Antigenicity and immunogenicity of RV144 vaccine AIDSVAX clade E envelope immunogen is enhanced by a gp120 N-terminal deletion

Abstract

An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity.

Fig 1

Diagram of HIV-1 gp120 proteins constructs with modifications. Designs for each of the A244 proteins either with no modifications (gp120), with both the HSV gD tag (first 27 aa of mature HSV gD protein) and N-terminal 11-aa deletion (gDΔ11 gp120), or with only the 11-aa deletion (Δ11 gp120) are outlined. The gDΔ11 gp120 constructs for all three proteins are similar with respect to modifications to the design used in the RV144 vaccine trial for A244-rgp120 and MN-rgp120. The Env gp120 proteins were expressed and purified as described in Materials and Methods. A similar design was used to construct gp120 proteins with or without N-terminal deletion and included the clade B Env MN, 63521, and 6240, the clade C 1086, and the E clade 92TH023. LLE, linear linkage encoding.

Fig 2

Binding of C1, V2, and V3 antibodies to RV144 immunogen gp120 proteins. RV144 Env proteins MN-rgp120 and A244-rgp120 binding at various concentrations (0.5, 1.0, 2.0, 3.0, 4.0, and 5 μg/ml for A32; 2.0, 5.0, 10, 20, 40, and 60 μg/ml and 5.0, 10.0, 25.0, 50.0, 75.0, and 100 μg/ml for 697D MAb binding to MN gp120 and A244 gp120, respectively; and 0.5, 1.0, 2.0, 3.0, 4.0, and 5 μg/ml for 19b) to A32, 697D, and 19b are shown. The calculated K_d values and fitted curves (black line) for each of the binding interactions are shown. Each MAb was captured on anti-Fc antibody immobilized surfaces, and gp120 monomeric proteins were injected as analytes, as described in Materials and Methods. The data are representative of three measurements.

Fig 3

Relative proportions of monomer and dimer in A244 gp120 proteins. (A) SDS-PAGE analysis (under reduced and nonreduced conditions) of A244, A244 gD Δ11, and A244 Δ11 gp120 proteins showing the presence of disulfide-linked dimers in various proportions. Each of the gp120 preparations was analyzed by size exclusion chromatography (SEC), which showed a relatively larger proportion of monomer in A244 gp120 (B) than in either A244 Δ11 (C) or A244 gD Δ11 (D). Peak volume analysis of the monomer and dimer fractions gave the following proportions of monomer and dimer: in A244 gp120-A244 gp120, dimer, 58%, and monomer, 38%; in A244 Δ11 gp120, dimer, 30%, and monomer, 66%; in A244 gD Δ11 gp120, dimer, 33%, and monomer, 63%. Similarly, for B.63521 gp120, the proportion of monomer was improved by N-terminal deletion: for 63521 gp120, dimer, 23%, and monomer, 77%; and for B.63251 Δ11 gp120, there was no resolved peak for the dimer and the result for monomer was >95%. For C.1086, a modest improvement was observed with C.1086 gp120 (dimer, 33%; monomer, 67%) and C.1086Δ7 gp120 (dimer, 27%; monomer, 73%). The standard deviation of the measurements of percentage of monomer and dimer was within 1%.

Fig 4

Enhanced binding of C1, V2, and V1/V2 antibodies to E.A244gp120 proteins with Δ11 deletion. Each of the analyte gp120 proteins (left panel, A244 gp120; middle panel, A244gDΔ11; right panel, A244Δ11 gp120) was injected over the listed antibodies captured on an anti-Fc immobilized surface. Each gp120 protein was titrated at 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 μg/ml for 19b (A) and A32 (B); A244 gp120 at 10, 20, 30, 40, and 50 μg/ml for 697D (C); 10, 25, 50, 75, and 100 μg/ml on CH01(D) and PG9 (E); A244gDΔ11 at 5, 10, 20, 30, and 40 μg/ml for 697-D and 10, 25, 50, 75, and 100 μg/ml for CH01 and at 10, 20, 30, and 40 μg/ml for PG9; A244Δ11 at 2, 4, 6, 10, 25, and 50 μg/ml for 697-D, 10, 25, 50, 75, and 100 μg/ml for CH01 and 10, 25, 50, 75, and 100 μg/ml for PG9 MAb captured surfaces. Data are representative of at least 3 measurements made on individual flow cells with equivalent amounts of captured antibodies. All SPR binding experiments were carried out using purified monomeric gp120 as assessed by size exclusion chromatography.

Fig 5

Binding of CH01_RUA1 and CH01_RUA2 to A244Δ11 gp120. Each of the CH01 RUAs (RUA1 and RUA2 data are shown in top and bottom panels, respectively) was captured as described in the legend for Fig. 4. Each of the three forms of A244 gp120 was flowed at concentrations of 10, 25, 50, 75, and 100 μg/ml. Kinetic rate constants and K_d were derived as described in Materials and Methods. Data are representative of at least 3 measurements made on individual flow cells with equivalent amounts of captured antibodies.

Fig 6

RV144 vaccinee sera antibody responses. (A) RV144 vaccinee IgG binding to A244 gp120 proteins show higher avidity for A244 gp120 with Δ11. RV144 visit 8 (week 26, 2 weeks following the final immunization) IgG (n = 97) binding was measured for A244 gp120, A244 gD Δ11, and A244 Δ11 gp120 proteins. Binding responses and dissociation rate constants for avidity score measurements were calculated as described in Materials and Methods. The mean avidity of binding to A244 gp120, A244 gD Δ11, and A244 Δ11 gp120 were 1.0 ± 1.5, 10.0 ± 0.5, and 5.7 ± 0.7 RU · s (× 10⁵), respectively. The differences in avidity were significant for A244 gp120 versus A244gDΔ11 (Student's t test, P < 0.001) and A244 gp120 versus A244Δ11 gp120 (Student's t test, P < 0.001). The binding of plasma IgG samples from the placebo group is shown as open circles. (B) RV144 vaccinee plasma IgG binding to A244 gp120 proteins show higher relative binding to A244 gp120 with Δ11. RV144 visit 8 (week 26, 2 weeks following the final immunization) plasma antibody was measured against A244 gp120, A244 gD Δ11, and A244 Δ11 gp120 proteins in a binding antibody multiplex assay, and the mean fluorescence intensity (MFI) values were plotted. The differences in binding responses were significant for A244 gp120 versus A244 Δ11 gp120 (Student's t test, P < 0.001). (C) Blocking of RV144 induced IgG binding to A244 gD Δ11 gp120 by conformational C1 (A32), V2 (697D), and V1/V2 (CH01) antibodies. RV144 IgG samples (n = 109) with high levels (>80 RU measured at 200 μg/ml) of binding to A244 Δ11 gp120 were selected for antibody blocking studies. A control group (n = 19, open circles) showing no binding to A244 Δ11 gp120 was included to assess nonspecific signal in IgG samples. The mean blocking rates of RV144 plasma IgG by MAbs A32, 697-D, and CH01 were 67.4% ± 11.4%, 34.1% ± 14.5%, and 22.1% ± 12.5%, respectively. (D) ELISAs showing high levels of A32 blocking (mean = 39.6% ± 19.2%) by RV144 IgG and low levels of CD4 blocking antibodies (mean = 13% ± 8.9%). Blocking of IgG from visit 1 were 6.7% ± 4.2% and 8.9% ± 7.6% for A32 and CD4, respectively.

Fig 7

RV144 MAbs CH51 and CH54 show higher-affinity binding to A244gp120 with Δ11 modification. A244, A244 gD Δ11, and A244 Δ11 gp120 were each injected at increasing concentrations of 5, 10, 15, 20, and 25 μg/ml over either CH51 (A) or CH54 (B) captured on anti-Fc immobilized surfaces. Binding of both CH51 and CH54 was 1 order of magnitude higher for both A244 gp120 with the Δ11 modification than for A244 gp120 (left panel) with no modification.

Fig 8

Higher levels of V1/V2 MAb blocking antibodies induced in rhesus macaques immunized with A244 Δ11 gp120 protein. Binding ELISA titers (ED₅₀, half-maximal dilution titer) of NHP plasma antibodies to RV144 immunogen protein A244 gDΔ11 gp120 (A) and the V1/V2 protein B. Case A V1/V2 Tags (B) were measured for each animal in the three immunogen groups. Data from A244 gp120-, A244 gD Δ11 gp120-, and A244 Δ11 gp120-immunized animals are shown in open circles, filled circles, and filled diamonds, respectively. Bars in each plot indicate mean values for each of the indicated immunogen group. PI-2, PI-3, and PI-4 indicate postimmunization plasma samples harvested 2 weeks after immunization time points of 4, 8, and 16 weeks, respectively. The difference in titers between animal groups immunized with A244 gp120 (median ED₅₀ = 342 ± 125) versus A244 Δ11 gp120 (median ED₅₀ = 1,069 ± 583) for binding to B. Case V1/V2 Tags did not reach statistical significance (P = 0.09). (C) Blocking of NHP IgG with conformational V2 and V1/V2 MAbs. For 697-D blocking, the median values (± SD) for A244 gp120-, A244 gD Δ11-, and A244 Δ11 gp120-immunized NHP groups were 30.9 ± 4.2 (28.7), 37.0 ± 9.8 (33.6), and 56.6 ± 9.8 (51.7), respectively (*, P = 0.013 for A244 Δ11 versus A244 gp120). For CH01 blocking, the blocking rates were 20.3% ± 3.8% (24.1%), 18.6% ± 8.1% (21.8%), and 45.2% ± 7.4% (51.7%), respectively (*, P = 0.006 for A244 Δ11 versus A244 gp120; **, P = 0.026 for A244 Δ11 versus A244 gD gp120). For CH58, the blocking rates were 77.5% ± 9.4%, 90.5% ± 4.6%, and 74.1% ± 24.3%, respectively, for A244 gp120-, A244 gDΔ11-, and A244 Δ11 gp120-immunized groups. The median values in a second independent experiment were similar and are given in parentheses above. The measurement of blocking antibodies was carried out on NHP IgG samples harvested from PI-3 plasma of each immunogen group and was performed as described for data in Fig. 6 and in Materials and Methods.