How to assess the binding strength of antibodies elicited by vaccination against HIV and other viruses

Abstract

Vaccines that protect against viral infections generally induce neutralizing antibodies. When vaccines are evaluated, the need arises to assess the affinity maturation of the antibody responses. Binding titers of polyclonal sera depend not only on the affinities of the constituent antibodies but also on their individual concentrations, which are difficult to ascertain. Therefore an assay based on chaotrope disruption of antibody-antigen complexes was designed for measuring binding strength. This assay works well with many viral antigens but gives differential results depending on the conformational dependence of epitopes on complex antigens such as the envelope glycoprotein of HIV-1. Kinetic binding assays might offer alternatives, since they can measure average off-rate constants for polyclonal antibodies in a serum. Here, potentials and fallacies of these techniques are discussed.

Keywords: HIV Env; Vaccine; affinity; antibody; avidity; chaotrope; kinetic constants; neutralization; surface plasmon resonance; virus.

Figure 1

The Hofmeister series. In 1888 Franz Hofmeister described the ranking of salt solutions for efficacy in precipitating serum globulins (81). By comparing cations paired with the same anion and vice versa he elegantly dissected the effects of the individual ions and ranked them as illustrated in the series. Each series describes a spectrum from the first ion (left), which most decreases globulin solubility (“salting out”) to the last ion (right), which most increases it (“salting in”). Anionic effects tend to be stronger than the cationic ones. The series has also been called lyotropic and the left-hand extreme kosmotropic, whereas the righ-hand extreme is commonly referred to as chaotropic. Those terms are derived from a now much revised view of how the ions work. Thiocynate, the most chaotropic of the anions, is often used in the chaotrope-based avidity assay. Certain non-ionic molecules, e.g., urea, share some properties of the chaotropic ions and have also been used in the assay. The old theory suggested that the effect was general and attributable to how the ions interact with water: the propensity of water molecules to form shells around macromolecules would be diminished by the chaotrope. Part of the binding energy for two interacting protein molecules is explained by how the macromolecular interaction reduces the sizes of those combined water shells. Therefore the binding energy would be unfavorably affected by the chaotrope. The newer insights in Hofmeister solvation science suggest less general mechanisms: instead the solvation effects on proteins partly stem from ionic interactions with the peptidic backbone and the side chains, the latter in particular making the susceptibility of each antibody-antigen complex unique, like the amino-acid composition of the parts contributing to the epitope and the paratope. Already the old view suggested variation in chaotrope resistance according to the relative contributions to the paratope-epitope binding of van der Waals or hydrophobic versus polar interactions. But the new insights further undermine the theoretical basis for using chaotropes to measure binding strength.

Figure 2. Antibodies (Fabs) bound to neutralization epitopes of an HIV-1 Env trimer

(A) The Fabs (green) directed to various known neutralization epitopes on each of the three Env protomers nearly cover the trimer (beige). (B) Fabs directed to different epitopes are shown in different colors. Most antibodies bind with a stoichiometry of up three paratopes per trimer; some directed to quaternary-structural epitopes at the trimer apex (PG9) bind to only one epitope per trimer; but here only one Fab of each antibody is represented, thereby revealing the unoccupied surfaces on the other protomers (white). (C) The unoccupied epitopes, corresponding to those occupied by the Fabs in B, are shown in the same colors as the corresponding Fabs in B. The figure shows simultaneous binding of multiple paratopes, a situation that resembles the binding of polyclonal antibodies in a serum although in some sera antibodies to a single epitope dominate. Because the antibodies will have varying affinities and unknown concentrations, only the average off-rate constant can be assessed by kinetic techniques. Furthermore, the binding of one Fab may positively or negatively affect the binding of another (93). The images are reproduced from Derking et al. PLoSPath 2015 11: e1004767 (93).

Figure 3

The kinetics of antibody binding to immobilized trimeric HIV-1 envelope glycoprotein (Env) analyzed by SPR. The sensorgrams, i.e., response units (RU) after background subtraction as a function of time (s), show the binding curves for titrated antibodies as indicated in the legend. The same color code applies to all diagrams but the titration ranges start and end at different concentrations and also differ in the dilution steps. The data were fitted with a model for bivalent binding except in the case of the functionally monovalent antibody, 2G12, the binding of which was fitted with the simple Langmuir model. The curves generated by the modeling are depicted in black but are only visible where they diverge somewhat from the data. Note how the binding of the antibodies, all broadly neutralizing (bNAbs), differs both in on-rate (association phase, 5 min) and off-rate (dissociation phase, 10 min). The on-rate is the product of the on-rate constant, kon, and the antibody concentration; the off-rate is concentration-independent and hence its constant, koff, can be determined when the antibody concentrations are unknown, as is the case for polyconal sera. Therefore it has been possible to apply SPR and another kinetic method, biolayer interferometry, to the monitoring of increased binding strength of antibodies during affinity maturation (10, 104). The diagrams are reproduced from Yasmeen et al. 2014, Retroviology 11:41 (29).