Transcriptomic profiling facilitates classification of response to influenza challenge

Abstract

Despite increases in vaccination coverage, reductions in influenza-related mortality have not been observed. Better vaccines are therefore required and influenza challenge studies can be used to test the efficacy of new vaccines. However, this requires the accurate post-challenge classification of subjects by outcome, which is limited in current methods that use artificial thresholds to assign 'symptomatic' and 'asymptomatic' phenotypes. We present data from an influenza challenge study in which 22 healthy adults (11 vaccinated) were inoculated with H3N2 influenza (A/Wisconsin/67/2005). We generated genome-wide gene expression data from peripheral blood taken immediately before the challenge and at 12, 24 and 48 h post-challenge. Variation in symptomatic scoring was found amongst those with laboratory confirmed influenza. By combining the dynamic transcriptomic data with the clinical parameters this variability can be reduced. We identified four subjects with severe laboratory confirmed influenza that show differential gene expression in 1103 probes 48 h post-challenge compared to the remaining subjects. We have further reduced this profile to six genes (CCL2, SEPT4, LAMP3, RTP4, MT1G and OAS3) that can be used to define these subjects. We have used this gene set to predict symptomatic infection from an independent study. This analysis gives further insight into host-pathogen interactions during influenza infection. However, the major potential value is in the clinical trial setting by providing a more quantitative method to better classify symptomatic individuals post influenza challenge.

Key message: Differential gene expression signatures are seen following influenza challenge. Expression of six predictive genes can classify response to influenza challenge. The genomic influenza response classification replicates in an independent dataset.

Figures

Fig. 1

Clinical observations and classification of study subjects. Each individual is assigned a unique letter code (A-V). Vaccinated individuals are shown in red. Individuals are defined as having no symptoms, mild or moderate/severe based on the summed self-reported symptoms collected over 6 days. Positive shedding indicating viral shedding was detected on at least 1 day after challenge. Seroconversion was determined by HI titre using serum samples obtained at 26-days post-challenge (Online Resource, Table S1). LCI is defined as mild or moderate/severe symptoms and viral shedding (shown by grey shaded area)

Fig. 2

Principal components and hierarchical clustering plot. a The first three principal components are plotted with the proportion of variance explained by each component. The yellow oval highlights the samples taken from the four subjects with moderate/severe LCI 48 h post-challenge. N none or mild LCI, S moderate/severe LCI, V vaccinee, C control. b The hierarchical tree illustrates the relationship between clusters of samples. The height of the branches indicates the strength of the separation. The red oval highlights the samples taken from the four subjects with moderate/severe LCI 48 h post-challenge

Fig. 3

Heatmaps for differentially expressed probes. The top 100 most significant differentially expressed probes between the four moderate/severe LCI, and the remaining samples at 48 h post-challenge were used to generate heatmaps of gene expression. a Moderate/severe LCI subjects. b Mild LCI subjects. Individuals D and G were vaccinated; others are unvaccinated

Fig. 4

Pathway analysis of differential expression in LCI. The differentially expressed probes between the four moderate/severe LCI samples 48 h post-challenge and the remaining samples were used to determine pathway enrichment with IPA. a Pathways with significant enrichment based on Benjamini-Hochberg (B-H) multiple testing corrected p value <0.01 are shown. The blue bars show the −log(p value). Differentially expressed genes are plotted for each enriched pathway (red upregulated in moderate/severe LCI individuals, green downregulated) with numbers of enriched genes shown in brackets below x-axis labels. b The most significantly enriched pathway, interferon signalling, is shown with enriched genes shaded in colour