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

The four dengue virus serotypes (DENV1-4) are the most prevalent arboviruses in humans and a major public health concern worldwide. Identification of the immune mechanisms that modulate DENV infection outcome is critical for development of a safe and effective vaccine. Neutralizing antibodies (nAbs) are considered an essential component of the protective response, yet measurement of their potency is primarily performed using a single cellular substrate and partially mature virions. This approach does not capture the full breadth of neutralizing activity and may lead to biased estimations of nAb potency and repertoire. Here, we evaluated the nAb response associated with protection against dengue cases using samples collected after one or more DENV infections but prior to a subsequent symptomatic versus inapparent DENV1, DENV2 or DENV3 infection from a long-standing pediatric cohort study in Nicaragua. We compared nAb titers in pre-inapparent and pre-symptomatic infection samples in Vero cells with or without DC-SIGN expression, and with either mature or partially mature virions. This method allowed us to measure the magnitude and characteristics of the nAb response associated with outcome and revealed that correlation of nAb titers with protection from symptomatic infection is dependent on the individual's prior DENV immune status as well as the subsequent infecting DENV serotype. Further, the nAb potency and the protective NT 50 cutoff was greatly impacted by virion maturation state and cell substrate. These results have important implications for determination of antibody correlates of protection for vaccines and natural infections.