Early molecular correlates of adverse events following yellow fever vaccination

Abstract

The innate immune response shapes the development of adaptive immunity following infections and vaccination. However, it can also induce symptoms such as fever and myalgia, leading to the possibility that the molecular basis of immunogenicity and reactogenicity of vaccination are inseparably linked. To test this possibility, we used the yellow fever live-attenuated vaccine (YFLAV) as a model to study the molecular correlates of reactogenicity or adverse events (AEs). We analyzed the outcome of 68 adults who completed a YFLAV clinical trial, of which 43 (63.2%) reported systemic AEs. Through whole-genome profiling of blood collected before and after YFLAV dosing, we observed that activation of innate immune genes at day 1, but not day 3 after vaccination, was directly correlated with AEs. These findings contrast with the gene expression profile at day 3 that we and others have previously shown to be correlated with immunogenicity. We conclude that although the innate immune response is a double-edged sword, its expression that induces AEs is temporally distinct from that which engenders robust immunity. The use of genomic profiling thus provides molecular insights into the biology of AEs that potentially forms a basis for the development of safer vaccines.

Figure 1. Consort diagram of the study.

Subjects were vaccinated with yellow fever live-attenuated vaccine (YFLAV) with or without history of prior Japanese encephalitis (JE) vaccination. Subjects were followed up for development of adverse events (AEs) for 1 month. Each systemic AE was subcategorized by system organ class: CNS, musculoskeletal (MSK), respiratory (RESP), and gastrointestinal (GI). Blood was sampled for antibody levels, viremia, and gene expression at stipulated time points (see main text for details)

Figure 2. Delayed systemic AEs are not correlated with YF immunogenicity or vaccine viremia.

(A) Venn diagram showing number of subjects who reported only immediate adverse events (AEs) that occurred less than 24 hours after yellow fever live-attenuated vaccine (YFLAV) administration (n = 1), only delayed AEs that occurred more than 24 hours after vaccination (n = 26), and immediate AEs that fully resolved before new-onset delayed AEs (n = 16). (B) Histogram showing the number of subjects with reported AEs by day of onset after YF vaccination. (C) Box-and-whisker plot showing the day of onset of specific symptoms after YF vaccination (the line within the box indicates the median, the end of the box shows the 25th and 75th percentile, and ends of the whiskers are minimum and maximum). Red bars represent immediate AEs reported 24 hours or less after YF vaccination. Blue bars represent delayed AEs reported more than 24 hours after YF vaccination. Only events reported more than once are shown. n = number of events. (D) YF-neutralizing antibody titers at 1 month after vaccination in subjects with delayed AE (red) or without AE (blue) as measured by plaque neutralization reduction test (PRNT). Data are expressed as the PRNT titer that neutralized 50% of the viral inoculum (PRNT₅₀). (E) YFLAV RNA levels in peripheral blood measured by qPCR at days 3 and 7 after vaccination in subjects with delayed AEs or without AEs. In D and E, P values were obtained by 2-tailed Mann-Whitney test, and mean ± SEM is shown. Sample sizes are depicted in the figure. Dotted line depicts limit of detection.

Figure 3. Whole-genome expression reveals significant differences in innate immune signaling pathways in those with delayed AEs.

(A) Top 10 enriched gene sets identified by gene set enrichment analysis (GSEA) from venous blood microarray data in subjects with delayed adverse events (AEs) (n = 18) compared with subjects without AEs (n = 8) at day 1 after yellow fever (YF) vaccination, ranked by normalized enrichment scores (NES), with FDR q values less than 0.25. (B) Heatmap of the microarray data showing fold changes observed at day 1 versus day 0, for genes in the Toll-like receptor cascades, IFN, and interleukins (ILS) signaling. (C) Pearson’s correlation (r) of log-transformed fold change (day 1 versus day 0) of genes CSF2RB, IFITM3, MX2, IFIT3, IFIT1, and HCK, measured using nCounter assay. All 9 subjects shown here were independent of the data set from the microarray analysis. P values indicate significance of the slope. (D) Top 10 enriched gene sets from venous blood microarray data ranked by NES at day 3 after vaccination. Dotted line represents the cut-off FDR q value of 0.25.

Figure 4. Genes that are differentially expressed in vaccinees with MSK and CNS AEs.

The number of (A) upregulated gene sets, or (B) downregulated gene sets identified by GSEA (FDR q values < 0.25) from venous blood microarray data in subjects with delayed musculoskeletal (MSK) (n = 9) or CNS (n = 11) symptoms, at day 1 after vaccination. Overlap indicates the number of significantly enriched gene sets shared between subjects with MSK or CNS symptoms. (C) Top 10 enriched gene sets ranked by normalized enrichment scores (NES) associated with both MSK and CNS adverse event (AE) groups. Unique gene sets ranked by NES for subjects with (D and E) MSK and (F) CNS AEs. Heatmaps of genes in adenosine diphosphate (ADP) signaling, sphingolipid metabolism, anaphase-promoting complex/cyclosome (APC/C) regulation of the cell cycle, activation of prereplicative complex, signaling by stem cell factor (SCF) kit, and SEMA4D in semaphorin signaling for subjects with (G and H) MSK AEs and (I) CNS AEs.