Antibody-mediated neutralization of flaviviruses: a reductionist view

Abstract

Flaviviruses are a group of ~70 small RNA viruses responsible for significant morbidity and mortality across the globe. Efforts to develop effective vaccines for several clinically important flaviviruses are underway. Antibodies are a significant component of the host's protective response against flavivirus infection with the potential to contribute to immunity via several distinct mechanisms, including an ability to directly neutralize virus infection. Conversely, virus-reactive antibodies have been implicated in the increased risk of severe clinical manifestations following secondary dengue virus infection. In this review, we will discuss recent progress toward understanding the molecular basis of antibody-mediated neutralization of flaviviruses. Neutralization requires engagement of the virion with a stoichiometry that exceeds a required threshold. From this perspective, we will discuss viral and host factors that impact the number of antibody molecules bound to the virus particle and significantly modulate the potency of neutralizing antibodies.

Figure 1. Structure of the flavivirus E protein and its arrangement on the virion

A) Ribbon diagram of the anti-parallel flavivirus E protein dimer as seen from the top and side. Individual domains of the E protein are indicated, as is the fusion loop at the distal end of E-DII. The N-linked carbohydrate modifications on E are shown as brown spheres. B) Cryoelectron reconstruction of the flavivirus immature virion. Image created using Chimera (http://www.cgl.ucsf.edu/chimera/). C) Cryoelectron reconstruction of the flavivirus mature virion illustrating the arrangement of E proteins in a T=3 pseudo-icosahedron. Individual E proteins are colored according to their proximity to the 2-, 3-, or 5-fold symmetry axes (blue, red, and green, respectively). Image obtained from the VIPERdb Virus Particle Explorer (http://viperdb.scripps.edu/) (Shepherd et al., 2006).

Figure 2. Antibody affinity and epitope accessibility govern the neutralization potency of anti-flavivirus antibodies

Neutralization of flavivirus virions requires engagement of the virion by antibody with a stoichiometry that exceeds a threshold (estimated as 30 mAbs based on studies with two WNV E-DIII-LR-specific mAbs) (Pierson et al., 2007). Engagement of the virion with a stoichiometry below this threshold may support antibody-dependent enhancement of infection (ADE). A) Antibody affinity determines the percentage of epitopes bound by antibody at any non-saturating concentration. For example, incubation of a theoretical virus displaying 180 E protein epitopes with antibody at a concentration equal to its K_d results in engagement of the virion with a stoichiometry of an average of 90 antibody molecules. Reducing the concentration two-fold (0.5x of the antibody K_d) translates into engagement by 60 antibodies, as determined by the relationship (Bound/Bound_max)= ([Ab]/([Ab]+K_d)). (right panel) Mutations that change the affinity of antibody-virion interactions, such as the single T330I substitution introduced into the WNV E protein. B) Epitope accessibility governs the occupancy requirements for neutralization. For a theoretical flavivirus displaying 180 epitopes, an epitope occupancy of 17% is required to exceed a stoichiometric threshold of 30 antibody molecules. A reduction in epitope accessibility translates into increases in the fraction of epitopes that must be engaged in order to support virus neutralization. (right panel) Epitope accessibility varies considerably for antibodies that bind structurally distinct epitopes on the flavivirus. Antibodies that bind highly accessible determinants my completely neutralize infection at relatively modest occupancy (60%, green shading), whereas antibodies that bind poorly accessibly structures neutralize infection only at full occupancy (red shading). Figures and data were modified from (Pierson et al., 2008; Pierson et al., 2007).

Figure 3. Factors that govern the neutralizing activity of anti-flavivirus antibodies

Neutralization of flavivirus infection is a multiple hit phenomenon that requires engagement of the virion by antibody with a stoichiometry that exceeds a threshold. Whether an individual antibody can dock on the virion with a stoichiometry sufficient to exceed this threshold depends on its affinity for viral antigens and the total number of accessible epitopes displayed on the average virion (shown schematically on the x-axis). Changes in epitope accessibility significantly impact the potency of neutralizing antibodies. The dynamic motion of DENV appears to regulate exposure of the A-strand epitope recognized by mAb 1A1D-2 at equilibrium, and explains the temperature-dependence of binding by this antibody. The process of virion maturation also impacts accessibility of epitopes bound by neutralizing antibodies. Virion maturation reduces accessibility of several classes of antibodies to levels that no longer support neutralization. Interactions with the complement protein C1q increase the potency of neutralizing antibodies, even those specific for poorly accessible determinants. C1q augments neutralization by decreasing the stoichiometric threshold required for neutralization (shifted red dashed line, bottom). Figure modified from (Pierson et al., 2008).