West Nile virions incorporate 180 envelope (E) proteins that orchestrate the process of virus entry and are the primary target of neutralizing antibodies. The E proteins of newly synthesized West Nile virus (WNV) are organized into trimeric spikes composed of pre-membrane (prM) and E protein heterodimers. During egress, immature virions undergo a protease-mediated cleavage of prM that results in a reorganization of E protein into the pseudo-icosahedral arrangement characteristic of mature virions. While cleavage of prM is a required step in the virus life cycle, complete maturation is not required for infectivity and infectious virions may be heterogeneous with respect to the extent of prM cleavage. In this study, we demonstrate that virion maturation impacts the sensitivity of WNV to antibody-mediated neutralization. Complete maturation results in a significant reduction in sensitivity to neutralization by antibodies specific for poorly accessible epitopes that comprise a major component of the human antibody response following WNV infection or vaccination. This reduction in neutralization sensitivity reflects a decrease in the accessibility of epitopes on virions to levels that fall below a threshold required for neutralization. Thus, in addition to a role in facilitating viral entry, changes in E protein arrangement associated with maturation modulate neutralization sensitivity and introduce an additional layer of complexity into humoral immunity against WNV.

Neutralizing antibodies are a significant component of the host's protective response against flavivirus infection. Neutralization of flaviviruses occurs when individual virions are engaged by antibodies with a stoichiometry that exceeds a required threshold. From this “multiple-hit” perspective, the neutralizing activity of antibodies is governed by the affinity with which it binds its epitope and the number of times this determinant is displayed on the surface of the virion. In this study, we investigated time-dependent changes in the fate of West Nile virus (WNV) decorated with antibody in solution. Experiments with the well-characterized neutralizing monoclonal antibody (MAb) E16 revealed a significant increase in neutralization activity over time that could not be explained by the kinetics of antibody binding, virion aggregation, or the action of complement. Additional kinetic experiments using the fusion-loop specific MAb E53, which has limited neutralizing activity because it recognizes a relatively inaccessible epitope on mature virions, identified a role of virus “breathing” in regulating neutralization activity. Remarkably, MAb E53 neutralized mature WNV in a time- and temperature-dependent manner. This phenomenon was confirmed in studies with a large panel of MAbs specific for epitopes in each domain of the WNV envelope protein, with sera from recipients of a live attenuated WNV vaccine, and in experiments with dengue virus. Given enough time, significant inhibition of infection was observed even for antibodies with very limited, or no neutralizing activity in standard neutralization assays. Together, our data suggests that the structural dynamics of flaviviruses impacts antibody-mediated neutralization via exposure of otherwise inaccessible epitopes, allowing for antibodies to dock on the virion with a stoichiometry sufficient for neutralization.

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for 50 to 100 million human infections each year, highlighting the need for a safe and effective vaccine. In this study, we describe the production of pseudoinfectious DENV reporter virus particles (RVPs) using two different genetic complementation approaches, including the creation of cell lines that release reporter viruses in an inducible fashion. In contrast to studies with West Nile virus (WNV), production of infectious DENV RVPs was temperature-dependent; the yield of infectious DENV RVPs at 37 °C is significantly reduced in comparison to experiments conducted at lower temperatures or with WNV. This reflects both a significant reduction in the rate of infectious DENV RVP release over time, and the more rapid decay of infectious DENV RVPs at 37 °C. Optimized production approaches allow the production of DENV RVPs with titers suitable for the study of DENV entry, assembly, and the analysis of the humoral immune response of infected and vaccinated individuals.

Summary Virus neutralization is governed by the number of antibodies that bind a virion during the cellular entry process. Cellular and serum factors that interact with antibodies have the potential to modulate neutralization potency. Although the addition of serum complement can increase the neutralizing activity of antiviral antibodies in vitro, the mechanism and significance of this augmented potency in vivo remains uncertain. Herein, we show that the complement component C1q increases the potency of antibodies against West Nile virus by modulating the stoichiometric requirements for neutralization. The addition of C1q does not result in virolysis, but instead reduces the number of antibodies that must bind the virion to neutralize infectivity. For IgG subclasses that bind C1q avidly, this reduced stoichiometric threshold falls below the minimal number of antibodies required for antibody-dependent enhancement (ADE) of infection of K562 cells expressing Fc-γ receptors (CD32), and explains how C1q restricts the ADE of flavivirus infection.

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