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. 2014 May 29;11:41.
doi: 10.1186/1742-4690-11-41.

Differential Binding of Neutralizing and Non-Neutralizing Antibodies to Native-Like Soluble HIV-1 Env Trimers, Uncleaved Env Proteins, and Monomeric Subunits

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Free PMC article

Differential Binding of Neutralizing and Non-Neutralizing Antibodies to Native-Like Soluble HIV-1 Env Trimers, Uncleaved Env Proteins, and Monomeric Subunits

Anila Yasmeen et al. Retrovirology. .
Free PMC article

Abstract

Background: The trimeric envelope glycoproteins (Env) on the surface of HIV-1 virions are the targets for neutralizing antibodies (NAbs). No candidate HIV-1 immunogen has yet induced potent, broadly active NAbs (bNAbs). Part of the explanation may be that previously tested Env proteins inadequately mimic the functional, native Env complex. Trimerization and the proteolytic processing of Env precursors into gp120 and gp41 profoundly alter antigenicity, but soluble cleaved trimers are too unstable to serve as immunogens. By introducing stabilizing mutations (SOSIP), we constructed soluble, cleaved Env trimers derived from the HIV-1 subtype A isolate BG505 that resemble native Env spikes on virions both structurally and antigenically.

Results: We used surface plasmon resonance (SPR) to quantify antibody binding to different forms of BG505 Env: the proteolytically cleaved SOSIP.664 trimers, cleaved gp120-gp41ECTO protomers, and gp120 monomers. Non-NAbs to the CD4-binding site bound only marginally to the trimers but equally well to gp120-gp41ECTO protomers and gp120 monomers, whereas the bNAb VRC01, directed to the CD4bs, bound to all three forms. In contrast, bNAbs to V1V2 glycan-dependent epitopes bound preferentially (PG9 and PG16) or exclusively (PGT145) to trimers. We also explored the antigenic consequences of three different features of SOSIP.664 gp140 trimers: the engineered inter-subunit disulfide bond, the trimer-stabilizing I559P change in gp41ECTO, and proteolytic cleavage at the gp120-gp41ECTO junction. Each of these three features incrementally promoted native-like trimer antigenicity. We compared Fab and IgG versions of bNAbs and validated a bivalent model of IgG binding. The NAbs showed widely divergent binding kinetics and degrees of binding to native-like BG505 SOSIP.664. High off-rate constants and low stoichiometric estimates of NAb binding were associated with large amounts of residual infectivity after NAb neutralization of the corresponding BG505.T332N pseudovirus.

Conclusions: The antigenicity and structural integrity of cleaved BG505 SOSIP.664 trimers render these proteins good mimics of functional Env spikes on virions. In contrast, uncleaved gp140s antigenically resemble individual gp120-gp41ECTO protomers and gp120 monomers, but not native trimers. Although NAb binding to functional trimers may thus be both necessary and sufficient for neutralization, the kinetics and stoichiometry of the interaction influence the neutralizing efficacy of individual NAbs.

Figures

Figure 1
Figure 1
The effect of oligomerization on Env antigenicity. The sensorgrams show the binding (RU) of the listed IgGs to the BG505 SOSIP.664 trimer, gp120-gp41ECTO protomer (gp140), and monomeric gp120 over time (s) on the x axis. Association was followed for 5 min and dissociation for 10 min. The Env proteins were captured on the chip by amine-coupled D7324 antibody. For each Ab tested similar levels of Env were captured: RL values were ~ 500 RU for trimers and protomers for all Abs except for the V3-specific ones, where RL was ~200 RU for trimer and protomer, and in all cases ~15% lower for gp120 to yield approximately equal numbers of gp120 subunits for all three forms of Env. The antibodies tested as analytes bind to different clusters of epitopes: b12, b6, and VRC01 to the CD4bs; F240 to cluster I in gp41; PG9, PG16, and PGT145 to V1V2-glycan epitopes at the apex of the trimer; PGT151 to a newly discovered epitope that spans the interface between gp120 and gp41ECTO in one protomer and also makes contact with a second gp41ECTO subunit; 2G12 to a mannose-glycan-dependent epitope; PGT123 and PGT128 to composite V3-base and glycan epitopes; and 14e and 19b to V3 epitopes. MAbs b12, b6, F240, 14e, and 19b do not neutralize the corresponding BG505.T332N virus, whereas VRC01, 2G12, PGT123, PGT128, PG9, PG16, PGT145, and PGT151 do. All MAbs were injected at 1 μM. The sensorgrams show one of two replicates.
Figure 2
Figure 2
The effects of cleavage and trimer-stabilizing mutations on Env antigenicity. The sensorgrams show the binding (RU) of the listed MAbs to BG505 SOSIP.664 trimers (labeled “SOSIP.R6”) and five mutated variants thereof (see Results). MAbs b12, b6, F240, 14e, and 19b do not neutralize the corresponding BG505.T332N virus, whereas VRC01, 2G12, PG16, and PGT145 all do. The SPR method was the same as for Figure 1, except that the MAbs were injected at 500 nM. The sensorgrams show one of two replicates.
Figure 3
Figure 3
Comparisons of VRC01 and PGT122 binding to the Env SOSIP.664 trimer and gp120-gp41ECTO protomer. The sensorgrams show VRC01 (A) and PGT122 (B) IgG binding to BG505 SOSIP.664 trimers (top) and gp120-gp41ECTO protomers (bottom). The colored curves show the response at various analyte concentrations as indicated to the right. Note that the color code is the same for all diagrams but that the titration ranges start and end at different concentrations and also differ in the dilution steps. The modeled curves in black (bivalent model) become visible only when they diverge from the empirical data. The sensorgrams show one of two of replicates. In some experiments the dissociation phase had to be extended to 20 min to achieve significant values (T > 10) for kd1 (not shown).
Figure 4
Figure 4
The kinetics of bNAb interactions with SOSIP.664 trimers. The sensorgrams show binding titrations fitted with the bivalent model for all NAbs except 2G12 (Langmuir model). The colored curves show the response at analyte concentrations indicated to the right. Note that the color code is the same for all diagrams but that the titration ranges start and end at different concentrations and also differ in the dilution steps. The modeled curves in black are only visible when they diverge from the data. The sensorgrams show one of the multiples of replicates (n) given in Additional file 3: Table S5. In some experiments the dissociation phase had to be extended to 20 min to achieve significant values (T > 10) for kd1 (not shown).
Figure 5
Figure 5
Persistent fractions in neutralization assays. The infectivity of BG505.T332N pseudovirus was measured on Tzm-bl cells as luciferase activity (luminescence) after incubation with NAbs. The log [relative infectivity] is expressed on the y-axis as a function of the log NAb concentration [nM] on the x-axis. The data are fitted with a sigmoid function with variable slope and an unconstrained upper plateau; the lower plateau as a fitted parameter represents the persistent fraction, PF.

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