Mechanism and Significance of Cell Type-Dependent Neutralization of Flaviviruses

Abstract

The production of neutralizing antibodies (NAbs) is a correlate of protection for many human vaccines, including currently licensed vaccines against flaviviruses. NAbs are typically measured using a plaque reduction neutralization test (PRNT). Despite its extensive use, parameters that impact the performance of the PRNT have not been investigated from a mechanistic perspective. The results of a recent phase IIb clinical trial of a tetravalent dengue virus (DENV) vaccine suggest that NAbs, as measured using a PRNT performed with Vero cells, do not correlate with protection. This surprising finding highlights the importance of understanding how well the PRNT captures the complexity of the NAb response to DENV. In this study, we demonstrated that the structural heterogeneity of flaviviruses arising from inefficient virion maturation impacts the results of neutralization assays in a cell type-dependent manner. Neutralization titers of several monoclonal antibodies were significantly reduced when assayed on Vero cells compared to Raji cells expressing DC-SIGNR. This pattern can be explained by differences in the efficiency with which partially mature flaviviruses attach to each cell type, rather than a differential capacity of antibody to block infection. Vero cells are poorly permissive to the fraction of virions that are most sensitive to neutralization. Analysis of sera from recipients of live-attenuated monovalent DENV vaccine candidates revealed a strong correlation between the sensitivity of serum antibodies to the maturation state of DENV and cell type-dependent patterns of neutralization. Cross-reactive patterns of neutralization may be underrepresented by the "gold-standard" PRNT that employs Vero cells. IMPORTANCECell type-dependent patterns of neutralization describe a differential capacity of antibodies to inhibit virus infection when assayed on multiple cellular substrates. In this study, we established a link between antibodies that neutralize infection in a cell type-dependent fashion and those sensitive to the maturation state of the flavivirus virion. We demonstrated that cell type-dependent neutralization reflects a differential capacity to measure neutralization of viruses that are incompletely mature. Partially mature virions that most efficiently bind maturation state-sensitive antibodies are poorly represented by assays typically used in support of flavivirus vaccine development. The selection of cellular substrate for neutralization assays may significantly impact evaluation of the neutralization potency of the polyclonal response. These data suggest that current assays do not adequately capture the full complexity of the neutralizing antibody response and may hinder the identification of correlates of protection following flavivirus vaccination.

Context-Dependent Cleavage of the Capsid Protein by the West Nile Virus Protease Modulates the Efficiency of Virus Assembly

“…To study mechanisms of flavivirus neutralization, our laboratory has produced RVPs by complementation of a WNV replicon (WNVrep) with structural genes derived from WNV, DENV, and the tick-borne flavivirus Langat (34,35,44). In particular, high-titer RVP stocks have been generated by complementation of WNVrep with the structural genes of DENV2 strain 16681 (34,58).…”

Deconstructing the Antiviral Neutralizing-Antibody Response: Implications for Vaccine Development and Immunity

“…At least two factors have the potential to modulate flavivirus epitope accessibility. While steric constraints limit antibody accessibility on the mature virion, inefficient maturation of flaviviruses results in the release of partially mature virions on which epitope accessibility may differ (41,42,44,205). Decreasing the efficiency of virion maturation results in an increase in sensitivity to neutralization by many antibodies (41).…”

“…Partially mature virions can be infectious, although the extent of prM cleavage required for infectivity is not known (reviewed in reference 39). The efficiency of virion maturation has the potential to impact virus binding, environmental conditions required to trigger membrane fusion, cellular tropism, and sensitivity to antibodymediated neutralization (40)(41)(42)(43)(44).…”

New insights into the immunopathology and control of dengue virus infection

“…In dengue virus, prM protein cleavage is not complete in all the virions, leaving a proportion of intermediate forms, where viral particles contain a varying amount of cleaved and uncleaved prM protein 11 , [12][13][14] . prM protein cleavage is more efficient in certain cell types, particularly primary human cells such as dendritic cells compared to virus produced in insect cells or tumour cell lines such as Vero 15,16 .…”