Limits...
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.

Show MeSH

Related in: MedlinePlus

Escape from mAbs 19.3H-L1 and 19.3H-L3 by glycan addition and/or shifting.To investigate how longitudinal viruses, namely 7- and 10-month Envs, could harbor the humoral vulnerability associated with mutation D341N whilst maintaining neutralization resistant phenotypes, two potential compensatory mutations were investigated. I295N, which inserts a PNGS near the N-terminus of V3, was introduced by site-directed mutagenesis into two mAb-sensitive Envs, 0-A6 and 2-B12 (light orange). S335N, which shifts a PNGS closer to the N-terminus of the alpha2 helix, was similarly created in 0-A6, 2-B12, and 5-B52 (purple). Wild-type (solid lines) and site-directed mutant Envs (dashed lines) were pseudotyped and assayed with mAbs 19.3H-L1 (A) and 19.3H-L3 (B). To determine if mAb resistance was glycan-dependent, an S335Q substitution that destroyed the N333 PNGS was also created in Env 2-B12 (dark orange). Percent viral infectivity, as adjusted against wells containing no mAb, is depicted on the vertical axis; mAb concentrations (in µg/ml) are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-mAb combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells. In (C), the V3/alpha2 helix portion of the labeled Envs has been conceptualized with glycan forks, each of which represents a PNGS in the corresponding amino acid sequences. Glycans of particular interest (N295, N333, and N335) are designated using R880F-specific numbering. In (D), the proposed escape glycans N295 and N335 (red) have been modeled onto the R880F 0-B24 Env gp120 monomer (green) to illustrate how such masking could obscure underlying epitopes and prevent recognition by mAbs 19.3H-L1 and 19.3H-L3.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585129&req=5

ppat-1003173-g008: Escape from mAbs 19.3H-L1 and 19.3H-L3 by glycan addition and/or shifting.To investigate how longitudinal viruses, namely 7- and 10-month Envs, could harbor the humoral vulnerability associated with mutation D341N whilst maintaining neutralization resistant phenotypes, two potential compensatory mutations were investigated. I295N, which inserts a PNGS near the N-terminus of V3, was introduced by site-directed mutagenesis into two mAb-sensitive Envs, 0-A6 and 2-B12 (light orange). S335N, which shifts a PNGS closer to the N-terminus of the alpha2 helix, was similarly created in 0-A6, 2-B12, and 5-B52 (purple). Wild-type (solid lines) and site-directed mutant Envs (dashed lines) were pseudotyped and assayed with mAbs 19.3H-L1 (A) and 19.3H-L3 (B). To determine if mAb resistance was glycan-dependent, an S335Q substitution that destroyed the N333 PNGS was also created in Env 2-B12 (dark orange). Percent viral infectivity, as adjusted against wells containing no mAb, is depicted on the vertical axis; mAb concentrations (in µg/ml) are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-mAb combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells. In (C), the V3/alpha2 helix portion of the labeled Envs has been conceptualized with glycan forks, each of which represents a PNGS in the corresponding amino acid sequences. Glycans of particular interest (N295, N333, and N335) are designated using R880F-specific numbering. In (D), the proposed escape glycans N295 and N335 (red) have been modeled onto the R880F 0-B24 Env gp120 monomer (green) to illustrate how such masking could obscure underlying epitopes and prevent recognition by mAbs 19.3H-L1 and 19.3H-L3.

Mentions: As demonstrated in Figure 5, D341N appeared to be detrimental to the preservation of a neutralization-resistant phenotype, in the context of mAbs 19.3H-L1 and 19.3H-L3 during early infection. This mutation was, nonetheless, retained in later escape Envs. Inspection of the 7- and 10-month Env sequences containing D341N revealed that they had acquired additional substitutions, I295N (HXB2 residue 293) and/or S335N (HXB2 residue 334), absent from earlier Envs (Figure 2); each of these mutations affected a potential N-linked glycosylation site (PNGS). Accordingly, we hypothesized that these co-traveling mutations compensated for the vulnerability associated with D341N in a PNGS-dependent manner. To explore this, the I295N substitution, which created a PNGS, was introduced into two mAb-sensitive Envs: 0-A6 and 2-B12. The I295N versions of these two Envs displayed high-level resistance against mAbs 19.3H-L1 and 19.3H-L3 (Figure 8A–B, Table 2). Similarly, the S335N substitution, which also incorporated a PNGS, was inserted in three mAb-sensitive Envs: 0-A6, 2-B12, and 5-B52. The S335N versions of these three Envs also became highly resistant to 19.3H-L1 and 19.3H-L3 (Figure 8A–B, Table 2). The S335N substitution shifted a well-conserved PNGS sequon at position 333 (HXB2 residue 332; Figure 8C) that is targeted by broadly neutralizing mAbs PGT128 and 2G12 [8], [34], [35]. To determine if the observed mAb resistance was glycan-dependent, an S335Q substitution was created in Env 2-B12. Unlike S335N, which shifted the N333 sequon down two positions, S335Q destroyed the N333 sequon altogether (Figure 8C). The resulting mutant, 2-B12 S335Q, was two logs less sensitive to neutralization by mAb 19.3H-L1 than the parental Env 2-B12, but did not reach the high level of resistance achieved by 2-B12 S335N; in contrast, S335Q had only a slight effect on neutralization by mAb 19.3H-L3 (Figure 8A–B, Table 2). High-level resistance against mAbs 19.3H-L1 and 19.3H-L3, therefore, required the addition and/or shifting of PNGS sequons, but amino acid substitution S335Q also provided partial resistance that was much more effective against mAb 19.3H-L1. Together, the data strongly support a mechanism of mAb escape that was PNGS-dependent and may have introduced glycans capable of obscuring the V3-proximal space recognized by 19.3H-L1 and 19.3H-L3 (Figure 8D). Nevertheless, the two mAbs–common heavy chain notwithstanding–appear to recognize subtly distinct epitopes.


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)

Escape from mAbs 19.3H-L1 and 19.3H-L3 by glycan addition and/or shifting.To investigate how longitudinal viruses, namely 7- and 10-month Envs, could harbor the humoral vulnerability associated with mutation D341N whilst maintaining neutralization resistant phenotypes, two potential compensatory mutations were investigated. I295N, which inserts a PNGS near the N-terminus of V3, was introduced by site-directed mutagenesis into two mAb-sensitive Envs, 0-A6 and 2-B12 (light orange). S335N, which shifts a PNGS closer to the N-terminus of the alpha2 helix, was similarly created in 0-A6, 2-B12, and 5-B52 (purple). Wild-type (solid lines) and site-directed mutant Envs (dashed lines) were pseudotyped and assayed with mAbs 19.3H-L1 (A) and 19.3H-L3 (B). To determine if mAb resistance was glycan-dependent, an S335Q substitution that destroyed the N333 PNGS was also created in Env 2-B12 (dark orange). Percent viral infectivity, as adjusted against wells containing no mAb, is depicted on the vertical axis; mAb concentrations (in µg/ml) are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-mAb combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells. In (C), the V3/alpha2 helix portion of the labeled Envs has been conceptualized with glycan forks, each of which represents a PNGS in the corresponding amino acid sequences. Glycans of particular interest (N295, N333, and N335) are designated using R880F-specific numbering. In (D), the proposed escape glycans N295 and N335 (red) have been modeled onto the R880F 0-B24 Env gp120 monomer (green) to illustrate how such masking could obscure underlying epitopes and prevent recognition by mAbs 19.3H-L1 and 19.3H-L3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585129&req=5

ppat-1003173-g008: Escape from mAbs 19.3H-L1 and 19.3H-L3 by glycan addition and/or shifting.To investigate how longitudinal viruses, namely 7- and 10-month Envs, could harbor the humoral vulnerability associated with mutation D341N whilst maintaining neutralization resistant phenotypes, two potential compensatory mutations were investigated. I295N, which inserts a PNGS near the N-terminus of V3, was introduced by site-directed mutagenesis into two mAb-sensitive Envs, 0-A6 and 2-B12 (light orange). S335N, which shifts a PNGS closer to the N-terminus of the alpha2 helix, was similarly created in 0-A6, 2-B12, and 5-B52 (purple). Wild-type (solid lines) and site-directed mutant Envs (dashed lines) were pseudotyped and assayed with mAbs 19.3H-L1 (A) and 19.3H-L3 (B). To determine if mAb resistance was glycan-dependent, an S335Q substitution that destroyed the N333 PNGS was also created in Env 2-B12 (dark orange). Percent viral infectivity, as adjusted against wells containing no mAb, is depicted on the vertical axis; mAb concentrations (in µg/ml) are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-mAb combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells. In (C), the V3/alpha2 helix portion of the labeled Envs has been conceptualized with glycan forks, each of which represents a PNGS in the corresponding amino acid sequences. Glycans of particular interest (N295, N333, and N335) are designated using R880F-specific numbering. In (D), the proposed escape glycans N295 and N335 (red) have been modeled onto the R880F 0-B24 Env gp120 monomer (green) to illustrate how such masking could obscure underlying epitopes and prevent recognition by mAbs 19.3H-L1 and 19.3H-L3.
Mentions: As demonstrated in Figure 5, D341N appeared to be detrimental to the preservation of a neutralization-resistant phenotype, in the context of mAbs 19.3H-L1 and 19.3H-L3 during early infection. This mutation was, nonetheless, retained in later escape Envs. Inspection of the 7- and 10-month Env sequences containing D341N revealed that they had acquired additional substitutions, I295N (HXB2 residue 293) and/or S335N (HXB2 residue 334), absent from earlier Envs (Figure 2); each of these mutations affected a potential N-linked glycosylation site (PNGS). Accordingly, we hypothesized that these co-traveling mutations compensated for the vulnerability associated with D341N in a PNGS-dependent manner. To explore this, the I295N substitution, which created a PNGS, was introduced into two mAb-sensitive Envs: 0-A6 and 2-B12. The I295N versions of these two Envs displayed high-level resistance against mAbs 19.3H-L1 and 19.3H-L3 (Figure 8A–B, Table 2). Similarly, the S335N substitution, which also incorporated a PNGS, was inserted in three mAb-sensitive Envs: 0-A6, 2-B12, and 5-B52. The S335N versions of these three Envs also became highly resistant to 19.3H-L1 and 19.3H-L3 (Figure 8A–B, Table 2). The S335N substitution shifted a well-conserved PNGS sequon at position 333 (HXB2 residue 332; Figure 8C) that is targeted by broadly neutralizing mAbs PGT128 and 2G12 [8], [34], [35]. To determine if the observed mAb resistance was glycan-dependent, an S335Q substitution was created in Env 2-B12. Unlike S335N, which shifted the N333 sequon down two positions, S335Q destroyed the N333 sequon altogether (Figure 8C). The resulting mutant, 2-B12 S335Q, was two logs less sensitive to neutralization by mAb 19.3H-L1 than the parental Env 2-B12, but did not reach the high level of resistance achieved by 2-B12 S335N; in contrast, S335Q had only a slight effect on neutralization by mAb 19.3H-L3 (Figure 8A–B, Table 2). High-level resistance against mAbs 19.3H-L1 and 19.3H-L3, therefore, required the addition and/or shifting of PNGS sequons, but amino acid substitution S335Q also provided partial resistance that was much more effective against mAb 19.3H-L1. Together, the data strongly support a mechanism of mAb escape that was PNGS-dependent and may have introduced glycans capable of obscuring the V3-proximal space recognized by 19.3H-L1 and 19.3H-L3 (Figure 8D). Nevertheless, the two mAbs–common heavy chain notwithstanding–appear to recognize subtly distinct epitopes.

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