Dengue in Vietnamese infants--results of infection-enhancement assays correlate with age-related disease epidemiology, and cellular immune responses correlate with disease severity

Abstract

The pathogenesis of severe dengue is not well understood. Maternally derived subneutralizing levels of dengue virus-reactive IgG are postulated to be a critical risk factor for severe dengue during infancy. In this study, we found that, in healthy Vietnamese infants, there was a strong temporal association between the Fc-dependent, dengue virus infection-enhancing activity of neat plasma and the age-related epidemiology of severe dengue. We then postulated that disease severity in infants with primary infections would be associated with a robust immune response, possibly as a consequence of higher viral burdens in vivo. Accordingly, in infants hospitalized with acute dengue, the activation phenotype of peripheral-blood NK cells and CD8+ and CD4+ T cells correlated with overall disease severity, but HLA-A*1101-restricted NS3(133-142)-specific CD8+ T cells were not measurable until early convalescence. Plasma levels of cytokines/chemokines were generally higher in infants with dengue shock syndrome. Collectively, these data support a model of dengue pathogenesis in infants whereby antibody-dependent enhancement of infection explains the age-related case epidemiology and could account for antigen-driven immune activation and its association with disease severity. These results also highlight potential risks in the use of live attenuated dengue vaccines in infants in countries where dengue is endemic.

Figure 1

Dengue virus serotype 2 (DENV2) infection enhancement in diluted maternal plasma. A, n-Fold enhancement of infection of K562 cells by DENV2 (unbroken lines) after preincubation with dilutions of maternal plasma (n = 27), compared with plasma control samples (dashed lines) from flavivirus-naive individuals (n = 2), which did not enhance DENV2 infection at any dilution. B, Correlation, for each mother-infant pair, between the titer of maternal plasma that resulted in maximum DENV2-infection enhancement in K562 cells (from panel A) and the age of the infant when the pair presented to hospital with dengue caused by DENV2. There was a negative correlation between antibody-dependent enhancement of infection and the age of the infant (Spearman’s r = -0.39; P = .04).

Figure 2

A, Age-related case prevalence of dengue hemorrhagic fever (DHF) in infants (n = 73) enrolled in a prospective study at Children’s Hospitals 1 and 2 in Ho Chi Minh City, Vietnam, between November 2004 and March 2006. The median age of the infants was 7 months. B, Serial neat plasma samples collected, at birth and at 6, 9 and 12 months of age, from 42 healthy Vietnamese infants born to dengue-immune mothers, and then mixed with DENV2 and cultured with K562 cells for 3 days. Shown is the n-fold increase (median, interquartile, and maximum/minimum range) in DENV2-infected K562 cells in infants’ plasma samples at 6, 9 and 12 months, compared with that in the infant’s own cord-plasma sample. Plasma from 6-month-old infants provided significantly more enhancement than did plasma collected at any other time point (P < .001, by paired t test). C, Individual neat plasma samples collected, at birth and at 3, 6, 9 and 12 months of age, from 150 unrelated healthy Vietnamese infants (n = 30 at each time point), and then mixed with DENV2 and cultured with K562 cells for 3 days. Shown is the n-fold increase (median, interquartile, and maximum/minimum range) in DENV2-infected K562 cells in infants’ plasma samples, compared with that in flavivirus-naive control samples. Plasma from 6-month-old infants provided significantly more enhancement than did plasma collected at any other time point (P < .001, by Mann-Whitney test). D, n-Fold infection enhancement obtained when DENV2 and K562 cells were cultured in the presence of both plasma from 10 6-month-old infants and either monoclonal antibody (mAb) to CD32 (FcγIIa) or 1 of 2 different control mAbs (i.e., anti-FASL and anti-interferon [IFN]-γ). Anti-CD32 mAb significantly abrogated infection enhancement, compared with the control mAbs or no mAb (P < .001, by Mann-Whitney test).

Figure 3

Cellular activation during acute dengue. Shown are the median, interquartile, and 95th-percentile percentage ranges of (A) CD8⁺CD69⁺ T cells, (B) CD3^-CD16⁺CD56⁺CD69⁺ NK cells, and (C) CD4⁺CD69⁺ T cells, as a proportion of lymphocytes in peripheral blood from healthy infants (n = 6) and from infants who had either dengue hemorrhagic fever grade II (DHF II) (n = 15) or III (DHF III) (n = 2) or other febrile illness (OFI) (n = 8), at study enrollment (shaded boxes) and at discharge (unshaded boxes). At the time of study enrollment, infants with acute DHF II had significantly higher percentages of CD8⁺CD69⁺ T cells (P = .006, by Mann-Whitney test), CD16⁺CD56⁺CD69⁺ NK cells (P = .001, by Mann-Whitney test), and CD4⁺CD69⁺ T cells (P = .04, by Mann-Whitney test) than did infants with OFI.

Figure 4

A, Detection of NS3_133-142-specific CD8⁺ T cells in infants with dengue. Shown is the frequency, at different time points, of NS3_133-142-specific CD8⁺ T cells in plasma samples of HLA-A*11-positive infants with acute dengue hemorrhagic fever, compared with that at defervescence (denoted as day 0). B, Flow-cytometry plot showing detection, by pooled tetramer staining, of NS3_133-142-specific CD8⁺ T cells in whole blood.

Figure 5

Correlations between virus-host and host-host factors in acute dengue in infants. Shown is the relationship, in the same plasma sample, between (A) plasma viral load and interferon-inducible protein (IP)-10 (n = 72) (Spearman’s r = 0.48; P = .0001), (B) plasma concentrations of RANTES and aspartate aminotransferase (AST) (n = 62) (Spearman’s r = -0.38; P = .002), and (C) plasma concentrations of monokine induced by interferon-γ (MIG) and AST (n = 66) (r = 0.46; P = .0001).