Dengue virus IgG and neutralizing antibody titers measured with standard and mature viruses are protective

Abstract

The standard dengue virus (DENV) neutralization assay inconsistently predicts dengue protection. We compare how IgG ELISA, envelope domain III (EDIII), or non-structural protein 1 (NS1) binding antibodies, and titers from plaque reduction neutralization tests (PRNTs) using standard and mature viruses are associated with dengue. The ELISA measures IgG antibodies that bind to inactivated DENV1-4. The EDIII and NS1 assays measure binding antibodies, and the PRNTs measure neutralizing antibodies to each specific DENV serotype. Healthy children (n = 1206) in Cebu, Philippines were followed for 5 years. ELISA IgG≥3 was associated with reduced dengue probability relative to naïve children (3% vs. 10%, p = 0.007). Serotype-specific antibodies binding EDIII or NS1 had no association with dengue risk. Standard virus PRNT geometric mean titers (GMT) > 200 and mature GMT > 100 were associated with reduced dengue disease overall (p < 0.01). High DENV2 and DENV3 titers against either standard or mature viruses protected against the matched serotype (p < 0.01). While 43% of dengue cases had standard virus PRNT titers>100, only 2% of cases had mature virus PRNT titers>100 (p < 0.001), indicating a lower, more consistent threshold for protection. These assays may serve as correlates of protection.

Fig. 1. Probability of dengue by baseline antibody measure.

The probability of disease is shown separately for baseline IgG ELISA (A–C) and baseline GMT measured by PRNT with standard reference (D–F) or mature clinical (G–I) strains. The probability of each disease outcome was modeled as a function of baseline antibody titer on both discrete and continuous scales. All continuous relationships were modeled using Poisson generalized additive models (continuous black line) with 95% confidence intervals (gray shading). Point estimates and confidence intervals correspond to predicted probabilities from logistic regression models. P-values were generated from the logistic regression models using two-sided tests comparing the dengue risk for each antibody bin with that in the naïve group. Only statistically significant p-values are shown. The dashed line indicates the disease probability in naïve individuals. DENV IgG ELISA was measured on all participants (n = 1,206), and GMTs were measured on random subsets using standard reference (n = 823) and mature clinical strains (n = 293). Inverse probability weighting was used to adjust for GMT subset size. All models were adjusted for age, sex, and enrollment site, and model estimates are shown for the average study participant (female, age 10, from Bogo). N for each row indicates the number of dengue cases and non-cases included in each analysis. Source data are provided as a Source Data file.

Fig. 2. Fitted linear regression line with 95% confidence intervals between GMTs as measured using PRNTs to standard reference and mature clinical strains.

GMTs are plotted on a log scale, and the correlation coefficient (R) was calculated using Pearson’s test with 95% confidence intervals. Only those with no dengue (open circles) or dengue (black diamond) and GMTs measured by both the standard and mature PRNTs are included (n = 256). The dashed red line indicates GMT = 100 by mature assay. Source data are provided as a Source Data file.

Fig. 3. Comparison of inapparent infection and symptomatic dengue to non-cases by baseline GMT and serotype-specific nAbs.

The odd ratios and 95% confidence intervals of inapparent infection (circles) and symptomatic dengue (triangle) by baseline GMT and serotype-specific nAbs measured with PRNT to standard reference (A) or mature clinical (B) strains. N = number of individuals evaluated with and without symptomatic or inapparent infection in each model. All analyses were logistic regression models adjusted for age, sex, and enrollment site, and inverse probability weighting was used to adjust for GMT and serotype-specific nAb subset sizes. Source data are provided as a Source Data file.

Fig. 4. The impact of standard and mature nAbs on cases caused by DENV2 and DENV3.

The odd ratios and 95% confidence intervals of experiencing a case of DENV2 (diamonds) or DENV3 (inverted triangles) by baseline titer were measured with standard reference (A) or mature clinical (B) strains. N = number of individuals with and without DENV2 or DENV3 disease evaluated in each model. All analyses were logistic regression models adjusted for age, sex, and enrollment site, and inverse probability weighting was used to adjust for GMT and nAb subset sizes. Individual-level data and distributions (medians in red, boxes show interquartile ranges, whiskers extend to show the full range of the data) of baseline titers for monotypic and multitypic immune participants, stratified by those who experienced a case caused by each serotype or did not experience a case, shown separately for standard reference (C) or mature clinical (D) neutralization assay results. A Welch two-sample, two-sided t test was used to compare differences in titer means between cases and non-cases for each analysis, with p-values reported. The number of individuals who experienced a case with that serotype or did not have a case are shown as the denominator under each boxplot, while those in each category with titers above the assay threshold (shown as a dotted line, 1:20 for the mature and 1:40 for the standard assay) are indicated in the numerator. Source data are provided as a Source Data file.

Fig. 5. Comparison of nAb titers by maturation state and virus strain.

Geometric mean (A) and serotype-specific (B–E) titers of each strain and maturation state combination. Horizontal lines connecting points track measurements from the same individual. Boxes indicate the interquartile range and bold lines indicate the median. N = 36, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; two-sided paired t test with Bonferroni correction. Source data are provided as a Source Data file.