Protection against symptomatic dengue infection by neutralizing antibodies varies by infection history and infecting serotype

Abstract

Dengue viruses (DENV1-4) are the most prevalent arboviruses in humans and a major public health concern. Understanding immune mechanisms that modulate DENV infection outcome is critical for vaccine development. Neutralizing antibodies (nAbs) are an essential component of the protective immune response, yet their measurement often relies on a single cellular substrate and partially mature virions, which does not capture the full breadth of neutralizing activity and may lead to biased estimations of nAb potency. Here, we analyze 125 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 DC-SIGN and with mature or partially mature virions, we find that nAb potency and the protective NT50 cutoff are greatly influenced by cell substrate and virion maturation state. Additionally, the correlation between nAb titer and protection from disease depends on prior infection history and infecting serotype. Finally, we uncover variations in nAb composition that contribute to protection from symptomatic infection differently after primary and secondary prior infection. These findings have important implications for identifying antibody correlates of protection for vaccines and natural infections.

Fig. 1. Participant serological sampling schematic.

Plasma samples from 125 DENV-immune participants were collected before a subsequent DENV1, 2, or 3 infection and were stratified into two separate groups according to disease outcome: Pre-inapparent (purple, n = 63) and pre-symptomatic (yellow, n = 62).

Fig. 2. Cross-reactive nAb titers across assay conditions.

a Schematic of the different neutralization assay conditions. b Neutralizing titers (NT₅₀) of 125 participants measured against the incoming DENV serotype using virions of different maturation state and different cell substrate. Median NT₅₀ values are indicated below each boxplot. Shown are median NT₅₀ (middle line), 25th to 75th percentile (box), and 5th to 95th percentile (whiskers) as well as the raw data (points). Diamond indicates mean NT₅₀. Asterisks indicate Benjamini–Hochberg adjusted p-values for paired Wilcoxon test. p-values: ns >0.05; *<0.05; **<0.01; ***<0.001; ****<0.0001. Part. Mature partially mature. Source Data are provided as a Source Data file.

Fig. 3. Cross-reactive nAb titers of pre-inapparent and pre-symptomatic DENV infection samples.

a NT₅₀ of pre-inapparent (purple, n = 63) and pre-symptomatic (yellow, n = 62) DENV infection groups measured against the incoming serotype across FRNT assay conditions, and b NT₅₀ of the same pre-inapparent and pre-symptomatic DENV infection group stratified by participants’ infection history at the time of sample collection. Shown are median NT₅₀ (middle line), 25th to 75th percentile (box), and 5th to 95th percentile (whiskers) as well as the raw data (points). Diamond indicates mean NT₅₀. Asterisks indicate Benjamini–Hochberg adjusted p-values for Wilcoxon test (two-sided). p-values: ns >0.05; *<0.05; **<0.01; ***<0.001; ****<0.0001. Post-1° post-primary infection, Post-2° post-secondary infection, pre-inapp. pre-inapparent DENV infection group (purple), pre-sympt. pre-symptomatic DENV infection group (yellow). Source Data are provided as a Source Data file.

Fig. 4. Comparison of nAb responses between pre-inapparent post-primary and post-secondary versus pre-symptomatic post-secondary infection samples.

a Schematic of the groups compared. Sample size indicated for each group. b Magnitude of the nAb response using different virion maturation states and cell substrates. Shown are median NT₅₀ (middle line), 25th to 75th percentile (box), and 5th to 95th percentile (whiskers) as well as the raw data (points). Diamond indicates mean NT₅₀. c Impact of change in assay condition on the neutralization titers of participants. Asterisks indicate Benjamini–Hochberg adjusted p-values for two-sided Wilcoxon test (b) and paired Wilcoxon test (c). p-values: ns >0.05; *<0.05; **<0.01; ***<0.001; ****<0.0001. Post-1° post-primary infection, Post-2° post-secondary infection, pre-inapp. pre-inapparent DENV infection group (purple), pre-sympt. pre-symptomatic DENV infection group (yellow), Part. Mature partially mature. Source Data are provided as a Source Data file.

Fig. 5. Cross-reactive nAb titers of pre-inapparent and pre-symptomatic DENV infection participants stratified by incoming serotype and infection history.

a NT₅₀ of pre-inapparent and pre-symptomatic DENV infection groups across FRNT assay conditions stratified by incoming serotype (total pre-DENV1 n = 19, pre-DENV2 n = 60, pre-DENV3 n = 46). b NT₅₀ of pre-DENV2 infection participants stratified by infection history (Post-1° n = 29, Post-2° n = 31). Shown are median NT₅₀ (middle line), 25th to 75th percentile (box), and 5th to 95th percentile (whiskers) as well as the raw data (points). Diamond indicates mean NT₅₀. Asterisks indicate Benjamini–Hochberg adjusted p-values for Wilcoxon test (two-sided). p-values: ns >0.05; *<0.05; **<0.01; ***<0.001; ****<0.0001. Post-1° post-primary infection, Post-2° post-secondary infection, pre-inapp. pre-inapparent DENV infection group (purple), pre-sympt. pre-symptomatic DENV infection group (yellow). Source Data are provided as a Source Data file.

Fig. 6. Serotype-specific characteristics of the nAb response associated with DENV infection outcome.

Impact of change in assay condition on the nAb response of pre-inapparent and pre-symptomatic infection participants from the a post-primary pre-DENV1 infection group (n = 19), b post-primary pre-DENV2 infection group (n = 29), c post-primary pre-DENV3 infection group (n = 26), and d post-secondary pre-DENV2 infection group (n = 31). Asterisks indicate Benjamini–Hochberg adjusted p-values for paired Wilcoxon tests (two-sided). p-values: ns >0.05; *<0.05; **<0.01; ***<0.001; ****<0.0001. Post-1° post-primary infection, Post-2° post-secondary infection, pre-inapp. pre-inapparent DENV infection group (purple), pre-sympt. pre-symptomatic DENV infection group (yellow), Part. Mature partially mature. Source Data are provided as a Source Data file.