4). Indeed analysis of the functional annotations of genes in the previously published single-gene level predictor Fulvestrant mouse of influenza vaccine response [16] did not include terms related to B-cell biology or proliferation (Supporting Information Table 4). Thus a gene-set based approach can identify networks of predictive genes and biological responses not otherwise detected by conventional, single-gene level approaches. The simplest explanation for the predictive power of gene sets containing proliferation and immunoglobulin genes in individuals with high HAI response to vaccination is that it represents the increased frequency

of proliferating B cells in postvaccination samples. To test this hypothesis, we compared the frequency of antibody-producing B cells in the peripheral blood of vaccinated subjects at day 7 postvaccination with the enrichment score for the top scoring proliferation

and immunoglobulin clusters. We BEZ235 in vivo found that the enrichment score of both gene sets was correlated significantly with the frequency of IgG antibody spot-forming cells (Fig. 5) but not IgM or IgA (data not shown). This is most consistent with the interpretation that enrichment of these gene sets was caused by increased representation of proliferating plasmablasts in PBMC samples from vaccinated subjects with high antibody responses. In this study, we applied a gene set enrichment-based approach to developing predictors of vaccine outcome and showed that enrichment of signatures corresponding to proliferating

B cells accurately segregate vaccine responders to TIV with an Anidulafungin (LY303366) AUC of 0.94 in a training set and an accuracy of 88% in an independent clinical trial. Our approach uses the differential enrichment of sets of biologically related genes rather than single genes as predictive features. This allows subtle biological changes manifest over networks of genes to be captured in a way that conventional gene expression predictors do not because they focus on small numbers of highly differentially expressed genes. Rapid expansion of plasmablasts following influenza vaccination has been previously observed [20], and it is intuitive that the magnitude of the plasmablast response would correlate with the humoral response to vaccination. However even at their peak, proliferating plasmablasts represent only a tiny fraction of the cells present in the PBMC samples analyzed by microarray in this study. As result, although detailed analysis of gene expression data from influenza vaccinated subjects had revealed that genes related to B-cell biology were related to the HAI response, the magnitude of change in these B-cell genes was not sufficiently large for them to be incorporated into the previously published gene expression predictor [16].