The Stoichiometry of Antibody-Mediated Neutralization and Enhancement of West Nile Virus Infection

Abstract

Antibody binding to the icosahedral arrangement of envelope proteins on the surface of flaviviruses can result in neutralization or enhancement of infection. We evaluated how many antibodies must bind to a given epitope on West Nile virus (WNV) to achieve neutralization. The most potent monoclonal antibodies (mAbs) block infection at concentrations that result in low occupancy of accessible sites on the virion, with neutralization occurring when as few as 30 of 180 envelope proteins are bound. In contrast, weakly neutralizing mAbs recognize fewer sites on the virion and require almost complete occupancy to inhibit WNV infection. For all mAbs studied, enhancement of infection is possible in cells bearing activating Fc-gamma receptors when the number of mAbs docked to the virion is not sufficient for neutralization. Thus, neutralization is best described by a model requiring "multiple hits" with the cumulative functional outcome determined by interplay between antibody affinity and epitope accessibility.

Antigenic cooperation among intrahost HCV variants organized into a complex network of cross-immunoreactivity

“…Fourth, Ab-mediated neutralization of viruses from the Flaviviridae family requires a stoichiometric Ab binding that exceeds a certain threshold (35)(36)(37). This threshold was estimated to be up to 30 Ab molecules per virion for West Nile virus (53,54) and is represented in our model with the relation u i,j = v K j,i between the probabilities u i,j and v j,i of neutralization and immune stimulation. The neutralization effectiveness of the Ab may be further hampered by dilution with low-affinity Abs resulting from boosting of different memory B cells by a viral variant or from polyclonal antigen-independent activation of naive B cells by HCV (55).…”

Genetic and Phenotypic Properties of Vero Cell-Adapted Japanese Encephalitis Virus SA14-14-2 Vaccine Strain Variants and a Recombinant Clone, Which Demonstrates Attenuation and Immunogenicity in Mice

“…47 The JEV SA14-14-2 RVPs expressing green fluorescent protein (GFP) were produced in HEK 293T cells, as described previously. 47,50,51 The RVP titers were determined by serial dilution of JEV RVP stocks and infecting Raji cells in 96-well plates. The RVP-infected cells were fixed with paraformaldehyde and the percentage of GFP expressing cells was determined by flow cytometry.…”

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

“…Previous studies have suggested that Abs can neutralize flaviviruses at a stoichiometric threshold of~30 bound Abs (14). Given a series of M uncorrelated states of the Ab-virus complex, and counting the number of states, k, where N bound < 30; the ratio k/M is a measure of infectivity, whereas (M -k)/ M provides a measure of neutralization; these measurements can be used to compute theoretical neutralization or infectivity curves.…”

“…This stoichiometric threshold of~30 Abs corresponds to the coating theory of neutralization (12,15,16), which postulates that a threshold density of Abs bound, as a function of virus surface area, is sufficient for neutralization and predicts that flaviviruses, with a surface area of~7000 nm 2 , should be neutralized by~20-50 Abs. Subsequent experiments have shown that subneutralizing Ab binding stoichiometries facilitate antibody-dependent enhancement of infection (14), underscoring the critical role of antibody binding stoichiometry on flavivirus immunity and pathogenicity.…”

“…The stochastic, heterogeneous nature of this process is even more pronounced at subsaturating Ab concentrations, where there are significant numbers of unbound binding sites. Given that the stoichiometric threshold for neutralization in flavivirus Abs can be achieved at concentrations far below saturation (14,22), characterizing the ensemble of Ab binding configurations at these concentrations is critical to capturing the microscopic details of neutralization.…”