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Differential binding of neutralizing and non-neutralizing antibodies to native-like soluble HIV-1 Env trimers, uncleaved Env proteins, and monomeric subunits.

Yasmeen A, Ringe R, Derking R, Cupo A, Julien JP, Burton DR, Ward AB, Wilson IA, Sanders RW, Moore JP, Klasse PJ - Retrovirology (2014)

Bottom Line: 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.We compared Fab and IgG versions of bNAbs and validated a bivalent model of IgG binding.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, Cornell University, New York, USA. pek2003@med.cornell.edu.

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.

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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).
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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).

Mentions: The kinetic profiles of VRC01 and PGT122, two NAbs that bound well to both SOSIP.664 trimers and gp120-gp41ECTO protomers, are displayed in Figure 3. (As shown in Figure 1, VRC01 only marginally differentiated among trimer, protomer, and gp120 in single-concentration qualitative analysis, whereas two NAbs, PGT123 and PGT128, closely related to PGT122, showed a slight trimer preference). The comparison by full kinetic modeling revealed differences in how VRC01 and PGT122 bind to the two forms of Env. VRC01 bound to the SOSIP.664 trimers with moderately fast association and markedly slow dissociation; but as it both associated faster with and dissociated more slowly from the protomer, its affinity was 10-fold higher for the protomer than the trimer (Additional file 3: Table S5). This affinity difference between trimer and protomer is in line with the results in Figure 2, which suggests that the formation of stable, native-like trimers disfavors VRC01 binding. The affinity difference is explained mechanistically by the three-dimensional cryo-EM structure of the same trimer in complex with the Fab of the CD4bs NAb PGV04. Thus, one protomer restricts access of the Fab to the CD4bs on the neighboring protomer [19].


Differential binding of neutralizing and non-neutralizing antibodies to native-like soluble HIV-1 Env trimers, uncleaved Env proteins, and monomeric subunits.

Yasmeen A, Ringe R, Derking R, Cupo A, Julien JP, Burton DR, Ward AB, Wilson IA, Sanders RW, Moore JP, Klasse PJ - Retrovirology (2014)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4067080&req=5

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).
Mentions: The kinetic profiles of VRC01 and PGT122, two NAbs that bound well to both SOSIP.664 trimers and gp120-gp41ECTO protomers, are displayed in Figure 3. (As shown in Figure 1, VRC01 only marginally differentiated among trimer, protomer, and gp120 in single-concentration qualitative analysis, whereas two NAbs, PGT123 and PGT128, closely related to PGT122, showed a slight trimer preference). The comparison by full kinetic modeling revealed differences in how VRC01 and PGT122 bind to the two forms of Env. VRC01 bound to the SOSIP.664 trimers with moderately fast association and markedly slow dissociation; but as it both associated faster with and dissociated more slowly from the protomer, its affinity was 10-fold higher for the protomer than the trimer (Additional file 3: Table S5). This affinity difference between trimer and protomer is in line with the results in Figure 2, which suggests that the formation of stable, native-like trimers disfavors VRC01 binding. The affinity difference is explained mechanistically by the three-dimensional cryo-EM structure of the same trimer in complex with the Fab of the CD4bs NAb PGV04. Thus, one protomer restricts access of the Fab to the CD4bs on the neighboring protomer [19].

Bottom Line: 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.We compared Fab and IgG versions of bNAbs and validated a bivalent model of IgG binding.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Weill Cornell Medical College, Cornell University, New York, USA. pek2003@med.cornell.edu.

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.

Show MeSH
Related in: MedlinePlus