Purification and characterization of oligomeric envelope glycoprotein from a primary R5 subtype B human immunodeficiency virus

Abstract

Human immunodeficiency virus (HIV) continues to be a major public health problem throughout the world, with high levels of mortality and morbidity associated with AIDS. Considerable efforts to develop an effective vaccine for HIV have been directed towards the generation of cellular, humoral, and mucosal immune responses. A major emphasis of our work has been toward the evaluation of oligomeric (o-gp140) forms of the HIV type 1 (HIV-1) envelope protein for their ability to induce neutralizing antibody responses. We have derived stable CHO cell lines expressing o-gp140 envelope protein from the primary non-syncytium-inducing (R5) subtype B strain HIV-1(US4). We have developed an efficient purification strategy to purify oligomers to near homogeneity. Using a combination of three detectors measuring intrinsic viscosity, light scattering, and refractive index, we calculated the molecular mass of the oligomer to be 474 kDa, consistent with either a trimer or a tetramer. The hydrodynamic radius (R(h)) of o-gp140 was determined to be 8.40 nm, compared with 5.07 nm for the monomer. The relatively smaller R(h) of the oligomer suggests that there are indeed differences between the foldings of o-gp140 and gp120. To assess the structural integrity of the purified trimers, we performed a detailed characterization of the glycosylation profile of o-gp140, its ability to bind soluble CD4, and also its ability to bind to a panel of monoclonal antibodies with known epitope specificities for the CD4 binding site, the CD4 inducible site, the V3 loop, and gp41. Immunogenicity studies with rabbits indicated that the purified o-gp140 protein was highly immunogenic and induced high-titer, high-avidity antibodies directed predominantly against conformational epitopes. These observations confirm the structural integrity of purified o-gp140 and its potential as a vaccine antigen.

FIG. 1.

Structure, expression, and stabilization of HIV-1 US4 envelope glycoprotein in the oligomeric conformation. (A) Linear map of the HIV-1 gp120 and gp41 envelope glycoproteins. The gp120 variable regions (V1 to V5) are indicated as solid squares, and the mutation of arginine 509 to serine 509 in the protease cleavage site between gp120 and gp41 is indicated by an arrow. The gp41 portion includes the N and C α-helical regions known as oligomerization domains. tPA, human tissue-type plasminogen activator. (B) Effect of codon optimization on expression of o-gp140. Total protein expression obtained from native and codon-optimized constructs by using capture ELISA in different fractions is presented. (C) Effect of protease cleavage site mutation upon oligomer stabilization. 293 cells were transfected with gp140 protease cleavage site-mutated and nonmutated constructs by using the LT-1 transfection reagent as described for CHO cells in Materials and Methods. Supernatants were collected on day 5, and partial purification of the proteins was performed. These partially purified proteins were tested in a CD4 binding assay using a Bio Sil SEC-250 sizing column to separate monomers from oligomers as described in Materials and Methods. Peaks representing oligomer (O), monomer (M), and CD4 are indicated.

FIG. 2.

Analysis of gp140 purification profile after column purification. (A) SDS-PAGE analysis of various fractions: starting material (lane 2); flowthrough after elution through DEAE (lane 3), CHAP (lane 4), and protein G (lane 6) columns; and elution of CHAP (lane 5) and GNA (lanes 7 and 8) columns. Numbers on the left are molecular masses in kilodaltons. (B) Immunoblot analysis of gp140 before (lane 1) and after (lane 2) incubation with GNA.

FIG. 3.

Purification and analysis of o-gp140. (A) o-gp140 was separated on a high-resolution sizing column from the dimeric and monomeric forms of gp140 in presence of a higher salt concentration (500 mM NaCl). Fractions corresponding to different conformational states of the protein, such as oligomer (peak A), dimer, and monomer (peak B), are indicated. (B) Polyacrylamide gel analysis of the sizing fractions under reducing and denaturing conditions. Lane 1, gp120 SF2; lane 2, oligomer; lane 3, dimer; lane 4, monomer. (C) Polyacrylamide gel analysis of the sizing fractions under native conditions. Lane 1, oligomer; lane 2, dimer; lane 3, monomer. (D) Immunodetection of o-gp140 using a MAb (20-2-C8.5F3) directed against the C4 domain of gp120 SF2. Lane 1, o-gp140; lane 2, gp140 monomer. Numbers on the right are molecular masses in kilodaltons.

FIG. 4.

Biophysical characterization of purified o-gp140 using a triple-detector array system. (A and B) Relative responses obtained for light scattering (thick lines), refractive index (thin lines), and viscosity (dashed lines) for gp120 US4 (A) and o-gp140 US4 (B). The majority of the purified protein is homogeneous. Maximum protein is in peak B as indicated by the refractive index detector signal. The lack of a refractive index signal in peak A indicates that a smaller fraction of the protein is in the aggregated state. (C) Summary table of biophysical properties, including molecular mass, intrinsic viscosity, and hydrodynamic radius of o-gp140. BSA, bovine serum albumin.

FIG. 5.

Carbohydrate analysis of purified o-gp140. (A) Immunoblot detection of biotinylated gp120 US4 (lane 1) and o-gp140 US4 (lane 2) using streptavidin-HRP. (B) Carbohydrate linkage analysis of o-gp140 using different enzymes. o-gp140 was digested with NANase (lane 2), O-glycosidase (lane 3), and PNGF (lane 4) separately and together (lane 5). o-gp140 without any enzyme was used as control (lane 1), and molecular mass standards (in kilodaltons) are also indicated. (C) endo-H digestion of gp120 US4 (lanes 2 and 3) and o-gp140 US4 (lanes 4 and 5). + and −, proteins with and without endo-H, respectively.

FIG. 6.

Carbohydrate profiling and sequencing analysis of purified o-gp140. (A) Heterogeneity of carbohydrates associated with gp120 US4 (lanes 3 and 4) and o-gp140 US4 (lanes 5 and 6). Two predominant oligosaccharides are indicated by arrows. (B) Carbohydrate sequencing analysis of oligosaccharide 1 (Oligo-1) and oligosaccharide 2. Purified oligosaccharides 1 and 2 were digested with NANase (lanes 3), GALase (lanes 4), HEXase (lanes 5), and MANase (lanes 6). Glucose ladder and fucosylated trisaccharide core structures were run in lanes 1 and 7, respectively. (C) Structure-based carbohydrate analysis.

FIG. 7.

(B and C) Binding of purified gp120 US4 (B) and o-gp140 US4 (C) to CD4 as determined by an HPLC-based assay. (A) Profile of unbound CD4. FITC, fluorescein isothiocyanate.

FIG. 8.

Immunochemical characterization of purified gp120 US4 and o-gp140 US4 by using a panel of MAbs, i.e., IgGCD4 (A), IgG1b12 (B), 17b (C), 2F5 (D), 447D (E), and T4 (F). OD, optical density.

FIG. 9.

Magnitude (A) and quality (B) of antibody responses induced by o-gp140 in rabbits at different time points after immunization. Animals were immunized at 0, 4, 12, and 24 weeks as indicated by the arrows.