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Structural and Thermodynamic Basis of Epitope Binding by Neutralizing and Nonneutralizing Forms of the Anti-HIV-1 Antibody 4E10.

Rujas E, Gulzar N, Morante K, Tsumoto K, Scott JK, Nieva JL, Caaveiro JM - J. Virol. (2015)

Bottom Line: The conclusions of our structure-function analysis strengthen the idea that to exert effective neutralization, the hydrophobic apex of the solvent-exposed CDR-H3 loop must recognize an antigenic structure more complex than just the linear α-helical epitope and likely constrained by the viral membrane lipids.However, 4E10 (or 4E10-like) antibodies are rarely found in HIV-1-infected individuals or elicited through vaccination.We conclude that the difference between neutralizing and nonneutralizing antibodies of 4E10 is neither structural nor energetic but is related to the capacity to recognize the HIV-1 gp41 epitope inserted in biological membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan Biophysics Unit (CSIC, UPV/EHU) and Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Bilbao, Spain.

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Alterations in the CDR H3 of 4E10 Fab impacts the binding of Fab-s to its cognate epitope in the context of the plasma membrane. (A) Cell vesicles displaying the MPER-TM1 protein product (solid line), and “empty” control vesicles (dashed lines) were probed with 4E10 IgG and Fabs (WT, WDWD, and ΔLoop) at the indicated concentrations. (B) Cell vesicles bearing recombinant MPER proteins (MPER-TM1, black columns; MPER-PDGFR, gray columns) were probed with 20 nM 4E10 IgG and Fabs and 20 nM 17/9 IgG (which binds to an HA tag located at the N terminus of the MPER constructs and is used to assess cell surface expression). Absorbance values were normalized to the cell surface expression of the constructs (i.e., the absorbance of 4E10IgG or Fabs/the absorbance of 17/9IgG). (C) Binding of 20 nM 4E10 IgG and Fabs to recombinant gp41, the MPER667–683 peptide, and assay controls (“empty” 293T cell lysates, BSA, and ovalbumin). For all experiments, the absorbance at 405 to 490 nm was recorded. Error bars represent standard error of the mean. The data shown are from one of two (see Fig. 8B and C) or three (see Fig. 8A) experiments, all of which yielded similar results.
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Figure 8: Alterations in the CDR H3 of 4E10 Fab impacts the binding of Fab-s to its cognate epitope in the context of the plasma membrane. (A) Cell vesicles displaying the MPER-TM1 protein product (solid line), and “empty” control vesicles (dashed lines) were probed with 4E10 IgG and Fabs (WT, WDWD, and ΔLoop) at the indicated concentrations. (B) Cell vesicles bearing recombinant MPER proteins (MPER-TM1, black columns; MPER-PDGFR, gray columns) were probed with 20 nM 4E10 IgG and Fabs and 20 nM 17/9 IgG (which binds to an HA tag located at the N terminus of the MPER constructs and is used to assess cell surface expression). Absorbance values were normalized to the cell surface expression of the constructs (i.e., the absorbance of 4E10IgG or Fabs/the absorbance of 17/9IgG). (C) Binding of 20 nM 4E10 IgG and Fabs to recombinant gp41, the MPER667–683 peptide, and assay controls (“empty” 293T cell lysates, BSA, and ovalbumin). For all experiments, the absorbance at 405 to 490 nm was recorded. Error bars represent standard error of the mean. The data shown are from one of two (see Fig. 8B and C) or three (see Fig. 8A) experiments, all of which yielded similar results.

Mentions: The results in Fig. 8 strengthen the conclusion that availability of the WT CDR-H3 apex contributes to additional interactions with membrane components as previously suggested (10, 14, 22, 45–48). The DNA vaccine candidates, MPER-TM1 and MPER-PDGFR (22), express fusion proteins comprising: (i) an N-terminal HA tag, (ii) the MPER, (iii) either the transmembrane domain of gp41 followed by 27-AA of the cytoplasmic domain (TM1) or that of the platelet-derived growth-factor receptor (PDGFR). These fusions are expressed in the context of the plasma membrane; however, the PDGFR transmembrane domain has been shown to reduce exposure of the 4E10 epitope (22) and, to a lesser extent, the 10E8 epitope (N. Gulzar, unpublished data). The titrations shown in Fig. 8A indicate that IgG and Fab WT engaged effectively with the 4E10 epitope presented in the context of the plasma membrane (EC50s of ca. 0.1 and 1 nM, respectively). In comparison, in the range of measured concentrations, binding to MPER-TM1 was negligible for the WDWD and ΔLoop mutants (Fig. 8A, bottom panels). None of the antibodies bound directly to the plasma membrane of cells devoid of an MPER construct (squares and dotted lines), demonstrating that the binding scored for the WT IgG and Fab under our experimental conditions was strictly dependent on the presence of the MPER epitope.


Structural and Thermodynamic Basis of Epitope Binding by Neutralizing and Nonneutralizing Forms of the Anti-HIV-1 Antibody 4E10.

Rujas E, Gulzar N, Morante K, Tsumoto K, Scott JK, Nieva JL, Caaveiro JM - J. Virol. (2015)

Alterations in the CDR H3 of 4E10 Fab impacts the binding of Fab-s to its cognate epitope in the context of the plasma membrane. (A) Cell vesicles displaying the MPER-TM1 protein product (solid line), and “empty” control vesicles (dashed lines) were probed with 4E10 IgG and Fabs (WT, WDWD, and ΔLoop) at the indicated concentrations. (B) Cell vesicles bearing recombinant MPER proteins (MPER-TM1, black columns; MPER-PDGFR, gray columns) were probed with 20 nM 4E10 IgG and Fabs and 20 nM 17/9 IgG (which binds to an HA tag located at the N terminus of the MPER constructs and is used to assess cell surface expression). Absorbance values were normalized to the cell surface expression of the constructs (i.e., the absorbance of 4E10IgG or Fabs/the absorbance of 17/9IgG). (C) Binding of 20 nM 4E10 IgG and Fabs to recombinant gp41, the MPER667–683 peptide, and assay controls (“empty” 293T cell lysates, BSA, and ovalbumin). For all experiments, the absorbance at 405 to 490 nm was recorded. Error bars represent standard error of the mean. The data shown are from one of two (see Fig. 8B and C) or three (see Fig. 8A) experiments, all of which yielded similar results.
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Figure 8: Alterations in the CDR H3 of 4E10 Fab impacts the binding of Fab-s to its cognate epitope in the context of the plasma membrane. (A) Cell vesicles displaying the MPER-TM1 protein product (solid line), and “empty” control vesicles (dashed lines) were probed with 4E10 IgG and Fabs (WT, WDWD, and ΔLoop) at the indicated concentrations. (B) Cell vesicles bearing recombinant MPER proteins (MPER-TM1, black columns; MPER-PDGFR, gray columns) were probed with 20 nM 4E10 IgG and Fabs and 20 nM 17/9 IgG (which binds to an HA tag located at the N terminus of the MPER constructs and is used to assess cell surface expression). Absorbance values were normalized to the cell surface expression of the constructs (i.e., the absorbance of 4E10IgG or Fabs/the absorbance of 17/9IgG). (C) Binding of 20 nM 4E10 IgG and Fabs to recombinant gp41, the MPER667–683 peptide, and assay controls (“empty” 293T cell lysates, BSA, and ovalbumin). For all experiments, the absorbance at 405 to 490 nm was recorded. Error bars represent standard error of the mean. The data shown are from one of two (see Fig. 8B and C) or three (see Fig. 8A) experiments, all of which yielded similar results.
Mentions: The results in Fig. 8 strengthen the conclusion that availability of the WT CDR-H3 apex contributes to additional interactions with membrane components as previously suggested (10, 14, 22, 45–48). The DNA vaccine candidates, MPER-TM1 and MPER-PDGFR (22), express fusion proteins comprising: (i) an N-terminal HA tag, (ii) the MPER, (iii) either the transmembrane domain of gp41 followed by 27-AA of the cytoplasmic domain (TM1) or that of the platelet-derived growth-factor receptor (PDGFR). These fusions are expressed in the context of the plasma membrane; however, the PDGFR transmembrane domain has been shown to reduce exposure of the 4E10 epitope (22) and, to a lesser extent, the 10E8 epitope (N. Gulzar, unpublished data). The titrations shown in Fig. 8A indicate that IgG and Fab WT engaged effectively with the 4E10 epitope presented in the context of the plasma membrane (EC50s of ca. 0.1 and 1 nM, respectively). In comparison, in the range of measured concentrations, binding to MPER-TM1 was negligible for the WDWD and ΔLoop mutants (Fig. 8A, bottom panels). None of the antibodies bound directly to the plasma membrane of cells devoid of an MPER construct (squares and dotted lines), demonstrating that the binding scored for the WT IgG and Fab under our experimental conditions was strictly dependent on the presence of the MPER epitope.

Bottom Line: The conclusions of our structure-function analysis strengthen the idea that to exert effective neutralization, the hydrophobic apex of the solvent-exposed CDR-H3 loop must recognize an antigenic structure more complex than just the linear α-helical epitope and likely constrained by the viral membrane lipids.However, 4E10 (or 4E10-like) antibodies are rarely found in HIV-1-infected individuals or elicited through vaccination.We conclude that the difference between neutralizing and nonneutralizing antibodies of 4E10 is neither structural nor energetic but is related to the capacity to recognize the HIV-1 gp41 epitope inserted in biological membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan Biophysics Unit (CSIC, UPV/EHU) and Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), Bilbao, Spain.

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Related in: MedlinePlus