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

Comparison of post-electroporation growth kinetics of yellow fever viruses (YFV) and deletion mutants in BHK-21 cells.

<p>A) YFV 17D (▪ solid line), YFV 17D GFP (▪ dashed line), and YFV 17D Cherry (○ solid line); B) YFV 17D 5′ ΔE (▴), YFV 17D 3′ ΔE (•), YFV 17D 5′ΔE Cherry (Δ), and YFV 17D 3′ ΔE GFP (○). Titers expressed as plaque forming units/mL.</p

Schematic representation of flavivirus and alphavirus recombinant crosses.

<p>A) Yellow fever virus 17D deletion mutant replicon recombinant cross, B) chikungunya virus deletion mutant replicon/defective helper recombinant cross.</p

Schematic representation of parental replicon/defective helper recombinant cross (CHIK-LR-3′Δ-Replicon and CHIK-LR-5Δ′-Helper) along with sequence topology analysis of the cross-over region and resulting recombinant topology of clonally isolated recombinant genomes.

<p>Genes not necessarily shown to scale. aa-amino acid, E-envelope glycoprotein, ns-nonstructural, nt-nucleotide, LR-LaReunion, and UTR-untranslated region, @LR location of sequence deletion.</p

Northern blot analysis of chikungunya virus (CHIKV) intra-genic recombinant isolates.

<p>Blot was hybridized with CHIKV-5′UTR BioProbe. Lane 1: CHIKV RNA ladder (size markers indicate CHIKV RNAs of full length genomic, replicon, full length structural gene helper, and capsid only structural helper respectively), Lane 2: CHIKV-LR-3′Δ-Replicon only electroporation, Lane 3: CHIKV-LR-5′Δ-Helper only electroporation, Lanes 4 and 8: CHIKV-LR-3′Δ-Replicon/ CHIKV-LR-5′Δ-Helper co-electroporations, and Lanes 5, 6, 7, 9, and 10: clonal recombinant isolates.</p

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

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

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

Immunization accelerates virus clearance.

<p>Mice were immunized subcutaneously with RV16 VP0 protein plus IFA/CpG or with IFA/CpG adjuvant only and infected intranasally with RV1B or sham PBS-challenged. RV RNA in lung tissue was measured by Taqman qPCR. n = 4 mice/group. n.d., not detected.</p