Characterization of human immunodeficiency virus type 1 monomeric and trimeric gp120 glycoproteins stabilized in the CD4-bound state: antigenicity, biophysics, and immunogenicity

Abstract

The human immunodeficiency virus type 1 exterior gp120 envelope glycoprotein is highly flexible, and this flexibility may contribute to the inability of monomeric gp120 immunogens to elicit broadly neutralizing antibodies. We previously showed that an S375W modification of a critical interfacial cavity central to the primary receptor binding site, the Phe43 cavity, stabilizes gp120 into the CD4-bound state. However, the immunological effects of this cavity-altering replacement were never tested. Subsequently, we screened other mutations that, along with the S375W alteration, might further stabilize the CD4-bound state. Here, we define a selected second cavity-altering replacement, T257S, and analyze the double mutations in several gp120 envelope glycoprotein contexts. The gp120 glycoproteins with the T257S-plus-S375W double mutation (T257S+S375W) have a superior antigenic profile compared to the originally identified single S375W replacement in terms of enhanced recognition by the broadly neutralizing CD4 binding-site antibody b12. Isothermal titration calorimetry measuring the entropy of the gp120 interaction with CD4 indicated that the double mutant was also stabilized into the CD4-bound state, with increasing relative fixation between core, full-length monomeric, and full-length trimeric versions of gp120. A significant increase in gp120 affinity for CD4 was also observed for the cavity-filling mutants relative to wild-type gp120. The most conformationally constrained T257S+S375W trimeric gp120 proteins were selected for immunogenicity analysis in rabbits and displayed a trend of improvement relative to their wild-type counterparts in terms of eliciting neutralizing antibodies. Together, the results suggest that conformational stabilization may improve the ability of gp120 to elicit neutralizing antibodies.

FIG. 1.

Immunoprecipitation and ELISA analysis of WT and mutant (T257S+S375W) variants of core and gp120 proteins to characterize 17b binding in the presence or absence of sCD4. (A) Immunoprecipitation and gel analysis. Supernatants from transfection cultures of envelope glycoproteins were incubated with 17b monoclonal antibody and protein A-agarose beads with or without prior incubation with sCD4. The immunoprecipitated proteins were analyzed by SDS-PAGE and visualized with Coomassie blue staining. Lane 1, unmodified core without sCD4; lane 2, unmodified core with sCD4; lane 3, mutant core without sCD4; lane 4, mutant core with sCD4; lane 5, WT gp120 without sCD4; lane 6, WT gp120 with sCD4; lane 7, mutant gp120 without sCD4; lane 8, mutant gp120 with sCD4. Migration of core proteins (open arrow) and gp120 monomers (solid arrow) is indicated. (B) ELISA. Equal amounts of affinity-purified unmodified and mutant proteins were coated onto ELISA plates in duplicates and reacted with fivefold serial dilutions of 17b antibody in the presence (solid symbols) or absence (open symbols) of 20 μg/ml of sCD4. 17b binding to various Envs was detected by reacting the wells with anti-human IgG conjugated to peroxidase. Upper left (circles), WT core; lower left (diamonds), mutant core; upper right (squares), WT gp120; lower right (triangles), mutant gp120. Margins of error from duplicate reactions were negligible in all cases.

FIG. 2.

Reducing SDS-PAGE, blue native gels, and gel filtration profiles of WT and mutant (S375S+T257S) YU2 envelope proteins. (A) Reducing SDS-PAGE of purified envelope glycoproteins. Lane 1, unmodified core; lane 2, mutant core; lane 3, WT gp120; lane 4, mutant gp120; lane 5, WT gp120 trimer purified by gel filtration; lane 6, mutant gp120 trimer before gel filtration; lane 7, mutant gp120 trimer after gel filtration. Molecular weight markers (in thousands) are shown on the left. (B) Blue native gel. Lane 1, WT gp120 monomer; lane 2, mutant gp120 monomer; lane 3, WT trimer purified by gel filtration; lane 4, mutant trimer before gel filtration; lane 5, mutant trimer after gel filtration. Molecular weight markers (in thousands) are indicated on the left. (C) Gel filtration profile of the affinity-purified mutant trimer. (D) Gel filtration profile of the purified mutant trimeric fraction.

FIG. 3.

Antigenicity of WT and mutant glycoproteins. (A) Binding to three CD4 binding-site antibodies was measured by ELISA analysis of the following proteins: WT proteins (open symbols) and T257S+S375W mutant proteins (closed symbols). Top panel, binding to b12; middle panel, binding to F105; bottom panel, binding to b6. (B) Effect of a second-site mutation (T257S) on b12 recognition. Top panel, comparison of binding to b12 by WT gp120 (open square), S375W single mutant gp120 (cross), and T257S+S375W double mutant gp120 (closed square); bottom panel, binding to HIV IgG was used as an internal control for protein concentration. Margins of error from duplicate reactions were negligible in all cases.

FIG. 4.

Entry and b12 neutralization of pseudotyped WT and mutant HIV-1. (A) Entry of HIV-1 pseudotyped with WT, the S375W single mutant, and the T257S+S375W double mutant YU2 Env. Percent relative entries compared to WT virus are indicated in the graph. The average values of four replicates from one representative experiment have been plotted. y-axis error bars indicate standard deviations of the replicate values. (B) Neutralization IC₅₀ values of the three viruses with selected antibodies are shown.

FIG. 5.

Isothermal titration calorimetry analyses of unmodified and mutant (T257S+S375W) YU2 core proteins. ITC experiments representing the interactions of sCD4 with unmodified (A) and mutant (B) core proteins at 37°C are shown. The top panels represent the raw data as power versus time. The area under each spike is proportional to the heat produced for each injection. The bottom panels represent integrated areas per mole of injected ligand (sCD4) as a function of the molar ratio. The solid line represents the best nonlinear fit to the experimental data. (A) ΔH = −50 kcal/mol; K_d = 29 nM. (B) ΔH = −49 kcal/mol; K_d = 1 nM.

FIG. 6.

IC₅₀ neutralization values of different groups of immunized rabbit sera tested against a panel of HIV-1 isolates. Low-dose values were obtained with sera collected after three inoculations (post-3), and high-dose values were obtained with sera collected after three inoculations (post-3) as well as after four inoculations (post-4). IC₅₀ values between 100 and 249 are coded in yellow, and those exceeding 250 are highlighted in red. Virus pseudotyped with murine leukemia virus (MuLV) was used as a negative control for specificity.

FIG. 7.

Mapping of gp120 and stabilized trimer-elicited sera tested for the capacity to affect sCD4 binding and for the presence of CD4i antibodies. (A, B, and C) Binding of sCD4 to YU2 core protein was measured by ELISA analysis in the presence of fivefold serial dilutions of the following reagents: the human anti-gp120 antibodies b12, 2G12, and 17b (A); rabbit antisera obtained after four inoculations with WT gp120 proteins (samples 3 to 7) and WT trimer proteins (samples 8 to 11) (B); and antisera obtained after four inoculations with stabilized gp120 trimer protein (C). Each data point is an average of duplicate samples with negligible margins of errors that are not visible on the graphs. Two negative control curves are shown; in A and B, the binding of CD4 in the absence of any inhibitor (marked as no inhibitor) indicates 100% binding; in C, the average values obtained in the presence of BSA-immunized serum samples 1 and 2 are shown. (D) Neutralization IC₅₀ values of rabbit sera tested against HIV-2 isolate 7312A/V434M. The pseudovirus was treated with media lacking CD4 (−CD4) or containing 0.5 μg/ml of sCD4 (+CD4) for 1 h prior to adding antisera.

FIG. 8.

Binding of WT gp120 and stabilized trimer-elicited sera to unmodified YU2 core and cysteine-constrained HXBc2 core proteins detected by ELISA. (A and C) Binding to YU2 core proteins by antisera elicited by WT gp120 proteins (A) and sera elicited by the stabilized trimers (C). (B and D) Binding to the cysteine-constrained HXBc2 core protein Ds12F123 by antisera elicited by WT gp120 proteins (B) and sera elicited by the stabilized trimers (D). Prebleed antisera and antisera from rabbits inoculated with BSA are included as negative controls.