The association of neutralizing antibodies with protection against symptomatic dengue virus infection varies by serotype, prior infection history, and assay condition.

The four dengue virus serotypes (DENV1-4) are the most prevalent arboviruses in humans and a major public health concern worldwide. Understanding immune mechanisms that modulate DENV infection outcome is critical for epidemic preparedness and development of a safe and effective vaccine. Neutralizing antibodies (nAbs) are an essential component of the protective response, yet their measurement often relies on a single cellular substrate and partially mature virions, which do not capture the full breadth of neutralizing activity and may lead to biased estimations of nAb potency. Here, we investigated the characteristics of nAbs associated with protection against dengue cases using samples collected after one or more DENV infections but prior to subsequent symptomatic or inapparent DENV1, DENV2, or DENV3 infections from a long- standing pediatric cohort study in Nicaragua. By assessing nAb responses using Vero cells with or without the attachment factor DC-SIGN and with mature or partially mature virions, we found that nAb potency and the protective NT 50 cutoff were greatly influenced by cell substrate and virion maturation state. Additionally, the correlation between nAb titer and protection from disease depended on an individual's prior infection history and the subsequent infecting DENV serotype. Finally, we uncovered variations in nAbs composition that contributed to protection from symptomatic DENV infection differently after primary and secondary prior infection. These findings have important implications for identifying antibody correlates of protection in the context of vaccines and natural infections.