Characterization of the outer domain of the gp120 glycoprotein from human immunodeficiency virus type 1

Abstract

The core of the gp120 glycoprotein from human immunodeficiency virus type 1 (HIV-1) is comprised of three major structural domains: the outer domain, the inner domain, and the bridging sheet. The outer domain is exposed on the HIV-1 envelope glycoprotein trimer and contains binding surfaces for neutralizing antibodies such as 2G12, immunoglobulin G1b12, and anti-V3 antibodies. We expressed the outer domain of HIV-1(YU2) gp120 as an independent protein, termed OD1. OD1 efficiently bound 2G12 and a large number of anti-V3 antibodies, indicating its structural integrity. Immunochemical studies with OD1 indicated that antibody responses against the outer domain of the HIV-1 gp120 envelope glycoprotein are rare in HIV-1-infected human sera that potently neutralize the virus. Surprisingly, such outer-domain-directed antibody responses are commonly elicited by immunization with recombinant monomeric gp120. Immunization with soluble, stabilized HIV-1 envelope glycoprotein trimers elicited antibody responses that more closely resembled those in the sera of HIV-1-infected individuals. These results underscore the qualitatively different humoral immune responses elicited during natural infection and after gp120 vaccination and help to explain the failure of gp120 as an effective vaccine.

FIG. 1.

Schematic illustration of the OD1 and ID proteins. The Cα tracing of the gp120 core of HIV-1_YU2 is derived from the structure of the CD4-gp120 core-17b Fab complex (35). The gp120 core structures comprising the OD1 protein are colored yellow; the hypothetical location of the V3 loop, which is missing from the gp120 core, is shown. The ID protein (red) includes the ID of the gp120 core and the gp120 V1/V2 loops (not shown), the C1 and C5 regions that are not in the gp120 core, and the gp41 ectodomain.

FIG. 2.

Expression of OD1 proteins in 293T cells. 293T cells transfected with a total of 10 μg of envelope glycoprotein expression plasmids were radiolabeled with [³⁵S]cysteine-methionine, and the supernatants were incubated with a mixture of sera from HIV-1-infected persons (P.S.). The precipitates were analyzed on SDS-10% polyacrylamide gels. For OD1 and ID coexpression, 5 μg of each expressor plasmid DNA was transfected. Different species of the OD1 proteins are labeled on the right, and the molecular mass markers are indicated on the left. For deglycosylation, the protein-antibody-Protein A-agarose beads were boiled for 5 min in 1× sample buffer without β-mercaptoethanol. After incubation on ice for a few minutes, 500 U of PNGase F (New England Biolabs) and 0.5 mU of O-glycosidase (Boehringer Mannheim) were added to each sample, followed by incubation for 30 min in a 37°C water bath. The sample was boiled for 5 min before loading on a SDS-10% polyacrylamide gel.

FIG. 3.

Binding of MAbs to OD1. Immunoprecipitations of radiolabeled supernatants of transfected 293T cells were performed as in the Fig. 2 legend with either 3 μl of pooled sera from HIV-1-infected persons (P.S.) or 1 μg of purified MAbs.

FIG. 4.

Binding of human anti-V3 MAbs to the OD1 protein. (A and B) The human MAbs tested were originally selected either with a V3_JR-CSF-fusion protein (V3-FP) or gp120_LAI (MAbs 694/98 and 1334) (A) or with V3 peptides representing the strains HIV-1_MN and HIV-1_RF (B). The curves in red represent antibodies with 50% neutralizing activity against HIV-1_SF162, and those in blue represent antibodies that achieved <50% neutralization of the same isolate (21). The curves for the negative control antibody, which is directed against the parvovirus B19 protein, are shown in green.

FIG. 5.

Expression and purification of recombinant envelope glycoproteins. The OD1, OD1-PADRE, and OD1-ΔV3-PADRE proteins were stably expressed in Drosophila S2 cells and purified by using the C-terminal His₆ tags. The gp120 and gp140(-/FT) glycoproteins were expressed in CHO-K1 cell clones selected for high-level expression. The proteins were first purified by a nickel affinity column and then by size exclusion chromatography. The purified proteins were quantified by OD₂₈₀ measurements and snap-frozen on dry ice-ethanol. The proteins were added to 1× SDS sample buffer without boiling, run on a 10% SDS-polyacrylamide gel, and visualized by Coomassie blue staining. The OD1, OD1-PADRE, and OD1-ΔV3-PADRE proteins exhibited the same pattern of migration after being boiled in sample buffer with 2% β-mercaptoethanol (data not shown).

FIG. 6.

Binding of immunized rabbit sera to the OD1 protein or the HIV-1_YU2 gp120 glycoprotein. Four New Zealand White rabbits in each group were immunized with the purified proteins indicated in the legend. Rabbits were immunized with 120 μg of protein in 1× Ribi adjuvant (Sigma) three times, and serum samples were harvested at 2 weeks after the last injection. Serial dilution of the antisera is indicated on the x axis. The captured antigen on the ELISA plates was either the purified OD1 glycoprotein (A) or the purified HIV-1_YU2 gp120 glycoprotein (B).

FIG. 7.

Profiles of antibody responses in rabbits immunized with monomeric gp120 glycoprotein. Four rabbits were immunized with 120 μg of the purified HIV-1_YU2 gp120 glycoprotein three times, and serum samples were harvested 2 weeks after the last injection. The antibodies in immunized rabbit sera were detected by an ELISA with the OD1 protein (blue lines) or the gp120 glycoprotein (red lines) as detecting antigen. In one set of experiments, the rabbit antisera were first incubated with 100 μg of the OD1 protein (A), the deglycosylated OD1 proteins (C), or the OD1-ΔV3-PADRE protein (D)/ml, and the glycoprotein-serum mixture was tested in the ELISA by using either the OD1 or gp120 protein as the detecting antigen. (B) To deglycosylate the OD1 protein, 600 μg of the OD1 protein was incubated with 5 μl of PNGase F (New England Biolabs) in 1× G7 buffer at 37°C overnight. Then, 5 μg of the deglycosylated OD1 protein was run on a SDS-14% polyacrylamide gel and visualized after Coomassie blue staining. The experiments shown in panel C, in which the deglycosylated OD1 was added to sera, were conducted on ice to inhibit PNGase F activity.

FIG. 8.

Profiles of antibody responses in rabbits immunized with the trimeric gp140(-/FT) glycoprotein. Four rabbits were immunized with 120 μg of the purified gp140(-/FT) glycoprotein three times and serum samples were harvested 2 weeks after the last injection. The serum antibodies were measured by an ELISA with the OD1 protein (blue lines), the gp120 glycoprotein (red lines), or the gp140(-/FT) glycoprotein (green lines) as detecting antigens. In some experiments, the antisera were incubated with 100 μg of the OD1 protein (▵) or the purified gp120 protein (□)/ml at 37°C for 1 h, and the glycoprotein-antiserum mixture was then tested in the ELISA (dashed lines).

FIG. 9.

Profiles of antibodies in HIV-Ig and potently neutralizing sera from HIV-1-infected persons. Antibodies in HIV-Ig and sera (anti-HIV-1#1 and anti-HIV-1#2) from HIV-1-infected persons reactive with the OD1 protein (blue lines), the gp120 monomer (red lines), and the gp140(-/FT) trimer (green lines) were measured in an ELISA. In some experiments, 100 μg of the OD1 protein/ml was used to adsorb the OD1-specific antibodies from the antibodies reactive with the gp120 or gp140(-/FT) proteins (dashed lines with “▵”). Similarly, 100 μg of the purified gp120 protein/ml was used to adsorb antibodies from the pool of gp140(-/FT)-reactive specific antibodies (dashed lines with “□”). The experiments were done twice, and similar results were achieved; the data shown are from a single set of experiments.