The existence of high-resolution structures of the E proteins of flaviviruses, and their arrangement on the surface of the virion at different stages of the viral lifecycle, have been a powerful tool for studying how these viruses interact with the host (reviewed in 
). Interpretation of these structures in the context of biological systems is complicated by the fact that they represent the average state of what is likely a very dynamic ensemble of conformations sampled by virions at equilibrium. The structural dynamics of non-enveloped viruses have been studied extensively (reviewed in 
). Limited proteolysis studies of the Flock house virus strongly suggest the capsid proteins “breathe”, allowing proteases access to internal structures predicted to be inaccessible on an intact and non-dynamic virion 
. Furthermore, drugs that prevent the dynamic movement of the picornavirus human rhinovirus 14 have been shown to effectively inhibit infectivity 
. While the dynamics of enveloped viruses have been studied less extensively, examples of temperature- and time-dependent antibody reactivity have been described for several viruses 
. The DENV group-reactive neutralizing MAb 1A1D-2 binds an epitope on the A-strand of E-DIII that is poorly accessible on proteins proximal to each of the three symmetry axes of the mature virion. Binding of this MAb is temperature-dependent (does not occur at 4°C) and appears to trap E proteins on the virion surface in a conformation distinct from the herringbone icosahedral arrangement of the mature virion 
Overall, prior studies implicating a kinetic component of antibody-mediated neutralization have focused on either a single MAb (DENV MAb 1A1D-2, influenza MAb Y8-10C2) or a panel of MAbs that recognizes a similar epitope (MAbs specific for the membrane-proximal external region (MPER) of HIV gp41), with the implication that such antibodies were atypical in their ability to bind buried or inaccessible epitopes 
. In this study we provide functional evidence identifying the widespread impact of the dynamic movement of flaviviruses on neutralization by all antibodies that bind the E protein; a kinetic aspect of neutralization appears to be the rule rather than the exception.
Our data suggest that the structural dynamics of virions has the potential to modulate the potency of all antibodies that bind E proteins arrayed on the surface of the virion through changes in epitope accessibility. This is illustrated most dramatically by antibodies that bind poorly accessible determinants on the mature virion, such as the WNV DII-fusion loop-reactive MAb E53. Neutralization of mature WNV by E53 is restricted by the number of times the antibody can bind the virion. E53 binds the E protein only when associated with prM as heterotrimeric spikes that project off the surface of the immature virus particle 
. While homogeneous populations of mature WNV are not efficiently neutralized by E53 when assayed using conventional approaches, increasing the time the virus is incubated with antibody resulted in a marked increase in neutralization activity (>100-fold) (). Because the virus cannot revert to an immature configuration once prM cleavage occurs and the virion is released from the cell 
, this dramatic increase in neutralization can only be explained by exposure of the DII-fusion loop epitope through dynamic motion of viral E proteins.
The neutralization activity of every monoclonal (n
20) and polyclonal (n
5) antibody assayed in this study was enhanced by increasing the time the virion was exposed to antibody prior to the addition of target cells, including antibodies previously demonstrated to be non- or weakly-neutralizing using standard assays. This reflects the fact that as the number of accessible epitopes on the individual virion increases as a consequence of dynamic motion, the fraction of them that must be bound in order to exceed the stoichiometric threshold (percent occupancy) is reduced; neutralization can then occur at lower concentrations of antibody. However, the magnitude of this kinetic effect was not uniform among antibodies localizing to different epitopes. This may reflect differences in the number of times an antibody binds the average state of the virion relative to the threshold number of antibodies required for neutralization, as well as the rate at which additional epitopes are made available for binding (Figure S3
). In contrast to the cryptic nature of the DII-fusion loop epitope recognized by E53, the potently neutralizing MAb E16 is specific for a relatively accessible determinant displayed on the lateral ridge of DIII 
. Cryo-electron microscopy studies indicate this antibody binds 120 out of 180 E proteins incorporated into mature virions; the remaining 60 E proteins proximal to the five-fold symmetry axis of the virion cannot be bound due to steric conflicts among the tightly clustered DIII epitopes 
. An increase in the accessibility of these additional epitopes on the virion through dynamic motion would translate into a modest reduction in the occupancy requirements for neutralization by E16, in agreement with the 4.0-fold increase in antibody potency (n
11, range 2.3–6.8) observed in our studies after ~24 hours incubation. A similar 3.8-fold increase after ~24 hours was observed when E16 Fab fragments were used (n
2, range 3.2–4.5).
The dynamic motion of virions has the potential to increase antibody potency by providing access to otherwise cryptic antibody-binding determinants. Of note, mapping studies suggest that many epitopes on the mature virion are poorly accessible for antibody binding 
. Antibodies do not induce viral breathing, but rather stabilize conformations of the E protein that exist as part of the ensemble of conformations sampled by the virion at equilibrium. The longer the virion remains exposed to antibody, the more opportunities exist for engagement of an otherwise inaccessible epitope, allowing for time-dependent increases in the stoichiometry with which antibodies decorate virions. If changes in epitope accessibility are the underlying mechanism of the kinetic aspects of neutralization, there should be a limit to the increase in potency observed over time. Eventually, dynamic virion structures should expose all potential epitopes, and these will become fixed in place by antibody binding, yielding a neutralization profile determined by the relationship between antibody occupancy and the stoichiometric threshold. In support of this, increases in neutralization and changes in the ADE curves for E16 no longer occurred when incubations longer than 24 hours were performed (Figure S5
In addition to exposing more epitopes for antibody binding, time-dependent changes in the antigenic surface of the virus particle may also allow engagement of the virion with increased affinity, via bivalent interactions among E proteins in conformations not present in the average state, as well as cooperative effects during antibody binding. That the kinetic impact on neutralization by E16 was observed using both intact antibodies and Fab fragments incapable of cross-linking virions indicates that increases in antibody potency do not reflect antibody-mediated aggregation among virions. Importantly, all of the experiments included in our study were performed using conditions of antibody excess, and yielded results that were independent of the concentration of virus in the assay. In contrast, the aggregation of antigens by antibodies is dependent on the antibody-antigen ratio.
Our results suggest that changes in the antibody-mediated neutralization of DENV occur more rapidly than with WNV. One interpretation of this result is that DENV virions are more dynamic than those of WNV, allowing more rapid access to otherwise inaccessible determinants. In the absence of antibody, preparations of DENV become less infectious at a faster rate than observed with WNV, consistent with prior studies 
. Additionally, kinetic changes in neutralization with the cross-reactive MAb E60 occur at a faster rate with DENV-1 than WNV RVPs when compared in parallel studies (Figure S4
). While we do not yet understand, in molecular terms, why viruses lose infectivity over time, one possibility is that the intrinsic decay of flaviviruses is a consequence of structural dynamics. Viruses sampling multiple conformations in dynamic equilibrium may not always return to the average state because moving backwards may no longer be the most energetically favorable path. Additional evidence of time-dependent structural changes to the virus population is demonstrated by differences in the intrinsic decay rate of WNV observed when different cell types are used to measure infectivity. The rate of decay of viruses was ~2.7-fold more rapid when assayed on Raji-DC-SIGNR cells as compared to a K562 cell line expressing the same attachment protein (n
6, p<0.0001). Thus, the observed intrinsic decay cannot be attributed solely to the physical destruction of the virus, and suggests the additional possibility that not all conformations in a heterogeneous ensemble of virions are equally infectious on different cell types. Of interest, E proteins on individual virions in conformations that may no longer contribute functionally to fusion may also stably expose a different array of epitopes.
Our data suggest that the circumstances of antibody-virion interactions may significantly impact the fate of the virion immune complex. Standard in vitro
neutralization assays for flaviviruses generally include a short pre-incubation (~1 hr) of antibody and virus prior to infection of target cells; this incubation presumably allows the binding reaction between antibody and cognate epitope to reach steady-state. However, depending on the extent to which a virion is structurally dynamic (which controls the rate at which epitopes may become transiently accessible), the target cell type, and the volume of infection in vitro
, this presumption may be inaccurate. Because increases in the neutralization activity of DENV-reactive antibodies that bind dynamically exposed epitopes occur rapidly (within two hours) (), the interaction of DENV with antibodies may never truly reach steady-state. From this perspective, the length of time antibody is incubated with DENV is a variable that cannot be ignored. While antibody-mediated neutralization activity measured in vitro
using standard plaque reduction neutralization tests (PRNT) generally correlates with protection in vivo 
, this is an imperfect relationship. Antibodies with limited neutralization activity have been shown to protect in animal models of flavivirus infection 
. While this may reflect the direct contributions of effector functions of antibodies in vivo
, it is also possible that existing assays of the functional properties of antibodies have limitations. Considering the contribution of the structural dynamics of the virion when designing neutralization studies merits a systematic evaluation.
The impact of the dynamic exposure of viral epitopes in vivo
remains uncertain. Virtually nothing is known about the relevant concentrations and volumes that govern antibody-virion interactions in the tissues where many of the key events in the pathogenesis of these viruses occur. Kinetic changes in neutralization occur gradually over time as dynamic motion provides new opportunities for engagement of virions with a stoichiometry sufficient for neutralization (Figure S3
). Thus the rate of virus entry in vivo
is also an important, yet unknown, parameter that defines the extent to which this phenomenon will contribute to protection of the host. Of interest, the kinetics of WNV binding to target cells in vitro
occurred rather slowly (with maximal binding requiring ~3 hours) even in the presence of the high affinity attachment factor DC-SIGNR (Figure S6
). As DENV appears to be extremely dynamic, with kinetic effects on neutralization observed almost immediately, the impact of viral breathing on neutralization in vivo
cannot be discounted. Because the kinetics of neutralization are increased by an elevated temperature, it is interesting to speculate that certain classes of antibodies, such as those recognizing the fusion loop epitope commonly observed in infected individuals, may function better than previously anticipated in the context of the febrile response. Resolving this question awaits the development of approaches to quantitatively and directly measure antibody-mediated neutralization in vivo
Neither proteins, nor intact virions, are static structures. Our findings are consistent with a model in which the dynamic motion of flaviviruses provides an opportunity for antibodies to engage virions at otherwise inaccessible epitopes to reach a stoichiometry sufficient for neutralization. Given time, all of the E protein-reactive antibodies investigated were able to block virus infection, even those described originally as non-neutralizing using conventional assays 
. These results add to the complexity of our understanding of the functional properties of antibodies and suggest new avenues of investigation and analysis into the widespread and unappreciated impact of the dynamic motion of virions as moving targets for antibody recognition.