glycoproteins from HIV-1 and SIV in ELISA. (A) Immune sera at weeks 0, 5, and 10 from rabbit 1137 (top), 1138 (middle), and 1139 (bottom) were tested by ELISA at 1:500 dilution against the indicated Env glycoproteins. (B) Comparison of ELISA binding by postimmune sera (week 10) from TM-immunized rabbit 1141 and DM-immunized rabbit 1139 against Env glycoproteins. All proteins were produced in mammalian cells. OD, optical density.

Fig. 3

Titration of immune sera. Titers of gp120-specific IgG in rabbit serum samples were measured using purified JR-FL (top), R2 (middle), and R3A (bottom) in ELISA. Sera tested include DM-immunized rabbits 1137, 1138, and 1139 at week 10, and a positive control serum from a rabbit immunized with JR-FL gp120 (gp120 serum). The values are the averages ± standard errors of the means (error bars) of three independent experiments. All gp120 proteins were produced in 293-T cells. OD, optical density.

Fig. 4

Glycan binding analyses of DM-induced antibodies. (A) The indicated gp120 proteins were digested with PNGase F (PNF) or mock digested. Samples were run on SDS–PAGE gels for blotting with rabbit 1139 serum (top) and control anti-gp120 IgG (bottom). (B) The indicated gp120 proteins were digested with PNF, Endo H, and jack bean and A. saitoi mannosidases, with mock digestion using the same denatured conditions as PNGase F digestion but without the enzyme. Western blot was conducted using purified rabbit 1139 IgG (top) and control anti-gp120 IgG (bottom). (C) Glycan array analysis of DM-induced antibodies. Purified IgG from rabbit 1139 at week 0 and week 10, post-immune IgG from a WT yeast-immunized rabbit, and 2G12 were used to probe a microarray of 320 glycans. The glycan numbers (x-axis) correspond to the depicted high-mannose-type glycans. Group 1 contains synthetic glycans without terminal α1,2-linked mannose residues, group 2 with terminal Manα1,2-Man disaccharides, and group 3 with terminal Manα1,2-Manα1,2-Man trisaccharides. Group 4 contains natural high-mannose glycans with 2 GlcNAc residues at the base.