PGV04, an HIV-1 gp120 CD4 binding site antibody, is broad and potent in neutralization but does not induce conformational changes characteristic of CD4

Abstract

Recently, several broadly neutralizing monoclonal antibodies (bnMAbs) directed to the CD4-binding site (CD4bs) of gp120 have been isolated from HIV-1-positive donors. These include VRC01, 3BNC117, and NIH45-46, all of which are capable of neutralizing about 90% of circulating HIV-1 isolates and all of which induce conformational changes in the HIV-1 gp120 monomer similar to those induced by the CD4 receptor. In this study, we characterize PGV04 (also known as VRC-PG04), a MAb with potency and breadth that rivals those of the prototypic VRC01 and 3BNC117. When screened on a large panel of viruses, the neutralizing profile of PGV04 was distinct from those of CD4, b12, and VRC01. Furthermore, the ability of PGV04 to neutralize pseudovirus containing single alanine substitutions exhibited a pattern distinct from those of the other CD4bs MAbs. In particular, substitutions D279A, I420A, and I423A were found to abrogate PGV04 neutralization. In contrast to VRC01, PGV04 did not enhance the binding of 17b or X5 to their epitopes (the CD4-induced [CD4i] site) in the coreceptor region on the gp120 monomer. Furthermore, in contrast to CD4, none of the anti-CD4bs MAbs induced the expression of the 17b epitope on cell surface-expressed cleaved Env trimers. We conclude that potent CD4bs bnMAbs can display differences in the way they recognize and access the CD4bs and that mimicry of CD4, as assessed by inducing conformational changes in monomeric gp120 that lead to enhanced exposure of the CD4i site, is not uniquely correlated with effective neutralization at the site of CD4 binding on HIV-1.

Fig 1

Neutralization activity of PGV04 on a 162- and on a 97-virus panel. (A) Potency of neutralization. Boxes are color coded for neutralization by donor serum as follows: yellow, median potency between 1:150 and 1:500, and orange, median potency between 1:501 and 1:1,000. Boxes are color coded for neutralization by MAbs as follows: orange, median potency between 0.2 and 2.0 μg/ml, and red, median potency <0.2 μg/ml. CRF07_BC, CRF08_BC, and AC viruses were not included in the clade analysis but are counted toward the total number of neutralized viruses in the 162-virus panel because there was only one virus tested for each of these clades. Clade D and E viruses were not included in the clade analysis but are counted toward the total number of neutralized viruses in the 97-virus panel because there were only 2 and 3 viruses, respectively, tested for these clades. dil'n, dilution. (B) Breath of neutralization. Boxes are colored as follows: orange, 61 to 90% of viruses neutralized, and red, 91 to 100% of viruses neutralized. As in described for panel A, CRF07_BC, CRF08_BC, AC, D, and E viruses were not included in the clade analysis but are counted toward the total number of neutralized viruses for the respective panel. (C) Comparison of the MAb PGV04 IC₅₀ and the donor serum NT₅₀ for the 162 viruses tested. The Spearman's rank correlation coefficient was calculated as −0.71 and the correlation was significant with a P value of <0.0001. IC₅₀s and NT₅₀s of viruses that did not neutralize were entered at the limit of the assay as 50 μg/ml for the MAb or 100 (1/dilution) for the serum. The anomalous data points are colored pink for those viruses that were neutralized by PGV04 at < 1 μg/ml but not by the donor serum and blue for those viruses that were neutralized by the donor serum but not by PGV04.

Fig 2

Effect of MAb binding to monomeric gp120 on binding of CD4i MAbs. Saturating amounts of the MAbs listed in the key were added to either JR-FL (A) or YU2 (B) gp120-coated ELISA plates. Then, a dilution curve of biotinylated 17b or X5 was added. Binding was detected with a streptavidin-AP conjugate and absorbance was read at 405 nm. OD, optical density.

Fig 3

Effects of CD4 or CD4bs MAbs on inducing the MAb 17b epitope, assessed by SPR. YU2 core gp120 was flowed over anti-Fc antibody-captured IgG 17b in the absence or presence of a 10-fold molar excess of 2-domain CD4 or Fabs of CD4bs MAbs. The sensorgrams in black and red represent gp120 alone and gp120 with ligands, respectively. RU, relative units.

Fig 4

Induction of the MAb 17b binding site on cell surface-expressed trimers. Saturating amounts of the MAbs listed in the key were added to 293T cells expressing JR-FL (A) or YU2ΔCT (B) envelope on their surface. Then, a dilution curve of biotinylated 17b was added to the cells. A streptavidin MAb conjugated to PE was used for detection, and binding was measured using flow cytometry. (C) Serial dilutions of the MAbs listed in the key were added to 293T cells expressing YU2ΔCT envelope; a secondary MAb conjugated to PE was used for detection, and binding was measured using flow cytometry. MFI, mean fluorescence intensity.

Fig 5

Mapping of selected alanine substitutions on a gp120 core structure. A V1/V2 and V3 trimmed core HIV-1 gp120 in its PGV04-bound conformation is shown in surface representation. Selected alanine substitutions that affected neutralization by (A) or binding to (B) PGV04 are color coded according to their degree of neutralization or binding relative to wild-type JR-CSF binding as follows: blue, 0 to 5%; green, 6 to 40%; and yellow, 250 to 1,000%. Alanine substitutions in the V1/V2 and V3 loops that affect PGV04 neutralization or binding are not represented, as these variable loops are not present in the core gp120 structure. The PGV04 epitope on HIV-1 gp120 is highlighted with red borders.

Fig 6

Ability of PGV04 to bind to mock-treated BaL gp120 (solid lines) or endo-H and endo-F-treated BaL gp120 (dotted lines).

Fig 7

Antibody polyreactivity ELISA. PGV04 was tested for ELISA reactivity against a panel of antigens. The MAbs b12 and 4E10 were also included for comparison. The vertical dashed lines demarcate the results for each antigen.