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Analysis of human CD4-antibody interaction using the BIAcore system
F Velge-Roussel1, P Breton, F Lescure
1CJF INSERM 93-09 Immunologie des Maladies Infectieuses, Equipe Associée INRA d'Immunologie Parasitaire, UFR des Sciences Pharmaceutiques, Tours, France.
Insights
This study characterizes the binding kinetics of an anti-CD4 antibody (IOT4a) to soluble CD4, crucial for HIV entry. The interaction is enthalpy-driven with a positive entropy contribution, suggesting electrostatic interactions stabilize the complex.
Area of Science:
- Immunology
- Virology
- Biophysics
Background:
- The interaction between the CD4 protein and the HIV gp120 protein is a critical initial step for HIV entry into host cells.
- Certain anti-CD4 antibodies have demonstrated the ability to inhibit this essential interaction.
Purpose of the Study:
- To determine the kinetic and thermodynamic parameters of the interaction between the anti-CD4 antibody IOT4a and immobilized recombinant soluble CD4 (rsCD4).
Main Methods:
- Utilized biosensor studies with a BIAcore instrument to analyze the antibody-antigen interaction.
- Employed non-linear regression analysis of sensorgrams to model the binding kinetics.
- Calculated kinetic and thermodynamic parameters, including association constant (KA), enthalpy change, and entropy change.
Main Results:
- The interaction between IOT4a and rsCD4 exhibited a double exponential time curve.
- An association constant (KA) of 5.2 x 10^7 M^-1 was determined at 25°C.
- Complex formation was exothermic (-4.5 kcal/mol) and entropically favorable (+20 cal/mol·K), indicating an enthalpy-driven process with a significant positive entropy contribution, likely due to electrostatic interactions.
Conclusions:
- The binding kinetics and thermodynamics of IOT4a-rsCD4 interaction were quantitatively characterized.
- The observed thermodynamic profile, particularly the positive entropy contribution, differs from some antigen-antibody models and suggests a role for electrostatic interactions.
- The findings validate the theoretical approach used for analyzing the sensorgrams and provide insights into the molecular basis of antibody-antigen recognition in the context of HIV-CD4 interaction.
Abstract:
Interaction between CD4 cell surface protein and HIV-bearing gp120 has been described as the initial step for HIV entry into host cells. Some anti-CD4 antibodies were shown to inhibit this interaction. Biosensor studies using the BIAcore were performed to determine kinetic and thermodynamic parameters of the interaction of one of these antibodies (i.e. IOT4a, clone 13B8-2) with immobilized recombinant soluble CD4 (rsCD4). A non-linear regression method was used to analyze the sensorgrams, showing the existence of a double exponential time curve. A KA of 5.2 x 10(7) M-1 was calculated at 25 degrees C. The complex formation was exothermic (-4.5 kcal.mol-1( and entropically positive (+20 cal.mol-1.K-1). The reaction rate (0.234 x 10(5) M-1.s-1 at 25 degrees C) as well as the enthalpy change of the activated complex (+9.7 kcal.mol-1) are not compatible with a diffusion controlled reaction. The thermodynamic values calculated from equilibrium data corresponded to those calculated from kinetic data confirming the validity of the theoretical approach. As for most antigen-antibody interactions, complex formation was enthalpy driven. The overall positive entropy contribution to the stabilization of the complex is in contrast to that observed for the lysozyme-anti-lysozyme model and is probably due to electrostatic interaction between the epitope and the antibody combining site.