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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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Heterologous neutralization breadth in R880F.The two R880F mAbs, 19.3H-L1 (A) and 19.3H-L3 (B), in conjunction with 16-month (C) and 3-year (D) autologous plasma were evaluated for cross-neutralizing capacity against virions pseudotyped with fourteen heterologous HIV-1 Envs from three clades (A/C recombinant and subtype A Envs = lavender, subtype B Envs = coral, subtype C Envs = teal). Two 0-month Envs (0-A6/B24) representative of the transmitted/founder sequence are included in each of these panels. Percent viral infectivity, as adjusted against wells containing no mAb or test plasma, is depicted on the vertical axis; mAb concentrations (in µg/ml) or reciprocal plasma dilutions are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-mAb or Env-plasma combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells. V3/alpha2 helix amino acid sequences were aligned and examined using Sequencher v5.0 and Geneious v5.0.3 software (E). Dashes represent conserved positions; dots represent gaps. Significant PNGS sequons (N295, N333, N335) are highlighted in black at their points of origin.
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ppat-1003173-g009: Heterologous neutralization breadth in R880F.The two R880F mAbs, 19.3H-L1 (A) and 19.3H-L3 (B), in conjunction with 16-month (C) and 3-year (D) autologous plasma were evaluated for cross-neutralizing capacity against virions pseudotyped with fourteen heterologous HIV-1 Envs from three clades (A/C recombinant and subtype A Envs = lavender, subtype B Envs = coral, subtype C Envs = teal). Two 0-month Envs (0-A6/B24) representative of the transmitted/founder sequence are included in each of these panels. Percent viral infectivity, as adjusted against wells containing no mAb or test plasma, is depicted on the vertical axis; mAb concentrations (in µg/ml) or reciprocal plasma dilutions are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-mAb or Env-plasma combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells. V3/alpha2 helix amino acid sequences were aligned and examined using Sequencher v5.0 and Geneious v5.0.3 software (E). Dashes represent conserved positions; dots represent gaps. Significant PNGS sequons (N295, N333, N335) are highlighted in black at their points of origin.

Mentions: The VH, in particular the CDR H3, has generally been considered a major determinant of epitope recognition and nAb breadth. In our study, VL differences appreciably expanded the neutralization capacity of mAb 19.3H-L3 against autologous Envs. To probe whether this increase in breadth carried over to neutralization of heterologous Envs, mAbs 19.3H-L1 and 19.3H-L3 were tested against a panel of fourteen heterologous Env pseudotypes that included one A/C recombinant, four subtype A, three subtype B, and six subtype C Envs. The mAbs were unable to neutralize any of the heterologous Envs (Figure 9A–B). Thus, while mAb 19.3H-L3 possessed increased breadth against autologous Envs as compared to 19.3H-L1, this did not extend to genetically diverse Envs. Regardless of this restricted mAb cross-clade neutralization, R880F plasma collected at 16-months or 3-years post-infection did have similarly moderate breadth against heterologous Envs, which increased in potency over time (Figure 9C–D). An amino acid alignment of Envs from the heterologous breadth panel demonstrated that Envs neutralized with the greatest potency at 3-years post-seroconversion, A-Q461 and C-Z205F (IC50 values of approximately 1∶1000), contained the N335 (HXB2 residue 334) shifted glycan associated with viral escape from mAbs 19.3H-L1 and 19.3H-L3 (Figure 9E). Furthermore, Env A-Q461 also incorporated the N295 (HXB2 residue 293) substitution indicative of mAb escape. To investigate if the N295 glycan addition and/or the shifted N335 glycan in R880F Envs could have been partially responsible for the heterologous neutralization capacity that developed in this subject, several glycan knock-out mutants were created and tested with 3-year R880F plasma (Figure 10A). Within A-Q461, the N295 PNGS was eliminated either alone or in conjunction with the N335 PNGS; the N335 PNGS was also individually knocked out (Figure 10B). The positions of interest were reverted back to the amino acid present in the transmitted/founder Env 0-A6/B24. For C-Z205F, the N335 PNGS was similarly abolished (Figure 10B). Additionally, two heterologous Envs that were only modestly neutralized but that contained the highlighted glycans, C-Z109F and C-Z214M, were mutated as well. All six of the glycan knock-out mutants exactly mirrored their parental equivalents, suggesting that the particular glycans at positions 295 and 335 did not directly contribute to the breadth observed at 3-years post-infection. These data do suggest, however, that early viral escape events likely influenced how breadth developed in this subject, by expanding what was originally a narrow, regional response at the base of the V3 loop to recognize and neutralize distinct portions of Env across genetically diverse variants.


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)

Heterologous neutralization breadth in R880F.The two R880F mAbs, 19.3H-L1 (A) and 19.3H-L3 (B), in conjunction with 16-month (C) and 3-year (D) autologous plasma were evaluated for cross-neutralizing capacity against virions pseudotyped with fourteen heterologous HIV-1 Envs from three clades (A/C recombinant and subtype A Envs = lavender, subtype B Envs = coral, subtype C Envs = teal). Two 0-month Envs (0-A6/B24) representative of the transmitted/founder sequence are included in each of these panels. Percent viral infectivity, as adjusted against wells containing no mAb or test plasma, is depicted on the vertical axis; mAb concentrations (in µg/ml) or reciprocal plasma dilutions are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-mAb or Env-plasma combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells. V3/alpha2 helix amino acid sequences were aligned and examined using Sequencher v5.0 and Geneious v5.0.3 software (E). Dashes represent conserved positions; dots represent gaps. Significant PNGS sequons (N295, N333, N335) are highlighted in black at their points of origin.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003173-g009: Heterologous neutralization breadth in R880F.The two R880F mAbs, 19.3H-L1 (A) and 19.3H-L3 (B), in conjunction with 16-month (C) and 3-year (D) autologous plasma were evaluated for cross-neutralizing capacity against virions pseudotyped with fourteen heterologous HIV-1 Envs from three clades (A/C recombinant and subtype A Envs = lavender, subtype B Envs = coral, subtype C Envs = teal). Two 0-month Envs (0-A6/B24) representative of the transmitted/founder sequence are included in each of these panels. Percent viral infectivity, as adjusted against wells containing no mAb or test plasma, is depicted on the vertical axis; mAb concentrations (in µg/ml) or reciprocal plasma dilutions are plotted along the horizontal axis in a logarithmic fashion. Each curve represents a single Env-mAb or Env-plasma combination, and error bars demonstrate the standard error of the mean of two independent experiments using duplicate wells. V3/alpha2 helix amino acid sequences were aligned and examined using Sequencher v5.0 and Geneious v5.0.3 software (E). Dashes represent conserved positions; dots represent gaps. Significant PNGS sequons (N295, N333, N335) are highlighted in black at their points of origin.
Mentions: The VH, in particular the CDR H3, has generally been considered a major determinant of epitope recognition and nAb breadth. In our study, VL differences appreciably expanded the neutralization capacity of mAb 19.3H-L3 against autologous Envs. To probe whether this increase in breadth carried over to neutralization of heterologous Envs, mAbs 19.3H-L1 and 19.3H-L3 were tested against a panel of fourteen heterologous Env pseudotypes that included one A/C recombinant, four subtype A, three subtype B, and six subtype C Envs. The mAbs were unable to neutralize any of the heterologous Envs (Figure 9A–B). Thus, while mAb 19.3H-L3 possessed increased breadth against autologous Envs as compared to 19.3H-L1, this did not extend to genetically diverse Envs. Regardless of this restricted mAb cross-clade neutralization, R880F plasma collected at 16-months or 3-years post-infection did have similarly moderate breadth against heterologous Envs, which increased in potency over time (Figure 9C–D). An amino acid alignment of Envs from the heterologous breadth panel demonstrated that Envs neutralized with the greatest potency at 3-years post-seroconversion, A-Q461 and C-Z205F (IC50 values of approximately 1∶1000), contained the N335 (HXB2 residue 334) shifted glycan associated with viral escape from mAbs 19.3H-L1 and 19.3H-L3 (Figure 9E). Furthermore, Env A-Q461 also incorporated the N295 (HXB2 residue 293) substitution indicative of mAb escape. To investigate if the N295 glycan addition and/or the shifted N335 glycan in R880F Envs could have been partially responsible for the heterologous neutralization capacity that developed in this subject, several glycan knock-out mutants were created and tested with 3-year R880F plasma (Figure 10A). Within A-Q461, the N295 PNGS was eliminated either alone or in conjunction with the N335 PNGS; the N335 PNGS was also individually knocked out (Figure 10B). The positions of interest were reverted back to the amino acid present in the transmitted/founder Env 0-A6/B24. For C-Z205F, the N335 PNGS was similarly abolished (Figure 10B). Additionally, two heterologous Envs that were only modestly neutralized but that contained the highlighted glycans, C-Z109F and C-Z214M, were mutated as well. All six of the glycan knock-out mutants exactly mirrored their parental equivalents, suggesting that the particular glycans at positions 295 and 335 did not directly contribute to the breadth observed at 3-years post-infection. These data do suggest, however, that early viral escape events likely influenced how breadth developed in this subject, by expanding what was originally a narrow, regional response at the base of the V3 loop to recognize and neutralize distinct portions of Env across genetically diverse variants.

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