Figure 4. Gene expression responses to HSV529 that associate with sex differ significantly between subjects previously naive or exposed to herpes simplex virus (HSV), and the interferon (IFN) response associated with HSV naive women correlates inversely with HSV2 neutralizing antibody titers.

(A) Changes in expression of all genes between days 0 and 1 were compared between men and women separately for the three HSV serogroups of subjects based on prior exposure to HSV. For each gene those changes with sex were then used to compute a difference between the HSV naive group and either HSV exposed group (x-axis). These differences were used to rank genes for hypergeometric enrichment analysis of blood transcription modules (BTMs), and all pathways with significant positive enrichment in the HSV naive group are shown (FDR adjusted p < 0.05), with enrichment score and significance indicated by plotted color and size, respectively. (B) Comparison of sex differences between HSV naive and HSV1⁺/HSV2⁻ subjects identified seven gene sets significantly enriched in the IFN antiviral sensing module. For these seven gene sets, responses of the 33 different leading-edge genes in rows, for all 45 vaccine recipients in columns are shown with the change in expression at day 1 compared to day 0 with z-scores normalized within each gene set, where red indicates above and blue indicates below mean values. (C) Principal component analysis was performed using expression of the 33 leading-edge genes from panel B, at all nine timepoints with RNA-seq data. The first principal component is plotted for all 60 subjects colored by sex and separated by HSV serostatus for vaccine recipients, or with all serogroups combined for placebo recipients. (D) For HSV naive women that were vaccinated, variation in day 1 IFN responses was quantified by gene set variation analysis (GSVA) of the antiviral IFN signature (y-axis), and correlated with HSV2 neutralizing antibody titers observed at day 30 or 60 (x-axis). Pearson coefficient and significance values are shown for the linear correlation in blue, with 95% confidence interval shaded red.

Figure 4—figure supplement 1. Enrichment of interferon (IFN) module gene sets in association with sex and prior exposure in day 1 transcriptional responses to HSV529.

Seven gene sets from the IFN and antiviral sensing module were enriched when comparing sex differences between herpes simplex virus (HSV) naive and HSV1⁺/HSV2⁻ subjects in day 1 transcriptional responses (Figure 4A). For these enrichments, plots show the fraction of genes included from each set (y-axis) when all genes tested are ranked by decreasing difference between HSV naive compared to HSV1⁺/HSV2⁻ subjects for greater expression in women compared to men (x-axis), with bars on the x-axis showing the distribution of members of these gene sets as black marks.

Figure 4—figure supplement 2. Multiple interferon (IFN)-related gene sets enriched in day 1 responses by herpes simplex virus (HSV) naive women correlate inversely with HSV2 neutralizing antibody titers.

Multiple gene sets from the IFN and antiviral sensing module were highly enriched when comparing sex differences between HSV naive and exposed subjects in day 1 transcriptional responses (Figure 4A). For the gene set with most significant enrichment, antiviral IFN signature, variation between individuals was analyzed for correlation with HSV2 neutralizing antibody titers observed for HSV naive women on day 30 (A) and day 60 (B). Here, the next five gene sets based on significance of enrichment in Figure 4A are also analyzed for correlation with HSV2 neutralizing antibody titers. Plots are as described for Figure 4D, and show Pearson coefficients ranging from −0.68 to −0.53, with significance values from p = 0.04 to p = 0.14.