JGV Direct - P. J. Klasse and Q. J. Sattentau

Journal of General Virology

Fig. 1

Fig. 1. Ab binding to virions and soluble Ag. (a) The minimum requirement for neutralization is Ab binding to Ags as they are presented on the virion surface. The formation of complexes between Ab and viral Ag can be analysed according to the law of mass action as the reaction approaches equilibrium. The affinity of the Abs for the Ags can be quantitatively expressed as the association constant K_a (M^-¹), or its reciprocal, the dissociation constant, K_d (M). K_d is equal to the concentration of free ligand at half-maximal complex formation. Alternatively, the thermodynamic equilibrium constants K_a and K_d can be obtained from the on- and off-rate constants: k_on and k_off, as indicated. Both the thermodynamic and the kinetic constants are relevant to explanations of neutralization. (b) The top row illustrates functional and intrinsic affinity and valency of Ab binding. The virion to the left has a bivalent IgG bound and the one to the right a Fab fragment of the same Ab. The IgG and Fab binding to soluble and immobilized Ag is shown to the right in the top row. The concentration of a monovalent soluble Ag that yields half-maximal binding to paratopes approximates the K_d relating to the intrinsic affinity, which would be expected to be the same for IgG and Fab binding. The concentration of a Fab that gives half-maximal binding on virion-associated and soluble Ag, respectively, may not be the same because of conformational differences between the two forms of the Ag: the intrinsic affinities of the paratopes for the two antigenic forms would then differ. More relevant to neutralization is the functional affinity of a whole bi-, tetra- or decavalent Ab for the epitopes in their context on the virions. The functional affinity simplifies matters by the postulate that the Ab is bound or unbound; although, for example, an IgG can have one or both paratopes bound. When both can bind, their doing so will be thermodynamically favoured and thereby the functional affinity will be substantially higher than the intrinsic affinity. This is the avidity effect, which might be quantified as the ratio of the functional over the intrinsic affinity (Klasse, 1996). Bivalent binding would also be possible when the monovalent Ag is immobilized onto a solid phase (shown top right). If the conformation and oligomeric state, and thereby the intrinsic affinity, as well as the spacing and orientation, of the Ag on the solid phase resemble those on the virion, then the functional affinity will be similar in the two situations. Below to the left, the potential effect of Ab hinge flexibility on functional affinity is shown. The flexibility of the hinge region of human IgG₃ is greater than that of the other IgG subclasses, which may facilitate two-point binding to a polyvalent Ag and increase the functional affinity of the Ab for the virion and thereby its neutralization potency (Cavacini et al., 1995; Scharf et al., 2001). In addition, as shown to the right, the Fc portions of neighbouring IgG molecules can interact non-covalently (Greenspan, 2001a), which could effectively double the valency of the Ab in the bound state. A neighbour effect of this kind will produce deviations from the law of mass action in Ab binding: intermolecular cooperativity may enhance the degree of binding achieved on the virion surface above certain Ab densities. (c) The qualitative aspect of affinity as structural complementarity between paratope and epitope is depicted schematically. Charge–charge interactions (+/–), hydrogen bonds (stippled lines) and van der Waals interactions make contributions to the binding energy. Affinity can be translated into changes in free energy, which relate to the system and not merely to the epitope and paratope (Greenspan & Cooper, 1995; Greenspan & Di Cera, 1999; Epa & Colman, 2001): the solvent (water molecules excluded from the molecular interface are schematically indicated), solutes, the energetics of solvation and of conformational states of bound and free Ag and Ab, as well as spatial context, all contribute (Greenspan, 2001b). Thus contact residues in epitope and paratope sometimes make smaller energetic contributions to the affinity than non-contact residues. Furthermore, residues in the paratope that are most crucial to the discrimination between different Ags may not make the greatest energetic contributions nor even be in contact with the epitope (Greenspan, 2001a, b). A residue in the epitope (*) crucial to Ab binding may be remote from the contact surfaces (Parry et al., 1990). A subtle amino acid substitution that determines the sensitivity of HIV-1 to neutralization by certain Abs directed to the outer envelope glycoprotein, gp120 or SU, is located in gp41, the non-covalently associated transmembrane protein (TM) (Klasse et al., 1993; Thali et al., 1994). According to the three-dimensional structure of gp120 and knowledge of which parts of the molecule interact with gp41, the mutated residue would be distant from the epitopes (Kwong et al., 1998).

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