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. 2016 Oct 18;111(8):1641-1654.
doi: 10.1016/j.bpj.2016.09.003.

Modeling the Role of Epitope Arrangement on Antibody Binding Stoichiometry in Flaviviruses

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Free PMC article

Modeling the Role of Epitope Arrangement on Antibody Binding Stoichiometry in Flaviviruses

Daniel R Ripoll et al. Biophys J. .
Free PMC article

Abstract

Cryo-electron-microscopy (cryo-EM) structures of flaviviruses reveal significant variation in epitope occupancy across different monoclonal antibodies that have largely been attributed to epitope-level differences in conformation or accessibility that affect antibody binding. The consequences of these variations for macroscopic properties such as antibody binding and neutralization are the results of the law of mass action-a stochastic process of innumerable binding and unbinding events between antibodies and the multiple binding sites on the flavivirus in equilibrium-that cannot be directly imputed from structure alone. We carried out coarse-grained spatial stochastic binding simulations for nine flavivirus antibodies with epitopes defined by cryo-EM or x-ray crystallography to assess the role of epitope spatial arrangement on antibody-binding stoichiometry, occupancy, and neutralization. In our simulations, all epitopes were equally competent for binding, representing the upper limit of binding stoichiometry that results from epitope spatial arrangement alone. Surprisingly, our simulations closely reproduced the relative occupancy and binding stoichiometry observed in cryo-EM, without having to account for differences in epitope accessibility or conformation, suggesting that epitope spatial arrangement alone may be sufficient to explain differences in binding occupancy and stoichiometry between antibodies. Furthermore, we found that there was significant heterogeneity in binding configurations even at saturating antibody concentrations, and that bivalent antibody binding may be more common than previously thought. Finally, we propose a structure-based explanation for the stoichiometric threshold model of neutralization.

Figures

Figure 1
Figure 1
Overview of spatial stochastic model. (A) We use a whole-virus structural model, derived from x-ray or cryo-EM data along with homology modeling. (B) The epitope for a given antibody is mapped to the structure. (C) MC-based spatial stochastic simulations were carried out. (D) Number of Abs bound over the course of three example MC trajectories. (E) Distribution of viral bound states at three Ab concentrations. (F) Ab binding curve derived from MC simulations at a range of Ab concentrations. To see this figure in color, go online.
Figure 2
Figure 2
Monovalent and bivalent binding curve and neutralization curves for flavivirus Abs. (A) Absolute binding curve showing the number of Abs bound as a function of Ab concentration for monovalent (top) and bivalent (bottom) binding simulations. (B) Relative binding curve showing the percentage of Abs bound as a function of concentration relative to the maximum number of Abs bound for for monovalent (top) and bivalent (bottom) binding simulations. (C) Neutralization curve, represented by the percentage of the virus population with >30 Abs bound at each concentration for monovalent (top) and bivalent (bottom) binding simulations. To see this figure in color, go online.
Figure 3
Figure 3
Structure and relative occupancy of symmetry-related epitopes. (A) Whole-virion structure used for binding simulations for flavivirus Abs E106 (left), 1F4 (middle), and EDE2-A1 (right), showing epitopes colored with respect to their symmetry-related epitope environment for the threefold (epitope A, green), twofold (epitope B, cyan), and fivefold (epitope C, magenta) axes of symmetry. (B) Binding curves showing Ab occupancy at epitopes A, B, and C, as well as total Ab occupancy from monovalent binding simulations of the respective Abs. To see this figure in color, go online.
Figure 4
Figure 4
Comparison of simulated binding configuration with cryo-EM structures. Cryo-EM reconstructions for E16 (A), 1F4 (B), and EDE2-A11 (C) (top) are shown with Fabs colored with respect to binding to epitope A (threefold axis, green), epitope B (twofold axis, cyan), and epitope C (fivefold axis, magenta). Representative binding configurations from simulations run at a saturating Ab concentration (10−4 M) for E16, 1F4, and EDE2-A11 are shown in the bottom row. Colored circles represent bound Abs, colored with respect to binding to epitopes A, B, and C, as described above. The center of each epitope on the flavivirus envelope surface is shown as a solid sphere, also colored according to epitopes A, B, and C, as described above. Spheres that are covered with a Fab structure (top) or Ab circle (bottom) represent bound epitopes; uncovered spheres represent unbound epitopes. To see this figure in color, go online.
Figure 5
Figure 5
Modeling bivalent binding of IgG antibodies to flavivirus. (A) Neutralization curves from monovalent and bivalent binding simulations for Abs E106, EDE2-A11, and E16. Experimental data for Fab and MAb neutralization for E106 was derived from (45). (B) Molecular models of bivalent binding for IgG Abs E106, EDE2-A11, and E16. Epitopes A (threefold axis), B (twofold axis), and C (fivefold axis) on the flavivirus envelope are colored green, cyan, and magenta, respectively. Fabs are noted with a red dotted circle. To see this figure in color, go online.
Figure 6
Figure 6
Minimal fusogenic element model of flavivirus neutralization. (A) Representative fusogenic elements are shown as a red line for nearest-neighbor models 3NN and 4NN and symmetry-axis models 3FA and 5FA. Dotted lines represent degenerate fusogenic element definitions; straight lines indicate a single defined fusogenic element. (B) Percentage of infectivity, measured as the percentage of the viral population with at least one unbound fusogenic element according to the NN models (top) or FA models (bottom), are plotted against occupancy, the number of antibodies bound, for Ab E16. The infectivity and occupancy for the empirical neutralization model (n = 30) is also shown. The data points were collected from binding simulations at Ab concentrations ranging from 10−6 to 10−12 M. Dotted lines show 50% infectivity at an occupancy of 30 Ab, determined experimentally for E16 (14). To see this figure in color, go online.

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