Cytokine Profile of Children Hospitalized with Virologically-Confirmed Dengue during Two Phase III Vaccine Efficacy Trials

<div><p>Background</p><p>Two large-scale efficacy studies with the recombinant yellow fever-17D–dengue virus, live-attenuated, tetravalent dengue vaccine (CYD-TDV) candidate undertaken in Asia (NCT01373281) and Latin America (NCT01374516) demonstrated significant protection against dengue disease during two years’ active surveillance (active phase). Long-term follow up of participants for breakthrough disease leading to hospitalization is currently ongoing (hospital phase).</p><p>Methodology/Principal findings</p><p>We assessed the cytokine profile in acute sera from selected participants hospitalized (including during the active phase) up to the beginning of the second year of long-term follow up for both studies. The serum concentrations of 38 cytokines were measured in duplicate using the Milliplex Human Cytokine MAGNETIC BEAD Premixed 38 Plex commercial kit (Millipore, Billerica, MA, USA). Partial least squares discriminant analyses did not reveal any difference in the overall cytokine profile of CYD-TDV and placebo recipients hospitalized for breakthrough dengue regardless of stratification used. In addition, there was no difference in the cytokine profile for breakthrough dengue among those aged <9 years versus those aged ≥ 9 years.</p><p>Conclusions/Significance</p><p>These exploratory findings show that CYD-TDV does not induce a particular immune profile versus placebo, corroborating the clinical profile observed.</p></div

Differentially expressed cytokines in acute samples as compared to baseline.

<p>Median cytokine values in children at baseline (1 month post-dose 3) (N = 40) and during the acute phase of dengue illness (N = 33) irrespective of treatment group. Upper error bars represent the 75% (Q3) percentile; the lower error bars represent the 25% (Q1) percentile. Cytokines with a significant difference (p<0.05) are marked with an asterisk. Values below the lower limit of quantification were replaced by half of the limit of quantification.</p

Significant differences in IL-10 and MCP-1 levels between groups among <9 year old children.

<p>Box and whisker plots of circulating (A) IL-10 and (B) MCP-1 levels during the acute phase of dengue illness in CYD-TDV and placebo recipients in children <9 years of age (CYD-TDV N = 43, Placebo N = 27) and ≥9 years of age (CYD-TDV N = 56, Placebo N = 81). The boundary of the box closest to zero indicates the 25th percentile, the line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. Values below the lower limit of quantification were replaced by half of the limit of quantification. Significant differences are indicated by *p < 0.05.</p

IL-1Ra and MCP-1 levels are significantly different between groups in severe cases.

<p>Box and whisker plots of circulating IL-1Ra (left panel) and MCP-1 (right panel) levels during the acute phase of dengue illness in CYD-TDV and placebo recipients: (A) irrespective of severity (CYD-TDV N = 99, Placebo N = 108); (B) in severe cases (CYD-TDV N = 24, Placebo N = 28); and (C) in non-severe cases (CYD-TDV N = 74, Placebo N = 80). The boundary of the box closest to zero indicates the 25th percentile, the line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. The individual points represent outliers. Values below the lower limit of quantification were replaced by half of the limit of quantification. Significant differences are indicated by *p < 0.05 and **p ≤ 0.01.</p

Cytokines with significant differences between children aged < 9 years or ≥ 9 years in the CYD-TDV or placebo groups.

<p>Data shown are geometric mean (GM), median, min and max values and the p-values calculated with the Wilcoxon Test (if data were not distributed normally) or Student Test (if data were normally distributed). Significant differences are highlighted in bold.</p

Growth of RGΔM2-2 versus parental virus in Vero cells.

<p>(A) Vero cells were infected at an MOI of 0.001 and incubated at 37°C. Samples harvested at the times indicated were titrated on Vero cells.</p

IM boost immunization in NHP.

<p>(A) AGMs were infected as indicated. (B) Complement-dependent serum neutralizing titers, PRNT60. (C) Protective efficacy as determined from BAL samples after challenge.</p

Attenuation and immunogenicity in cotton rats.

<p>(A) Groups of cotton rats were infected as indicated. (B) The degree of attenuation as amount of virus present in the lungs of animals in (A) was determined. ****p < 0.0001 by unpaired t test. (C) Immunogenicity study in cotton rats. Animals were immunized as indicated. (D) Serum neutralization titers were determined. (E) Protective efficacy as amount of challenge virus present in the lungs of immunized animals in (C).</p

IN vs IM immunization in NHP.

<p>(A) AGMs were infected as indicated. (B) Serum F-binding antibodies were determined by ELISA. (C) Complement-dependent serum neutralizing titers were measure by PRNT60. (D) Protective efficacy determined as virus titers from BAL samples after challenge.</p