A prospective nested case-control study of Dengue in infants: rethinking and refining the antibody-dependent enhancement dengue hemorrhagic fever model

Abstract

Background: Dengue hemorrhagic fever (DHF) is the severe and life-threatening syndrome that can develop after infection with any one of the four dengue virus (DENV) serotypes. DHF occurs almost exclusively in individuals with secondary heterologous DENV infections and infants with primary DENV infections born to dengue immune mothers. The widely accepted explanation for the pathogenesis of DHF in these settings, particularly during infancy, is antibody-dependent enhancement (ADE) of DENV infection.

Methods and findings: We conducted a prospective nested case-control study of DENV infections during infancy. Clinical data and blood samples were collected from 4,441 mothers and infants in up to two pre-illness study visits, and surveillance was performed for symptomatic and inapparent DENV infections. Pre-illness plasma samples were used to measure the associations between maternally derived anti-DENV3 antibody-neutralizing and -enhancing capacities at the time of DENV3 infection and development of infant DHF. The study captured 60 infants with DENV infections across a wide spectrum of disease severity. DENV3 was the predominant serotype among the infants with symptomatic (35/40) and inapparent (15/20) DENV infections, and 59/60 infants had a primary DENV infection. The estimated in vitro anti-DENV3 neutralizing capacity at birth positively correlated with the age of symptomatic primary DENV3 illness in infants. At the time of symptomatic DENV3 infection, essentially all infants had low anti-DENV3 neutralizing activity (50% plaque reduction neutralizing titers [PRNT(50)] </=50) and measurable DENV3 ADE activity. The infants who developed DHF did not have significantly higher frequencies or levels of DENV3 ADE activity compared to symptomatic infants without DHF. A higher weight-for-age in the first 3 mo of life and at illness presentation was associated with a greater risk for DHF from a primary DENV infection during infancy.

Conclusions: This prospective nested case-control study of primarily DENV3 infections during infancy has shown that infants exhibit a full range of disease severity after primary DENV infections. The results support an initial in vivo protective role for maternally derived antibody, and suggest that a DENV3 PRNT(50) >50 is associated with protection from symptomatic DENV3 illness. We did not find a significant association between DENV3 ADE activity at illness onset and the development of DHF compared with less severe symptomatic illness. The results of this study should encourage rethinking or refinement of the current ADE pathogenesis model for infant DHF and stimulate new directions of research into mechanisms responsible for the development of DHF during infancy.

Trial registration: ClinicalTrials.gov NCT00377754.

Figure 1. Flowchart of study participation, October 2006–January 2008.

The ages at each study visit are presented as the median (range) in months. *, A subset of 250 infants who had completed study visits 1 and 2 were selected for a third study visit in November/December 2007. These infants did not have any previously reported febrile illnesses.

Figure 2. Age distribution of infants with symptomatic primary DENV infections.

Filled bars, hospitalized infants with DHF. Open bars, hospitalized and nonhospitalized infants without DHF.

Figure 3. World Health Organization (WHO) weight-for-age z-scores at study enrollment and acute illness in infants with primary DENV infections.

Closed circles, weight-for-age z-scores at illness presentation; open triangles, weight-for-age z-scores at study enrollment. Values are presented as the mean with 95% CIs. *, p = 0.04, weight-for-age z-scores at presentation in DHF infants versus symptomatic dengue, not DHF, infants. †, p = 0.03, weight-for-age z-scores at study enrollment in DHF infants versus symptomatic and inapparent dengue, not DHF, infants.

Figure 4. Estimated DENV3 neutralizing capacities at birth and illness onset in infants with symptomatic primary DENV3 infections.

(A) Log₁₀ transformed PRNT₅₀ to DENV3 in maternal plasma (estimate for birth time point) were positively correlated with infant age at onset of symptomatic primary DENV3 infection (n = 30). Mean (95% CI) of the Pearson correlation coefficient (r) is shown. The linear regression curve and 95% CI are shown as a solid line and dashed lines, respectively. (B) Endpoint DENV3 PRNT₅₀ at the time of symptomatic primary DENV3 infection were extrapolated from pre-illness plasma samples as described in the Results (n = 34). Closed squares, hospitalized infants with DHF; open triangles, hospitalized infants without DHF; open circles, nonhospitalized infants without DHF. The closed circles and error bars are the DENV3 PRNT₅₀ geometric mean titers (GMT) and 95% CI, respectively. There were no significant differences among the disease severity groups (p = 0.9).

Figure 5. ADE of DENV3 infection at illness onset in infants with symptomatic primary DENV3 infections.

The most proximal pre-illness plasma sample from each infant with symptomatic primary DENV3 infection (n = 34) was diluted to achieve the estimated neutralizing capacity at illness onset and used in the ADE assay, as described in the Methods. Values are log₁₀ transformed DENV3 genome eqs/ml in cell culture supernatants at 72 h, mean±standard deviation of individual plasma samples from three independent experiments. Mean DENV3 genome eqs/ml and 95% CI are shown for each of the disease severity groups (mean [95% CI]). Closed squares, hospitalized infants with DHF; open triangles, hospitalized infants without DHF; open circles, nonhospitalized infants without DHF. ^a p<0.01 compared to virus alone; ^b p<0.01 compared to flavivirus seronegative plasma control. There were no significant differences among the disease severity groups (see Results).

Figure 6. Early viremia levels and ADE activity at illness onset in infants with symptomatic primary DENV3 infections.

(A) Early viremia levels in acute-phase sera from infants with symptomatic primary DENV3 infections. Values are log₁₀ transformed DENV3 genome eqs/ml measured in single acute-phase serum samples collected between 1–3 d after illness onset. Closed squares, hospitalized infants with DHF; open triangles, hospitalized infants without DHF; open circles, nonhospitalized infants without DHF. Line represents the lower limit of quantitation (2.93 log₁₀ DENV3 genome eqs/ml). (B) Mean log₁₀ transformed DENV3 genome eqs/ml from ADE assay cell culture supernatants versus viremia levels in single acute-phase serum samples collected between 1–3 d after illness onset (n = 21). Mean [95% CI] of the Pearson correlation coefficient (r) is shown. The linear regression curve and 95% CI are shown as a solid line and dashed lines, respectively.