Fig. 5: RNA sequence analysis in blood, post-MVA-1 immunization.

a Line graphs showing the number of DEGs in both the groups at indicated time points, when compared to their expression on Day 0 of MVA-1 immunization. The cutoff criteria was defined as log₂fold change ≥1 or ≤−1 and p value ≤0.05. A multiple-test correction was performed with the Benjamini–Hochberg method and a false-discovery rate <0.05. The lines were color-coded, representing groups (Red: MVA-ID, Blue: MVA-IM, Purple: number of genes that are up/down-regulated in both the groups). b, c Enriched reactome pathways associated with DEGs at Day 1, post-MVA-1 immunization, using online tool network analyst (networkanalyst.ca)²⁵. The cutoff criteria used was p value <0.05 and FDR <0.1. P values are not adjusted for multiple comparisons. Bars were color-coded representing respective vaccine groups (MVA-ID – Red, MVA-IM – Blue), b pathway enrichment from upregulated DEGs, and c pathway enrichment from downregulated DEGs. d Longitudinal normalized gene expression of selected key genes along AIM2/IFI16 inflammasome pathway at Day 0, 1, 2, 4, and 7 post-MVA-1 immunizations. The bright color-coded lines represent the geometrical mean of the corresponding groups and light color-coded lines in the background represent individual animals in the corresponding groups. Asterisks denote statistically significant differences between the groups at a given time point, measured by Mann–Whitney rank-sum (two-tailed) test (AIM2: D1:***p = 0.0004, D2:*p = 0.02; IFI16: D1:**p = 0.003; CGAS: D1:*p = 0.01; IL1B: D2: **p = 0.0021; IL1RAP: D1:*p = 0.035; IFNAR1: D1:**p = 0.0057, D2:**p = 0.0044). e Correlation matrix with genes along AIM2/IFI16 inflammasome pathway as variables on the y-axis and circulating Tfh, Th1/Tfh ratio, frequencies of intermediate monocytes (IM), and frequencies of activated intermediate monocytes (86 + IM) as variables on the x-axis. The positive correlation between the variables was denoted by a gradient of red color and the negative correlation between the variables was denoted by a gradient of blue color. In all the correlations included in the matrix, r and p values were included in the respective matrix box. Asterisks denote statistical significance from a spearman correlation (two-sided)(AIM2 Vs IM *p = 0.017, 86 + IM*p = 0.033, cTfh*p = 0.027; IFI16 Vs cTfh*p = 0.035; cGAS Vs IM *p = 0.025, 86 + IM*p = 0.015, cTfh*p = 0.013; IL1B Vs cTfh *p = 0.026; IL1RAP Vs cTFh*p = 0.027, Th1/Tfh****p < 0.0001; IFNAR1 Vs IM*p = 0.011, 86 + IM**p = 0.006, cTfh*p = 0.031, Th1/Tfh****p < 0.0001). MVA-1: first MVA. Source data are provided as a Source Data file.