Project Summary Biological sex has profound effects on the effectiveness of vaccines that protect against pathogens of pandemic potential, including influenza A viruses. Following influenza vaccination, females of reproductive ages (18-49 years) produce greater antiviral antibody responses than age-matched males, which correlates with greater estradiol (E2) concentrations in females. How estrogens drive greater B cell activation and antibody production after vaccination is not known. Estrogen receptor signaling in B cells might alter metabolic signaling to cause sex differences in vaccine-induced immunity. Activation of mTOR, in particular, and a switch towards β-oxidation as a primary energy source has been identified as indispensable for B cell proliferation, differentiation, and antibody production. Prior studies have demonstrated that females show greater preference for the use β- oxidation for energy production when under high energy demand. Thus, I hypothesized that differential activation of mTOR and downstream utilization of lipids for β-oxidation may be a potential mediator of sex differences in vaccine-induced immunity. My preliminary data show that after H1N1 vaccination in mice, adult females have greater activation of mTOR and mTOR-related proteins in splenic B cells than age-matched males. I hypothesize that greater mTOR activation in B cells from females is partially regulated by E2 through an ERα-mTOR feedback loop. My central hypothesis is that sex steroid signaling causes distinct metabolic signatures in B cells and that differential regulation of mTOR is the central mechanism governing sex-specific differences in vaccine-mediated humoral immunity. In Aim 1, I will test whether manipulation of mTOR and selected downstream proteins using small molecule agonist and antagonist can reverse sex differences in immune responses using a mouse model of influenza vaccination. I will use immunofluorescence assays and seahorse analysis of mitochondrial respiration to dissect sex differences in metabolic activation in B cells following vaccination, which will be correlated with measures of protective immunity. In Aim 2, I will examine the effects of sex chromosome complement and sex steroid signaling on both mTOR activation and metabolic activation of B cells. Through these experiments, I seek to define the cellular mechanisms mediating sex differences in vaccine-induced immunity. These results have significant translational application. As a fellow, the research that I have outlined is crucial to extend my training in animal models, viral immunology, sex-based biology, and biomedical research to refine my expertise in using metabolomics-based approaches to gain mechanistic insight into biological phenomena.