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Viral escape from neutralizing antibodies in early subtype A HIV-1 infection drives an increase in autologous neutralization breadth.

Murphy MK, Yue L, Pan R, Boliar S, Sethi A, Tian J, Pfafferot K, Karita E, Allen SA, Cormier E, Goepfert PA, Borrow P, Robinson JE, Gnanakaran S, Hunter E, Kong XP, Derdeyn CA - PLoS Pathog. (2013)

Bottom Line: Crystal structures of the antigen-binding fragments (Fabs) revealed flat epitope contact surfaces, where minimal light chain mutation in 19.3H-L3 allowed for additional antigenic interactions.Our data demonstrate that this subject's first recognized nAb epitope elicited strain-specific mAbs, which incrementally acquired autologous breadth, and directed later B cell responses to target distinct portions of Env.This immune re-focusing could have triggered the evolution of cross-clade antibodies and suggests that exposure to a specific sequence of immune escape variants might promote broad humoral responses during HIV-1 infection.

View Article: PubMed Central - PubMed

Affiliation: Immunology and Molecular Pathogenesis Graduate Program, Emory University, Atlanta, Georgia, United States of America.

ABSTRACT
Antibodies that neutralize (nAbs) genetically diverse HIV-1 strains have been recovered from a subset of HIV-1 infected subjects during chronic infection. Exact mechanisms that expand the otherwise narrow neutralization capacity observed during early infection are, however, currently undefined. Here we characterized the earliest nAb responses in a subtype A HIV-1 infected Rwandan seroconverter who later developed moderate cross-clade nAb breadth, using (i) envelope (Env) glycoproteins from the transmitted/founder virus and twenty longitudinal nAb escape variants, (ii) longitudinal autologous plasma, and (iii) autologous monoclonal antibodies (mAbs). Initially, nAbs targeted a single region of gp120, which flanked the V3 domain and involved the alpha2 helix. A single amino acid change at one of three positions in this region conferred early escape. One immunoglobulin heavy chain and two light chains recovered from autologous B cells comprised two mAbs, 19.3H-L1 and 19.3H-L3, which neutralized the founder Env along with one or three of the early escape variants carrying these mutations, respectively. Neither mAb neutralized later nAb escape or heterologous Envs. Crystal structures of the antigen-binding fragments (Fabs) revealed flat epitope contact surfaces, where minimal light chain mutation in 19.3H-L3 allowed for additional antigenic interactions. Resistance to mAb neutralization arose in later Envs through alteration of two glycans spatially adjacent to the initial escape signatures. The cross-neutralizing nAbs that ultimately developed failed to target any of the defined V3-proximal changes generated during the first year of infection in this subject. Our data demonstrate that this subject's first recognized nAb epitope elicited strain-specific mAbs, which incrementally acquired autologous breadth, and directed later B cell responses to target distinct portions of Env. This immune re-focusing could have triggered the evolution of cross-clade antibodies and suggests that exposure to a specific sequence of immune escape variants might promote broad humoral responses during HIV-1 infection.

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gp120 binding by and competition of R880F mAbs 19.3H-L1 and 19.3H-L3.The baseline binding of four biotinylated mAbs, 19.3H-L1, 19.3H-L3, 6.4C, or PGT128, was evaluated by ELISA with three R880F gp120 proteins: (A) wild-type 0-A6/B24, (B) point mutant 0-A6/B24 I295R, and (C) point mutant 0-A6/B24 E338K. R880F mAbs 19.3H-L1 and 19.3H-L3 were then competed with themselves, each other, and the negative control antibody, 6.4C. For the competition ELISAs, plates were coated with wild-type R880F 0-A6/B24 gp120 protein, pre-incubated with serially-diluted 19.3H-L1, 19.3H-L3, or 6.4C, washed, and then incubated with 1 µg/ml biotinylated 19.3H-L1 (D) or 19.3H-L3 (E). From data in (A), 1 µg/ml was selected as a point of non-saturated binding. The horizontal dashed lines in (D) and (E) represent 100% binding of biotinylated 19.3H-L1 or 19.3H-L3, at 1 µg/ml in the absence of competitor, respectively. Optical density values at 450 nm are depicted on the vertical axis; mAb concentrations (in µg/ml) are plotted along the horizontal axis in a logarithmic fashion. Error bars demonstrate the standard error of the mean of two independent experiments.
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ppat-1003173-g006: gp120 binding by and competition of R880F mAbs 19.3H-L1 and 19.3H-L3.The baseline binding of four biotinylated mAbs, 19.3H-L1, 19.3H-L3, 6.4C, or PGT128, was evaluated by ELISA with three R880F gp120 proteins: (A) wild-type 0-A6/B24, (B) point mutant 0-A6/B24 I295R, and (C) point mutant 0-A6/B24 E338K. R880F mAbs 19.3H-L1 and 19.3H-L3 were then competed with themselves, each other, and the negative control antibody, 6.4C. For the competition ELISAs, plates were coated with wild-type R880F 0-A6/B24 gp120 protein, pre-incubated with serially-diluted 19.3H-L1, 19.3H-L3, or 6.4C, washed, and then incubated with 1 µg/ml biotinylated 19.3H-L1 (D) or 19.3H-L3 (E). From data in (A), 1 µg/ml was selected as a point of non-saturated binding. The horizontal dashed lines in (D) and (E) represent 100% binding of biotinylated 19.3H-L1 or 19.3H-L3, at 1 µg/ml in the absence of competitor, respectively. Optical density values at 450 nm are depicted on the vertical axis; mAb concentrations (in µg/ml) are plotted along the horizontal axis in a logarithmic fashion. Error bars demonstrate the standard error of the mean of two independent experiments.

Mentions: To ascertain if 19.3H-L1 and 19.3H-L3 would compete for Env binding, three R880F gp120 monomeric proteins (the 0-A6/B24 founder Env gp120, and mutants containing I295R or E338K) were synthesized, purified, and employed in a competition ELISA assay. To first establish a baseline level of binding, the R880F mAbs were biotinylated and incubated with wild-type 0-A6/B24 gp120 protein. 19.3H-L3 demonstrated more robust binding, as compared to 19.3H-L1; the negative control mAb 6.4C (directed against a highly specific epitope in V1V2 [31]), and the broadly neutralizing mAb PGT128 [8], which shares epitope space with the R880F mAbs, both failed to bind (Figure 6A). Consistent with the neutralization data in Figure 5, neither R880F mAb could bind detectably to the I295R or E338K mutant gp120 proteins (Figure 6B–C). Wild-type 0-A6/B24 gp120 protein was then pre-incubated with 19.3H-L1, 19.3H-L3, or the negative control antibody 6.4C, washed, and incubated with either biotinylated 19.3H-L1 (Figure 6D) or 19.3H-L3 (Figure 6E) to discern if initial pre-incubation could block secondary binding. 19.3H-L1 modestly competed with itself (Figure 6D) but could not effectively compete for binding with 19.3H-L3 (Figure 6E). Conversely, 19.3H-L3 strongly competed with both itself (Figure 6E) and 19.3H-L1 (Figure 6D). Thus, 19.3H-L3 neutralizes a greater number of R880F Envs than 19.3H-L1, binds more strongly to the founder 0-A6/B24 gp120, and neutralizes the Env 0-A6/B24 pseudovirus more potently, underscoring the significance of VL alterations where antigen recognition and neutralization efficacy are concerned.


Viral escape from neutralizing antibodies in early subtype A HIV-1 infection drives an increase in autologous neutralization breadth.

Murphy MK, Yue L, Pan R, Boliar S, Sethi A, Tian J, Pfafferot K, Karita E, Allen SA, Cormier E, Goepfert PA, Borrow P, Robinson JE, Gnanakaran S, Hunter E, Kong XP, Derdeyn CA - PLoS Pathog. (2013)

gp120 binding by and competition of R880F mAbs 19.3H-L1 and 19.3H-L3.The baseline binding of four biotinylated mAbs, 19.3H-L1, 19.3H-L3, 6.4C, or PGT128, was evaluated by ELISA with three R880F gp120 proteins: (A) wild-type 0-A6/B24, (B) point mutant 0-A6/B24 I295R, and (C) point mutant 0-A6/B24 E338K. R880F mAbs 19.3H-L1 and 19.3H-L3 were then competed with themselves, each other, and the negative control antibody, 6.4C. For the competition ELISAs, plates were coated with wild-type R880F 0-A6/B24 gp120 protein, pre-incubated with serially-diluted 19.3H-L1, 19.3H-L3, or 6.4C, washed, and then incubated with 1 µg/ml biotinylated 19.3H-L1 (D) or 19.3H-L3 (E). From data in (A), 1 µg/ml was selected as a point of non-saturated binding. The horizontal dashed lines in (D) and (E) represent 100% binding of biotinylated 19.3H-L1 or 19.3H-L3, at 1 µg/ml in the absence of competitor, respectively. Optical density values at 450 nm are depicted on the vertical axis; mAb concentrations (in µg/ml) are plotted along the horizontal axis in a logarithmic fashion. Error bars demonstrate the standard error of the mean of two independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585129&req=5

ppat-1003173-g006: gp120 binding by and competition of R880F mAbs 19.3H-L1 and 19.3H-L3.The baseline binding of four biotinylated mAbs, 19.3H-L1, 19.3H-L3, 6.4C, or PGT128, was evaluated by ELISA with three R880F gp120 proteins: (A) wild-type 0-A6/B24, (B) point mutant 0-A6/B24 I295R, and (C) point mutant 0-A6/B24 E338K. R880F mAbs 19.3H-L1 and 19.3H-L3 were then competed with themselves, each other, and the negative control antibody, 6.4C. For the competition ELISAs, plates were coated with wild-type R880F 0-A6/B24 gp120 protein, pre-incubated with serially-diluted 19.3H-L1, 19.3H-L3, or 6.4C, washed, and then incubated with 1 µg/ml biotinylated 19.3H-L1 (D) or 19.3H-L3 (E). From data in (A), 1 µg/ml was selected as a point of non-saturated binding. The horizontal dashed lines in (D) and (E) represent 100% binding of biotinylated 19.3H-L1 or 19.3H-L3, at 1 µg/ml in the absence of competitor, respectively. Optical density values at 450 nm are depicted on the vertical axis; mAb concentrations (in µg/ml) are plotted along the horizontal axis in a logarithmic fashion. Error bars demonstrate the standard error of the mean of two independent experiments.
Mentions: To ascertain if 19.3H-L1 and 19.3H-L3 would compete for Env binding, three R880F gp120 monomeric proteins (the 0-A6/B24 founder Env gp120, and mutants containing I295R or E338K) were synthesized, purified, and employed in a competition ELISA assay. To first establish a baseline level of binding, the R880F mAbs were biotinylated and incubated with wild-type 0-A6/B24 gp120 protein. 19.3H-L3 demonstrated more robust binding, as compared to 19.3H-L1; the negative control mAb 6.4C (directed against a highly specific epitope in V1V2 [31]), and the broadly neutralizing mAb PGT128 [8], which shares epitope space with the R880F mAbs, both failed to bind (Figure 6A). Consistent with the neutralization data in Figure 5, neither R880F mAb could bind detectably to the I295R or E338K mutant gp120 proteins (Figure 6B–C). Wild-type 0-A6/B24 gp120 protein was then pre-incubated with 19.3H-L1, 19.3H-L3, or the negative control antibody 6.4C, washed, and incubated with either biotinylated 19.3H-L1 (Figure 6D) or 19.3H-L3 (Figure 6E) to discern if initial pre-incubation could block secondary binding. 19.3H-L1 modestly competed with itself (Figure 6D) but could not effectively compete for binding with 19.3H-L3 (Figure 6E). Conversely, 19.3H-L3 strongly competed with both itself (Figure 6E) and 19.3H-L1 (Figure 6D). Thus, 19.3H-L3 neutralizes a greater number of R880F Envs than 19.3H-L1, binds more strongly to the founder 0-A6/B24 gp120, and neutralizes the Env 0-A6/B24 pseudovirus more potently, underscoring the significance of VL alterations where antigen recognition and neutralization efficacy are concerned.

Bottom Line: Crystal structures of the antigen-binding fragments (Fabs) revealed flat epitope contact surfaces, where minimal light chain mutation in 19.3H-L3 allowed for additional antigenic interactions.Our data demonstrate that this subject's first recognized nAb epitope elicited strain-specific mAbs, which incrementally acquired autologous breadth, and directed later B cell responses to target distinct portions of Env.This immune re-focusing could have triggered the evolution of cross-clade antibodies and suggests that exposure to a specific sequence of immune escape variants might promote broad humoral responses during HIV-1 infection.

View Article: PubMed Central - PubMed

Affiliation: Immunology and Molecular Pathogenesis Graduate Program, Emory University, Atlanta, Georgia, United States of America.

ABSTRACT
Antibodies that neutralize (nAbs) genetically diverse HIV-1 strains have been recovered from a subset of HIV-1 infected subjects during chronic infection. Exact mechanisms that expand the otherwise narrow neutralization capacity observed during early infection are, however, currently undefined. Here we characterized the earliest nAb responses in a subtype A HIV-1 infected Rwandan seroconverter who later developed moderate cross-clade nAb breadth, using (i) envelope (Env) glycoproteins from the transmitted/founder virus and twenty longitudinal nAb escape variants, (ii) longitudinal autologous plasma, and (iii) autologous monoclonal antibodies (mAbs). Initially, nAbs targeted a single region of gp120, which flanked the V3 domain and involved the alpha2 helix. A single amino acid change at one of three positions in this region conferred early escape. One immunoglobulin heavy chain and two light chains recovered from autologous B cells comprised two mAbs, 19.3H-L1 and 19.3H-L3, which neutralized the founder Env along with one or three of the early escape variants carrying these mutations, respectively. Neither mAb neutralized later nAb escape or heterologous Envs. Crystal structures of the antigen-binding fragments (Fabs) revealed flat epitope contact surfaces, where minimal light chain mutation in 19.3H-L3 allowed for additional antigenic interactions. Resistance to mAb neutralization arose in later Envs through alteration of two glycans spatially adjacent to the initial escape signatures. The cross-neutralizing nAbs that ultimately developed failed to target any of the defined V3-proximal changes generated during the first year of infection in this subject. Our data demonstrate that this subject's first recognized nAb epitope elicited strain-specific mAbs, which incrementally acquired autologous breadth, and directed later B cell responses to target distinct portions of Env. This immune re-focusing could have triggered the evolution of cross-clade antibodies and suggests that exposure to a specific sequence of immune escape variants might promote broad humoral responses during HIV-1 infection.

Show MeSH
Related in: MedlinePlus