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

Determination of the earliest R880F nAb escape signatures in Env.Suspected 2-month nAb escape mutations from four different amino acid positions were introduced by site-directed mutagenesis into the transmitted/founder Env 0-B24 or escape Env 2-A3, which differed from the founder only at the site of mutation introduction. The transmitted/founder and wild-type 2-month Envs (solid lines) along with site-directed mutant Envs (dashed lines) were pseudotyped and assayed with 2-month plasma to determine if substitutions at C2 295 (A), alpha2 helix 338 (B), alpha2 helix 341 (C), or V5 456 (D) (HXB2 residues 293, 337, 340, or 460) could individually confer resistance. Percent viral infectivity, as adjusted against wells containing no test plasma, is depicted on the vertical axis; reciprocal plasma dilutions are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-plasma combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells (0-month Envs = circles, 2-month Envs and representative point mutants = triangles). Colored lines (2-A9/2-A13/0-B24 I295R in magenta, 2-B31/0-B24 I295T in red, and 2-B12/0-B24 D341N in cyan) indicate Envs that succumbed to neutralization, in varying combinations, by the isolated R880F mAbs.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585129&req=5

ppat-1003173-g003: Determination of the earliest R880F nAb escape signatures in Env.Suspected 2-month nAb escape mutations from four different amino acid positions were introduced by site-directed mutagenesis into the transmitted/founder Env 0-B24 or escape Env 2-A3, which differed from the founder only at the site of mutation introduction. The transmitted/founder and wild-type 2-month Envs (solid lines) along with site-directed mutant Envs (dashed lines) were pseudotyped and assayed with 2-month plasma to determine if substitutions at C2 295 (A), alpha2 helix 338 (B), alpha2 helix 341 (C), or V5 456 (D) (HXB2 residues 293, 337, 340, or 460) could individually confer resistance. Percent viral infectivity, as adjusted against wells containing no test plasma, is depicted on the vertical axis; reciprocal plasma dilutions are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-plasma combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells (0-month Envs = circles, 2-month Envs and representative point mutants = triangles). Colored lines (2-A9/2-A13/0-B24 I295R in magenta, 2-B31/0-B24 I295T in red, and 2-B12/0-B24 D341N in cyan) indicate Envs that succumbed to neutralization, in varying combinations, by the isolated R880F mAbs.

Mentions: The potential escape mutations at I295, D341, and E456 were introduced into the founder Env 0-B24 by site-directed mutagenesis to determine if these alterations could individually switch the founder Env phenotype from sensitive to resistant when assayed for neutralization by 2-month plasma. In addition, amino acid changes were introduced into escape Env 2-A3 at K338 to determine whether these mutants maintained nAb resistance. The I295R and I295T substitutions in C2 independently conferred nAb escape, with I295R producing a slightly higher level of resistance that was most evident at the 1∶100 dilution of plasma (Figure 3A). For position 338, two naturally occurring substitutions (E338D from 2-A23/2-A24 and E338G from 2-B18, see Figure 2) and three artificially introduced mutations (K338I, K338Q, and K338R) independently reproduced escape Env 2-A3's wild-type level of resistance, arguing that any change at this position could provide full escape from neutralization (Figure 3B). Thus, the degree of neutralization resistance conferred by changes at I295, but not at E338, varied by the amino acid substitution. Introduction of D341N into the alpha2 helix of the founder Env 0-B24 also recapitulated the wild-type resistance level of 2-B12 (Figure 3C). Because the I295, E338, and D341 escape mutations occurred independently in the 2-month Envs, each represents a distinct lineage for escape (Figure 2). In addition, the potency of resistance was substitution-specific; I295R/T and E338D/G/K produced the highest levels of resistance, while D341N lagged somewhat behind and provided partial resistance. In contrast, the E456K mutation in V5 exerted no overt influence on neutralization phenotype when introduced into the founder Env 0-B24, despite being carried in nearly half of the 2-month escape Envs (Figure 3D). Overall, at 2-months, the viral population utilized a common amino acid substitution mechanism that diverged down three discrete escape pathways, each of which conferred nAb resistance.


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)

Determination of the earliest R880F nAb escape signatures in Env.Suspected 2-month nAb escape mutations from four different amino acid positions were introduced by site-directed mutagenesis into the transmitted/founder Env 0-B24 or escape Env 2-A3, which differed from the founder only at the site of mutation introduction. The transmitted/founder and wild-type 2-month Envs (solid lines) along with site-directed mutant Envs (dashed lines) were pseudotyped and assayed with 2-month plasma to determine if substitutions at C2 295 (A), alpha2 helix 338 (B), alpha2 helix 341 (C), or V5 456 (D) (HXB2 residues 293, 337, 340, or 460) could individually confer resistance. Percent viral infectivity, as adjusted against wells containing no test plasma, is depicted on the vertical axis; reciprocal plasma dilutions are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-plasma combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells (0-month Envs = circles, 2-month Envs and representative point mutants = triangles). Colored lines (2-A9/2-A13/0-B24 I295R in magenta, 2-B31/0-B24 I295T in red, and 2-B12/0-B24 D341N in cyan) indicate Envs that succumbed to neutralization, in varying combinations, by the isolated R880F mAbs.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003173-g003: Determination of the earliest R880F nAb escape signatures in Env.Suspected 2-month nAb escape mutations from four different amino acid positions were introduced by site-directed mutagenesis into the transmitted/founder Env 0-B24 or escape Env 2-A3, which differed from the founder only at the site of mutation introduction. The transmitted/founder and wild-type 2-month Envs (solid lines) along with site-directed mutant Envs (dashed lines) were pseudotyped and assayed with 2-month plasma to determine if substitutions at C2 295 (A), alpha2 helix 338 (B), alpha2 helix 341 (C), or V5 456 (D) (HXB2 residues 293, 337, 340, or 460) could individually confer resistance. Percent viral infectivity, as adjusted against wells containing no test plasma, is depicted on the vertical axis; reciprocal plasma dilutions are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-plasma combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells (0-month Envs = circles, 2-month Envs and representative point mutants = triangles). Colored lines (2-A9/2-A13/0-B24 I295R in magenta, 2-B31/0-B24 I295T in red, and 2-B12/0-B24 D341N in cyan) indicate Envs that succumbed to neutralization, in varying combinations, by the isolated R880F mAbs.
Mentions: The potential escape mutations at I295, D341, and E456 were introduced into the founder Env 0-B24 by site-directed mutagenesis to determine if these alterations could individually switch the founder Env phenotype from sensitive to resistant when assayed for neutralization by 2-month plasma. In addition, amino acid changes were introduced into escape Env 2-A3 at K338 to determine whether these mutants maintained nAb resistance. The I295R and I295T substitutions in C2 independently conferred nAb escape, with I295R producing a slightly higher level of resistance that was most evident at the 1∶100 dilution of plasma (Figure 3A). For position 338, two naturally occurring substitutions (E338D from 2-A23/2-A24 and E338G from 2-B18, see Figure 2) and three artificially introduced mutations (K338I, K338Q, and K338R) independently reproduced escape Env 2-A3's wild-type level of resistance, arguing that any change at this position could provide full escape from neutralization (Figure 3B). Thus, the degree of neutralization resistance conferred by changes at I295, but not at E338, varied by the amino acid substitution. Introduction of D341N into the alpha2 helix of the founder Env 0-B24 also recapitulated the wild-type resistance level of 2-B12 (Figure 3C). Because the I295, E338, and D341 escape mutations occurred independently in the 2-month Envs, each represents a distinct lineage for escape (Figure 2). In addition, the potency of resistance was substitution-specific; I295R/T and E338D/G/K produced the highest levels of resistance, while D341N lagged somewhat behind and provided partial resistance. In contrast, the E456K mutation in V5 exerted no overt influence on neutralization phenotype when introduced into the founder Env 0-B24, despite being carried in nearly half of the 2-month escape Envs (Figure 3D). Overall, at 2-months, the viral population utilized a common amino acid substitution mechanism that diverged down three discrete escape pathways, each of which conferred nAb resistance.

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