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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 R880F immunoglobulin heavy and light chain variable domains and neutralization by R880F mAbs 19.3H-L1 and 19.3H-L3.Germline heavy and light chain gene segment utilization was determined by SoDA, a somatic diversification analysis program [32], and amino acid sequences were aligned and examined using Sequencher v5.0 and Geneious v5.0.3 software. Dashes represent conserved positions. Complementarity-determining regions (CDRs) are highlighted in gray. The two R880F mAbs share a common heavy chain, 19.3H-HC (A), which utilizes V3-30*02, D1-7*01, and J4*02 gene families, while the somatically related light chains 19.3H-L1 and 19.3H-L3 (B) employ V2-14*01 and J2*01 gene families and differ from each other at five positions in and just downstream of CDR1. Heavy chain 19.3H-HC, when paired with either 19.3H-L1 (C) or 19.3H-L3 (D), was evaluated for neutralization against pseudotyped R880F wild-type (solid lines) and site-directed mutant Envs (dashed lines). 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 (0-month Envs = circles, 2-month Envs and representative point mutants = triangles, 5-month Envs and a representative point mutant = inverted triangles, 7-month Envs = squares, 10-month Envs = diamonds). Colored lines (2-A9/2-A13/0-B24 I295R in magenta, 2-B31/0-B24 I295T in red, 2-B12/0-B24 D341N in cyan, and 5-B52/2-A3 K338G D341N in green) indicate Envs that succumbed to neutralization, in varying combinations, by the isolated R880F mAbs.
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ppat-1003173-g005: Amino acid alignment of R880F immunoglobulin heavy and light chain variable domains and neutralization by R880F mAbs 19.3H-L1 and 19.3H-L3.Germline heavy and light chain gene segment utilization was determined by SoDA, a somatic diversification analysis program [32], and amino acid sequences were aligned and examined using Sequencher v5.0 and Geneious v5.0.3 software. Dashes represent conserved positions. Complementarity-determining regions (CDRs) are highlighted in gray. The two R880F mAbs share a common heavy chain, 19.3H-HC (A), which utilizes V3-30*02, D1-7*01, and J4*02 gene families, while the somatically related light chains 19.3H-L1 and 19.3H-L3 (B) employ V2-14*01 and J2*01 gene families and differ from each other at five positions in and just downstream of CDR1. Heavy chain 19.3H-HC, when paired with either 19.3H-L1 (C) or 19.3H-L3 (D), was evaluated for neutralization against pseudotyped R880F wild-type (solid lines) and site-directed mutant Envs (dashed lines). 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 (0-month Envs = circles, 2-month Envs and representative point mutants = triangles, 5-month Envs and a representative point mutant = inverted triangles, 7-month Envs = squares, 10-month Envs = diamonds). Colored lines (2-A9/2-A13/0-B24 I295R in magenta, 2-B31/0-B24 I295T in red, 2-B12/0-B24 D341N in cyan, and 5-B52/2-A3 K338G D341N in green) indicate Envs that succumbed to neutralization, in varying combinations, by the isolated R880F mAbs.

Mentions: During HIV-1 infection, the antibodies circulating in patient plasma could ostensibly represent a heterogeneous pool with varying epitope specificities. Although we were able to identify a single, early nAb target in subject R880F using autologous plasma and 3-dimensional modeling, this epitope could be recognized by a polyclonal nAb response mediated by more than one B cell [31]. To illuminate the characteristics of individual monoclonal effectors, we PCR amplified and cloned antibody VH and VL genes from memory B cells present in a cryopreserved R880F peripheral blood mononuclear cell (PBMC) sample collected at 16-months post-seroconversion (Table 1). Multiple VHs and VLs were obtained, but only one VH, named 19.3H-HC, neutralized the founder Env when combined with either of two highly related VLs. Sequence analysis revealed that the R880F VH utilized IGHV3-30*02, IGHD1-7*01, and IGHJ4*02 gene segments based on matching within the SoDA database [32] and demonstrated 23.2% mutation across its framework (FWR) and complementarity-determining regions (CDR), as compared with germline at the amino acid level (Figure 5A). The VLs, named 19.3H-L1 and 19.3H-L3, were clonal relatives, both using IGLV2-14*01 and IGLJ2*01 gene segments based on matching within the SoDA database [32] and exhibiting mutation rates of 13.6% and 14.5% from the putative germline, respectively (Figure 5B). Five total amino acid differences between the 19.3H-L1 and 19.3H-L3 VLs congregated in and around CDR1: 19.3H-L3 contained two threonines (T) and one phenylalanine (F) in CDR1 that were not present in 19.3H-L1, while arginine (R) and glutamic acid (E) residues arose just downstream of CDR1 in the FWR2 region of 19.3H-L1 that were not present in 19.3H-L3 (Figure 5B). The VL CDR3 domains of 19.3H-L1 and 19.3H-L3 were identical and contained five amino acid differences from the putative germline. The two R880F mAbs produced by combination of 19.3H-HC and 19.3H-L1 or 19.3H-L3 are hereafter referenced solely by their VL designations.


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 R880F immunoglobulin heavy and light chain variable domains and neutralization by R880F mAbs 19.3H-L1 and 19.3H-L3.Germline heavy and light chain gene segment utilization was determined by SoDA, a somatic diversification analysis program [32], and amino acid sequences were aligned and examined using Sequencher v5.0 and Geneious v5.0.3 software. Dashes represent conserved positions. Complementarity-determining regions (CDRs) are highlighted in gray. The two R880F mAbs share a common heavy chain, 19.3H-HC (A), which utilizes V3-30*02, D1-7*01, and J4*02 gene families, while the somatically related light chains 19.3H-L1 and 19.3H-L3 (B) employ V2-14*01 and J2*01 gene families and differ from each other at five positions in and just downstream of CDR1. Heavy chain 19.3H-HC, when paired with either 19.3H-L1 (C) or 19.3H-L3 (D), was evaluated for neutralization against pseudotyped R880F wild-type (solid lines) and site-directed mutant Envs (dashed lines). 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 (0-month Envs = circles, 2-month Envs and representative point mutants = triangles, 5-month Envs and a representative point mutant = inverted triangles, 7-month Envs = squares, 10-month Envs = diamonds). Colored lines (2-A9/2-A13/0-B24 I295R in magenta, 2-B31/0-B24 I295T in red, 2-B12/0-B24 D341N in cyan, and 5-B52/2-A3 K338G D341N in green) indicate Envs that succumbed to neutralization, in varying combinations, by the isolated R880F mAbs.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585129&req=5

ppat-1003173-g005: Amino acid alignment of R880F immunoglobulin heavy and light chain variable domains and neutralization by R880F mAbs 19.3H-L1 and 19.3H-L3.Germline heavy and light chain gene segment utilization was determined by SoDA, a somatic diversification analysis program [32], and amino acid sequences were aligned and examined using Sequencher v5.0 and Geneious v5.0.3 software. Dashes represent conserved positions. Complementarity-determining regions (CDRs) are highlighted in gray. The two R880F mAbs share a common heavy chain, 19.3H-HC (A), which utilizes V3-30*02, D1-7*01, and J4*02 gene families, while the somatically related light chains 19.3H-L1 and 19.3H-L3 (B) employ V2-14*01 and J2*01 gene families and differ from each other at five positions in and just downstream of CDR1. Heavy chain 19.3H-HC, when paired with either 19.3H-L1 (C) or 19.3H-L3 (D), was evaluated for neutralization against pseudotyped R880F wild-type (solid lines) and site-directed mutant Envs (dashed lines). 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 (0-month Envs = circles, 2-month Envs and representative point mutants = triangles, 5-month Envs and a representative point mutant = inverted triangles, 7-month Envs = squares, 10-month Envs = diamonds). Colored lines (2-A9/2-A13/0-B24 I295R in magenta, 2-B31/0-B24 I295T in red, 2-B12/0-B24 D341N in cyan, and 5-B52/2-A3 K338G D341N in green) indicate Envs that succumbed to neutralization, in varying combinations, by the isolated R880F mAbs.
Mentions: During HIV-1 infection, the antibodies circulating in patient plasma could ostensibly represent a heterogeneous pool with varying epitope specificities. Although we were able to identify a single, early nAb target in subject R880F using autologous plasma and 3-dimensional modeling, this epitope could be recognized by a polyclonal nAb response mediated by more than one B cell [31]. To illuminate the characteristics of individual monoclonal effectors, we PCR amplified and cloned antibody VH and VL genes from memory B cells present in a cryopreserved R880F peripheral blood mononuclear cell (PBMC) sample collected at 16-months post-seroconversion (Table 1). Multiple VHs and VLs were obtained, but only one VH, named 19.3H-HC, neutralized the founder Env when combined with either of two highly related VLs. Sequence analysis revealed that the R880F VH utilized IGHV3-30*02, IGHD1-7*01, and IGHJ4*02 gene segments based on matching within the SoDA database [32] and demonstrated 23.2% mutation across its framework (FWR) and complementarity-determining regions (CDR), as compared with germline at the amino acid level (Figure 5A). The VLs, named 19.3H-L1 and 19.3H-L3, were clonal relatives, both using IGLV2-14*01 and IGLJ2*01 gene segments based on matching within the SoDA database [32] and exhibiting mutation rates of 13.6% and 14.5% from the putative germline, respectively (Figure 5B). Five total amino acid differences between the 19.3H-L1 and 19.3H-L3 VLs congregated in and around CDR1: 19.3H-L3 contained two threonines (T) and one phenylalanine (F) in CDR1 that were not present in 19.3H-L1, while arginine (R) and glutamic acid (E) residues arose just downstream of CDR1 in the FWR2 region of 19.3H-L1 that were not present in 19.3H-L3 (Figure 5B). The VL CDR3 domains of 19.3H-L1 and 19.3H-L3 were identical and contained five amino acid differences from the putative germline. The two R880F mAbs produced by combination of 19.3H-HC and 19.3H-L1 or 19.3H-L3 are hereafter referenced solely by their VL designations.

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