Pre-infection antiviral innate immunity contributes to sex differences in SARS-CoV-2 infection

Abstract

Male sex is a major risk factor for SARS-CoV-2 infection severity. To understand the basis for this sex difference, we studied SARS-CoV-2 infection in a young adult cohort of United States Marine recruits. Among 2,641 male and 244 female unvaccinated and seronegative recruits studied longitudinally, SARS-CoV-2 infections occurred in 1,033 males and 137 females. We identified sex differences in symptoms, viral load, blood transcriptome, RNA splicing, and proteomic signatures. Females had higher pre-infection expression of antiviral interferon-stimulated gene (ISG) programs. Causal mediation analysis implicated ISG differences in number of symptoms, levels of ISGs, and differential splicing of CD45 lymphocyte phosphatase during infection. Our results indicate that the antiviral innate immunity set point causally contributes to sex differences in response to SARS-CoV-2 infection. A record of this paper's transparent peer review process is included in the supplemental information.

Keywords: COVID-19; SARS-CoV-2; alternative splicing; causal mediation; differential expression; interferon-stimulated genes; sex differences; viral infection.

Figure 1

Analysis of sex divergence in clinical measurements (A) The fraction of each reported symptom for infected females (n = 137) and males (n = 1,033). Statistical significance was tested using the chi² independence test. Statistical significance: ^∗∗∗p < 0.001, ^∗∗p <0.01, ^∗p < 0.05. (B) Initial viral load, measured by probe CT and averaged across the S, N, and ORF1ab genes, between females and males. Statistical significance was tested by the Kolmogorov-Smirnov test. (C) Clinical data and biospecimens were collected longitudinally for each subject. Samples were labeled “baseline” if they tested negative for SARS-CoV-2, the first PCR-positive sample from each infected individual was labeled “first,” and any subsequent PCR-positive sample was labeled “mid.” Subjects were followed throughout infection, and any PCR-negative samples taken after infection were labeled “post.”

Figure 2

Multi-omic integrative analysis of the sex-dependent molecular responses to SARS-CoV-2 infection (A) The number of sex-dependent molecular variations upon SARS-CoV-2 infections discovered using gene expression, alternative splicing, and proteomics. (B) Sex divergence of gene expression and alternative splicing visualized in the virus response module of a tissue-specific functional network. Each node represents a gene, with its size proportional to its connectedness in the network. Clusters shown here represent the immune response genes from males (left) and females (right) with differential expression (top) and differential alternative splicing events (bottom) during SARS-CoV-2 infection. This network is constructed by probabilistically integrating a compendium of thousands of public omics datasets to provide functional maps of biological processes and pathways. Projecting each sex-specific signature onto this network reveals significant enrichment of the immune response module in both gene expression and alternative splicing. (C) Box-and-whisker plot of exon inclusion changes of PTPRC exon 6 upon SARS-CoV-2 infection in females (n = 55) and males (n = 255) throughout the infection course. The white circles represent the mean, the center line shows the median, and the upper and lower edges of the boxes represent the upper and lower quartiles, respectively. The differential splicing of PTPRC will alter the protein product of PTPRC, known as CD45, in females. (D) ISGs are significantly enriched in both males and females during COVID-19 infection but significantly more so in females as shown by the higher normalized enrichment score (NES) from gene set enrichment analysis. Colored bars below the x axis represent ISG locations within the list of all differentially expressed genes, ordered by fold change from baseline samples. p value calculated using the Mann-Whitney U test.

Figure 3

Higher baseline ISG expression observed in females in CHARM and in reference datasets (A) The majority of ISGs are significantly different between sexes in baseline samples. A subset of ISGs that remain statistically significant after cell type correction is shown (see Table S3 for full list), colored by the sex with higher expression. An orange marker represents genes with higher expression in females and blue represents genes with higher expression among males. Point size is proportional to the log fold change of expression between sexes. (B–D) (B) ISGs are more highly expressed in females in CHARM baseline samples and two independent studies, (C) Depression Genes and Networks (DGN) (n = 922), and (D) Netherlands Study of Depression and Anxiety (NESDA) (n = 1,848). Vertical bars represent ISG locations among gene lists sorted by log fold change between females and males. Fold enrichment is a measure of the observed versus expected number of ISGs present at each gene rank. p values are computed from rank-based enrichment.

Figure 4

Mediation analysis of the relationship between sex, ISGs, symptoms, and CD45 splice variant pre- and post-infection (A) Causal mediation analysis was performed to test the mediation effects between pre-infection ISGs to post-infection molecular and clinical responses, as well as post-infection molecular responses to clinical responses. A stacked bar chart summarizes the full mediation results, with stars marking statistically significant mediation results (average causal mediation effect [ACME] p < 0.05). (B) Pre-infection molecular and clinical measurements were separated from post-infection measurements. Selected significant results of mediation analysis were presented as a directed graph. Edges represent mediation or suppression effects.