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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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Amino acid alignment of longitudinal R880F Envs.Longitudinal Env amino acid sequences from 20 R880F contemporaneous plasma escape variants were aligned and examined using Sequencher v5.0 and Geneious v5.0.3 software, with particular emphasis on the three mutational hot spots–C2, the alpha2 helix in C3, and V5–that developed during early infection, as compared to the transmitted/founder Env (0-A6/B24). Dashes represent conserved positions. Subject-specific amino acid numbering labels the demarcated regions at their points of origination. Five residues including 295, 335, 338, 341, and 456 (HXB2 residues 293, 334, 337, 340, and 460) were specifically interrogated to define their contributions to nAb evasion and have been highlighted in black in 0-A6/B24 for easy identification. Colored sequences (2-A9/2-A13 in magenta, 2-B31 in red, 2-B12 in cyan, and 5-B52 in green) indicate Envs that succumbed to neutralization, in varying combinations, by the isolated R880F mAbs. Note that additional differences from the transmitted/founder Env, which occurred over time in these sequences but are not diagrammed here, may be viewed in Figure S1.
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ppat-1003173-g002: Amino acid alignment of longitudinal R880F Envs.Longitudinal Env amino acid sequences from 20 R880F contemporaneous plasma escape variants were aligned and examined using Sequencher v5.0 and Geneious v5.0.3 software, with particular emphasis on the three mutational hot spots–C2, the alpha2 helix in C3, and V5–that developed during early infection, as compared to the transmitted/founder Env (0-A6/B24). Dashes represent conserved positions. Subject-specific amino acid numbering labels the demarcated regions at their points of origination. Five residues including 295, 335, 338, 341, and 456 (HXB2 residues 293, 334, 337, 340, and 460) were specifically interrogated to define their contributions to nAb evasion and have been highlighted in black in 0-A6/B24 for easy identification. Colored sequences (2-A9/2-A13 in magenta, 2-B31 in red, 2-B12 in cyan, and 5-B52 in green) indicate Envs that succumbed to neutralization, in varying combinations, by the isolated R880F mAbs. Note that additional differences from the transmitted/founder Env, which occurred over time in these sequences but are not diagrammed here, may be viewed in Figure S1.

Mentions: To localize the earliest nAb target and elucidate consequent mechanisms of viral escape, full-length amino acid Env sequences for all 2-month nAb escape variants shown in Figure 1B were aligned and inspected for the presence of mutational hot spots. Amino acid changes concentrated in three regions of gp120 at 2-months: in C2 immediately preceding the beginning of V3, in the alpha2 helix in C3, and in V5. Figure 2 specifically diagrams these segments of gp120; Figure S1 includes the full gp160 alignment of all 22 R880F Envs. The isoleucine at position 295 (I295; HXB2 residue 293) in C2 mutated to arginine (I295R) in two Envs or threonine (I295T) in one Env (Figure 2). Additionally, glutamic acid E338 in the alpha2 helix (HXB2 residue 337) became three different residues including aspartic acid (E338D), glycine (E338G), and lysine (E338K) in six Envs (Figure 2). Of note, compared to the founder Env sequence, E338K was the sole mutation in the entire 2-A3 Env sequence (Figure S1). We concluded, then, that this single mutation directly mediated nAb escape. The aspartic acid at position 341 (D341; HXB2 residue 340), also in the alpha2 helix, changed to asparagine (D341N) in one Env (Figure 2). Finally, the glutamic acid at position 456 (E456; HXB2 residue 460) in V5 switched to lysine (E456K) in four Envs (Figure 2).


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)

Amino acid alignment of longitudinal R880F Envs.Longitudinal Env amino acid sequences from 20 R880F contemporaneous plasma escape variants were aligned and examined using Sequencher v5.0 and Geneious v5.0.3 software, with particular emphasis on the three mutational hot spots–C2, the alpha2 helix in C3, and V5–that developed during early infection, as compared to the transmitted/founder Env (0-A6/B24). Dashes represent conserved positions. Subject-specific amino acid numbering labels the demarcated regions at their points of origination. Five residues including 295, 335, 338, 341, and 456 (HXB2 residues 293, 334, 337, 340, and 460) were specifically interrogated to define their contributions to nAb evasion and have been highlighted in black in 0-A6/B24 for easy identification. Colored sequences (2-A9/2-A13 in magenta, 2-B31 in red, 2-B12 in cyan, and 5-B52 in green) indicate Envs that succumbed to neutralization, in varying combinations, by the isolated R880F mAbs. Note that additional differences from the transmitted/founder Env, which occurred over time in these sequences but are not diagrammed here, may be viewed in Figure S1.
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Related In: Results  -  Collection

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

ppat-1003173-g002: Amino acid alignment of longitudinal R880F Envs.Longitudinal Env amino acid sequences from 20 R880F contemporaneous plasma escape variants were aligned and examined using Sequencher v5.0 and Geneious v5.0.3 software, with particular emphasis on the three mutational hot spots–C2, the alpha2 helix in C3, and V5–that developed during early infection, as compared to the transmitted/founder Env (0-A6/B24). Dashes represent conserved positions. Subject-specific amino acid numbering labels the demarcated regions at their points of origination. Five residues including 295, 335, 338, 341, and 456 (HXB2 residues 293, 334, 337, 340, and 460) were specifically interrogated to define their contributions to nAb evasion and have been highlighted in black in 0-A6/B24 for easy identification. Colored sequences (2-A9/2-A13 in magenta, 2-B31 in red, 2-B12 in cyan, and 5-B52 in green) indicate Envs that succumbed to neutralization, in varying combinations, by the isolated R880F mAbs. Note that additional differences from the transmitted/founder Env, which occurred over time in these sequences but are not diagrammed here, may be viewed in Figure S1.
Mentions: To localize the earliest nAb target and elucidate consequent mechanisms of viral escape, full-length amino acid Env sequences for all 2-month nAb escape variants shown in Figure 1B were aligned and inspected for the presence of mutational hot spots. Amino acid changes concentrated in three regions of gp120 at 2-months: in C2 immediately preceding the beginning of V3, in the alpha2 helix in C3, and in V5. Figure 2 specifically diagrams these segments of gp120; Figure S1 includes the full gp160 alignment of all 22 R880F Envs. The isoleucine at position 295 (I295; HXB2 residue 293) in C2 mutated to arginine (I295R) in two Envs or threonine (I295T) in one Env (Figure 2). Additionally, glutamic acid E338 in the alpha2 helix (HXB2 residue 337) became three different residues including aspartic acid (E338D), glycine (E338G), and lysine (E338K) in six Envs (Figure 2). Of note, compared to the founder Env sequence, E338K was the sole mutation in the entire 2-A3 Env sequence (Figure S1). We concluded, then, that this single mutation directly mediated nAb escape. The aspartic acid at position 341 (D341; HXB2 residue 340), also in the alpha2 helix, changed to asparagine (D341N) in one Env (Figure 2). Finally, the glutamic acid at position 456 (E456; HXB2 residue 460) in V5 switched to lysine (E456K) in four Envs (Figure 2).

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