Structural insights into the mechanisms of antibody-mediated neutralization of flavivirus infection: implications for vaccine development

Abstract

Flaviviruses are a group of small RNA viruses that cause severe disease in humans worldwide and are the target of several vaccine development programs. A primary goal of these efforts is to elicit a protective humoral response directed against the envelope proteins arrayed on the surface of the flavivirus virion. Advances in the structural biology of these viruses has catalyzed rapid progress toward understanding the complexity of the flavivirus immunogen and the molecular basis of antibody-mediated neutralization. These insights have identified factors that govern the potency of neutralizing antibodies and will inform the design and evaluation of novel vaccines.

Figure 1. Structure of the DENV E Protein and Its Arrangement on the Virion

(A) Ribbon diagram of the antiparallel DENV 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–D) Surface shaded representation of the DENV virion at varying stages of maturation as determined by cryo-EM reconstruction. The significant conformational differences in the organization of E proteins of the virion during maturation are evident by comparing structure of spiky immature virions at (B) neutral and (C) acidic pH. In the latter case, prM is represented in cyan and the icosahedral asymmetry unit is indicated as a gray triangle. In (D), the structure of the mature DENV virion is shown. The images presented in (B)–(D) were created using previously described structures of DENV (Kuhn et al., 2002; Yu et al., 2008; Zhang et al., 2003).

Figure 2. Antigenic Complexity of the Flavivirus E Protein

(A) All three domains of the E protein contain epitopes recognized by neutralizing antibodies. Domains of the WNV E protein are labeled as in Figure 1. Residues that impact antibody binding (shown in magenta) were identified using a yeast-display mapping approach for a large panel of antibodies, identifying structurally distinct epitopes in E-DIII (below), E-DI, and E-DII. Epitopes are labeled as described previously (Oliphant et al., 2006). (B) The structure of WNV DIII is shown displaying the epitope recognized by the type-specific mAb E16. Residues of E-DIII determined by X-ray crystallography to interact directly E16 are highlighted in blue. (C) The structure of DENV E-DIII is shown displaying the epitope recognized by the sub-complex-specific mAb 1A1D-2 (highlighted in cyan). Note that the epitope for 1A1D-2 is centered on the A strand and does not make contact with the residues in the FG loop that play a role in type-specific reactivity. (D) Comparison of the epitope recognized by 1A1D-2 and the DENV2 type-specific mAb 3H5. (E) E proteins exist on the mature flavivirus associated with three distinct symmetry environments. Epitopes recognized by mAbs may not be equally accessible in all three of these environments. The WNV-specific mAb E16 binds an epitope (shown in green) that is accessible in only two of these environments. As shown in dark green, the E-DIII-LR epitope recognized by E16 is partially obscured on E proteins proximal to the five-fold axis of symmetry, which limits binding to 120 of 180 possible E proteins on the mature virus. The 1A1D-2 epitope (shown in pink) on E-DIII is partially blocked on E proteins of mature virus particles regardless of their position on the virions (shown in red).

Figure 3. A Model of the Stoichiometric Requirements for Flavivirus Neutralization

Neutralization of flavivirus infection is a multiple-hit phenomenon that requires engagement of the virion by antibody with a stoichiometry that exceeds a threshold (modeled as 30 mAbs based on studies with WNV E-DIII-LR-specific mAbs; red dashed line) (Pierson et al., 2007). 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 on the average virion (shown schematically on the x axis). Epitopes that are differentially accessible on virions as a function of the extent of virion maturation or the structural dynamics of the virus particle add to the complexity of the model (shown schematically for maturation-dependent epitopes and those that bind selective conformations, respectively). Neutralization via highly accessible determinants can be achieved by engagement to a relatively low occupancy, whereas antibodies that bind cryptic determinants must bind a larger fraction of them. Not all epitopes appear to exist on the average virion at levels that exceed this threshold. Engagement of the virion with a stoichiometry below this threshold may support antibody-dependent enhancement of infection. Virion images were created using Chimera (http://www.cgl.ucsf.edu/chimera) or obtained from the VIPERdb Virus Particle Explorer (http://viperdb.scripps.edu) (Shepherd et al., 2006).