The single-cell epigenomic and transcriptional landscape of immunity to influenza vaccination

Abstract

Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we mapped the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza induced persistently diminished H3K27ac in monocytes and myeloid dendritic cells (mDCs), which was associated with impaired cytokine responses to Toll-like receptor stimulation. Single-cell ATAC-seq analysis revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs. This was associated with elevated expression of antiviral genes and heightened resistance to the unrelated Zika and Dengue viruses. These results demonstrate that vaccination stimulates persistent epigenomic remodeling of the innate immune system and reveal AS03's potential as an epigenetic adjuvant.

Trial registration: ClinicalTrials.gov NCT01910519 NCT02154061.

Keywords: adjuvant; antiviral immunity; epigenome; influenza; innate memory; monocyte; single cell; systems biology; trained immunity; vaccines.

Conflict of interest statement

Declaration of interests B.P. serves on the External Immunology Network of GSK and on the Scientific Advisory Board of Medicago and Boehringer Ingelheim. R.v.d.M. is an employee of the GSK group of companies and holds shares in the GSK group of companies. B.P. and F.W. are inventors on a provisional patent application (no. S20-530 63/138,163 [STAN-1821PRV]) submitted by the Board of Trustees of the Leland Stanford Junior University, Stanford, CA, that covers the use of “Modulating The Epigenome With Adjuvants To Stimulate Broad And Persistent Innate Immunity Against Diverse Viruses.” The remaining authors declare no competing interests.

Copyright © 2021 Elsevier Inc. All rights reserved.

Figures

Figure 1 -. TIV alters the global histone modification profile of immune cells.

(A) Study overview. (B) UMAP was used to create a dimensionality-reduced representation of the global histone mark profiles of all immune cell subset. (C) UMAP was used to visualize epigenomic profiles at the sample level. (D, E) The effect size of vaccine-induced changes to the global histone modification profiles at day 30 vs day 0 were calculated. D) Top-10 most significantly increased and reduced histone modifications. E) Heatmap showing histone modification changes in innate immune cells. Only changes with an FDR

Figure 2 -. TIV-induced histone modification changes correlate with cytokine production.

(A) Schematic overview of experiment. (B) Heatmap showing the relative change in cytokine levels at indicated time points compared to day 0. (C) Cytokine levels before (d0) and after (d30) vaccination for each investigated subject. Wilcoxon signed rank test was used for hypothesis testing. * p

Figure 3 -. TIV induces reduced chromatin accessibility in immune response genes and AP-1 controlled regions.

(A) Schematic overview of the experiment. (B) Differentially accessible chromatin regions (DARs) at day 30 vs day 0 were identified using DESeq2. Pval +10kbp. (D) Network representation of gene set enrichment analysis of DARs in C monos using the Reactome database. Only significantly enriched terms (p

Figure 4 -. Heterogeneity within monocyte population drives TIV induced epigenomic changes.

(A) Schematic overview of the experiment. (B) UMAP representation of scATAC-seq landscape after pre-processing and QC filtering. (C) Heatmap showing differences in chromatin accessibility at indicated time points for the top5 transcription factors per subset. (D) UMAP representation of epigenomic subclusters within the classical monocyte population. (E) Density plot showing the relative contribution of different epigenomic subclusters to the total monocyte population at a given vaccine time point. (F) Variability in TF accessibility within the monocyte population. Value indicates range of accessibility values in all single monocytes. (G) Heatmap showing differences in chromatin accessibility between monocyte subclusters subset. (H) UMAP representation of monocyte subclusters showing differences in AP-1 accessibility. (I) UMAP representation of monocyte subclusters showing difference in accessibility at Hotspot module 2,3 gene loci. (J) Enrichment analysis of genes associated with loci in Hotspot module 2,3. (K) UMAP representation of the transcriptional landscape of single monocytes. Color indicates expression of genes associated with Hotspot modules 2,3.

Figure 5 -. H5N1+AS03 induces repressive epigenomic state akin to TIV.

(A) Schematic overview of experiment. (B) UMAP representation of EpiTOF landscape. (C) Histone modification levels in classical monocytes at day 0 vs day 42 as measured by EpiTOF. (D) Cytokine levels in supernatant of TLR-stimulated PBMCs at day 0 and day 42 after vaccination with H5N1+AS03. (C, D) Wilcox signed rank test; *p

Figure 6 -. H5N1+AS03 induces epigenomic state of enhanced antiviral immunity.

(A) Heatmap showing the change in chromatin accessibility at day 42 vs day 0 for the top5 transcription factors per subset. Color indicates the difference in accessibility, grey fields indicate non-significant changes (fdr > 0.05). (B) Line graph showing the difference in transcription factor (TF) accessibility during vaccination. Each line represents a separate TF within the indicated family. (C) Volcano plot showing the change in gene expression for IRF/STAT TF genes. (D) Scatter plot showing chromatin accessibility values for IRF1 (x-axis) and FOS (y-axis) in single cells. Indicated statistics are based on Pearson correlation. (E) MA plot showing the average accessibility and log2(FC) accessibility for genomic regions containing an IRF1 binding motif. Red color indicates regions with significantly changed accessibility (P +/−0.03) in vaccine-induced gene expression at the booster vaccination compared to the prime vaccination (y-axis, Day22day21 vs Day1day0). Chi-square test was used to determine whether both variables were related. (K) Bubble plot showing enrichment results using the Encode TF target gene database. Color indicates the origin of the analyzed genes in J). See also Figure S7

Figure 7 -. H1N1+AS03 induces enhanced resistance to in-vitro infection with heterologous viruses.

(A) Schematic overview of the experiment. (B) Boxplot showing viral titers in Dengue-, Zika-, and mock-infected samples. (C) Line graph showing the viral growth curve for Dengue virus (red) and Zika virus (blue). Dots and lines indicate average, error bars indicate standard error of mean. n > 21 samples (D) Log2 fold change in viral titers relative to day 0 before vaccination. Wilcoxon signed rank test was used to compare changes within group; ** p