Impact of circulating SARS-CoV-2 variants on mRNA vaccine-induced immunity.

The emergence of SARS-CoV-2 variants with mutations in major neutralizing antibody-binding sites can affect humoral immunity induced by infection or vaccination1-6. Here we analysed the development of anti-SARS-CoV-2 antibody and T cell responses in individuals who were previously infected (recovered) or uninfected (naive) and received mRNA vaccines to SARS-CoV-2. While individuals who were previously infected sustained higher antibody titres than individuals who were uninfected post-vaccination, the latter reached comparable levels of neutralization responses to the ancestral strain after the second vaccine dose. T cell activation markers measured upon spike or nucleocapsid peptide in vitro stimulation showed a progressive increase after vaccination. Comprehensive analysis of plasma neutralization using 16 authentic isolates of distinct locally circulating SARS-CoV-2 variants revealed a range of reduction in the neutralization capacity associated with specific mutations in the spike gene: lineages with E484K and N501Y/T (for example, B.1.351 and P.1) had the greatest reduction, followed by lineages with L452R (for example, B.1.617.2). While both groups retained neutralization capacity against all variants, plasma from individuals who were previously infected and vaccinated displayed overall better neutralization capacity than plasma from individuals who were uninfected and also received two vaccine doses, pointing to vaccine boosters as a relevant future strategy to alleviate the effect of emerging variants on antibody neutralizing activity.

Longitudinal analyses reveal immunological misfiring in severe COVID-19.

Recent studies have provided insights into the pathogenesis of coronavirus disease 2019 (COVID-19)1-4. However, the longitudinal immunological correlates of disease outcome remain unclear. Here we serially analysed immune responses in 113 patients with moderate or severe COVID-19. Immune profiling revealed an overall increase in innate cell lineages, with a concomitant reduction in T cell number. An early elevation in cytokine levels was associated with worse disease outcomes. Following an early increase in cytokines, patients with moderate COVID-19 displayed a progressive reduction in type 1 (antiviral) and type 3 (antifungal) responses. By contrast, patients with severe COVID-19 maintained these elevated responses throughout the course of the disease. Moreover, severe COVID-19 was accompanied by an increase in multiple type 2 (anti-helminths) effectors, including interleukin-5 (IL-5), IL-13, immunoglobulin E and eosinophils. Unsupervised clustering analysis identified four immune signatures, representing growth factors (A), type-2/3 cytokines (B), mixed type-1/2/3 cytokines (C), and chemokines (D) that correlated with three distinct disease trajectories. The immune profiles of patients who recovered from moderate COVID-19 were enriched in tissue reparative growth factor signature A, whereas the profiles of those with who developed severe disease had elevated levels of all four signatures. Thus, we have identified a maladapted immune response profile associated with severe COVID-19 and poor clinical outcome, as well as early immune signatures that correlate with divergent disease trajectories.

No evidence of fetal defects or anti-syncytin-1 antibody induction following COVID-19 mRNA vaccination.

The impact of Coronavirus Disease 2019 (COVID-19) mRNA vaccination on pregnancy and fertility has become a major topic of public interest. We investigated 2 of the most widely propagated claims to determine (1) whether COVID-19 mRNA vaccination of mice during early pregnancy is associated with an increased incidence of birth defects or growth abnormalities; and (2) whether COVID-19 mRNA-vaccinated human volunteers exhibit elevated levels of antibodies to the human placental protein syncytin-1. Using a mouse model, we found that intramuscular COVID-19 mRNA vaccination during early pregnancy at gestational age E7.5 did not lead to differences in fetal size by crown-rump length or weight at term, nor did we observe any gross birth defects. In contrast, injection of the TLR3 agonist and double-stranded RNA mimic polyinosinic-polycytidylic acid, or poly(I:C), impacted growth in utero leading to reduced fetal size. No overt maternal illness following either vaccination or poly(I:C) exposure was observed. We also found that term fetuses from these murine pregnancies vaccinated prior to the formation of the definitive placenta exhibit high circulating levels of anti-spike and anti-receptor-binding domain (anti-RBD) antibodies to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) consistent with maternal antibody status, indicating transplacental transfer in the later stages of pregnancy after early immunization. Finally, we did not detect increased levels of circulating anti-syncytin-1 antibodies in a cohort of COVID-19 vaccinated adults compared to unvaccinated adults by ELISA. Our findings contradict popular claims associating COVID-19 mRNA vaccination with infertility and adverse neonatal outcomes.

Plasma collagen neoepitopes are associated with multiorgan disease in the ACCESS and GRADS sarcoidosis cohorts.

INTRODUCTION: The pathogenesis of sarcoidosis involves tissue remodelling mediated by the accumulation of abnormal extracellular matrix, which is partly the result of an imbalance in collagen synthesis, cross-linking and degradation. During this process, collagen fragments or neoepitopes, are released into the circulation. The significance of these circulating collagen neoepitopes in sarcoidosis remains unknown. METHODS: We employed plasma samples from patients with sarcoidosis enrolled in A Case Control Etiologic Study of Sarcoidosis (ACCESS) and Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS), and healthy control patients recruited from the Yale community. Plasma concentrations of type III and VI collagen degradation (C3M and C6M) and formation (PRO-C3 and PRO-C6) were quantified via neoepitope-specific competitive ELISA, and statistical associations were sought with clinical phenotypes. RESULTS: Relative to healthy controls, the plasma of both sarcoidosis cohorts was enriched for C3M and C6M, irrespective of corticosteroid use and disease duration. While circulating collagen neoepitopes were independent of Scadding stage, there was a significant association between multiorgan disease and PRO-C3, PRO-C6 and C3M in the ACCESS cohort; PRO-C3 and C6M displayed this property in GRADS. These findings were unrelated to plasma levels of interleukin-4 (IL-4), IL-5, IL-6, IL-9, IL-10 and IL-13. Moreover, PRO-C3 was associated with dermatological disease in both cohorts. DISCUSSION: In two well-characterised sarcoidosis cohorts, we discovered that the plasma is enriched for neoepitopes of collagen degradation (C3M and C6M). In multiorgan disease, there was an association with circulating neoepitopes of type III formation (PRO-C3), perhaps mediated by dermatological sarcoidosis. Further investigation in this arena has the potential to foster new insights into the pathogenic mechanisms of this complex disease.

Combining Cellular Immunology With RNAseq to Identify Novel Chlamydia T-Cell Subset Signatures.

Chlamydia trachomatis serovars A-L cause important diseases of the eyes and reproductive tract by infecting epithelium lining those organs. A major hurdle for vaccine trials is finding a surrogate biomarker for protective immunity. Investigational data argues for T-cell biomarker(s) reflecting mucosal adaption, cytokine polarization, B-cell help, antibacterial effector mechanisms, or some combination thereof. A human investigation and 2 mouse studies link IL-13 to protection from infection/immunopathology. We performed RNAseq on T cells resident in spleens and genital tracts of naturally immune mice. CD4 signatures were consistent with helper function that differed by site including a genital tract-specific Fgl2 signal. The genital tract CD8 signature featured IL-10 and promotion of healing/scarring with a unique transcription of granzyme A. The RNAseq data was used to refine previously published CD4γ13 and CD8γ13 transcriptomes derived from protective T-cell clones, potentially identifying practicable T-cell subset signatures for assessing Chlamydia vaccine candidates.

Seasonal Variability and Shared Molecular Signatures of Inactivated Influenza Vaccination in Young and Older Adults.

The seasonal influenza vaccine is an important public health tool but is only effective in a subset of individuals. The identification of molecular signatures provides a mechanism to understand the drivers of vaccine-induced immunity. Most previously reported molecular signatures of human influenza vaccination were derived from a single age group or season, ignoring the effects of immunosenescence or vaccine composition. Thus, it remains unclear how immune signatures of vaccine response change with age across multiple seasons. In this study we profile the transcriptional landscape of young and older adults over five consecutive vaccination seasons to identify shared signatures of vaccine response as well as marked seasonal differences. Along with substantial variability in vaccine-induced signatures across seasons, we uncovered a common transcriptional signature 28 days postvaccination in both young and older adults. However, gene expression patterns associated with vaccine-induced Ab responses were distinct in young and older adults; for example, increased expression of killer cell lectin-like receptor B1 (KLRB1; CD161) 28 days postvaccination positively and negatively predicted vaccine-induced Ab responses in young and older adults, respectively. These findings contribute new insights for developing more effective influenza vaccines, particularly in older adults.

Dissecting alterations in human CD8+ T cells with aging by high-dimensional single cell mass cytometry.

We investigated the effect of aging on the multi-dimensional characteristics and heterogeneity of human peripheral CD8+ T cells defined by the expression of a set of molecules at the single cell level using the recently developed mass cytometry or Cytometry by Time-Of-Flight (CyTOF) and computational algorithms. CD8+ T cells of young and older adults had differential expression of molecules, especially those related to cell activation and migration, permitting the clustering of young and older adults through an unbiased approach. The changes in the expression of individual molecules were collectively reflected in the altered high-dimensional profiles of CD8+ T cells in older adults as visualized by the dimensionality reduction analysis tools principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE). A combination of PhenoGraph clustering and t-SNE analysis revealed heterogeneous subsets of CD8+ T cells that altered with aging. Furthermore, intermolecular quantitative relationships in CD8+ T cells appeared to change with age as determined by the computational algorithm conditional-Density Resampled Estimate of Mutual Information (DREMI). The results of our study showed that heterogeneity, multidimensional characteristics, and intermolecular quantitative relationships in human CD8+ T cells altered with age, distinctively clustering young and older adults through an unbiased approach.

Multiple network-constrained regressions expand insights into influenza vaccination responses.

MOTIVATION: Systems immunology leverages recent technological advancements that enable broad profiling of the immune system to better understand the response to infection and vaccination, as well as the dysregulation that occurs in disease. An increasingly common approach to gain insights from these large-scale profiling experiments involves the application of statistical learning methods to predict disease states or the immune response to perturbations. However, the goal of many systems studies is not to maximize accuracy, but rather to gain biological insights. The predictors identified using current approaches can be biologically uninterpretable or present only one of many equally predictive models, leading to a narrow understanding of the underlying biology. RESULTS: Here we show that incorporating prior biological knowledge within a logistic modeling framework by using network-level constraints on transcriptional profiling data significantly improves interpretability. Moreover, incorporating different types of biological knowledge produces models that highlight distinct aspects of the underlying biology, while maintaining predictive accuracy. We propose a new framework, Logistic Multiple Network-constrained Regression (LogMiNeR), and apply it to understand the mechanisms underlying differential responses to influenza vaccination. Although standard logistic regression approaches were predictive, they were minimally interpretable. Incorporating prior knowledge using LogMiNeR led to models that were equally predictive yet highly interpretable. In this context, B cell-specific genes and mTOR signaling were associated with an effective vaccination response in young adults. Overall, our results demonstrate a new paradigm for analyzing high-dimensional immune profiling data in which multiple networks encoding prior knowledge are incorporated to improve model interpretability. AVAILABILITY AND IMPLEMENTATION: The R source code described in this article is publicly available at https://bitbucket.org/kleinstein/logminer . CONTACT: steven.kleinstein@yale.edu or stefan.avey@yale.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DNA Methylation Regulates the Differential Expression of CX3CR1 on Human IL-7Rαlow and IL-7Rαhigh Effector Memory CD8+ T Cells with Distinct Migratory Capacities to the Fractalkine.

DNA methylation is an epigenetic mechanism that modulates gene expression in mammalian cells including T cells. Memory T cells are heterogeneous populations. Human effector memory (EM) CD8(+) T cells in peripheral blood contain two cell subsets with distinct traits that express low and high levels of the IL-7Rα. However, epigenetic mechanisms involved in defining such cellular traits are largely unknown. In this study, we use genome-wide DNA methylation and individual gene expression to show the possible role of DNA methylation in conferring distinct traits of chemotaxis and inflammatory responses in human IL-7Rα(low) and IL-7Rα(high) EM CD8(+) T cells. In particular, IL-7Rα(low) EM CD8(+) T cells had increased expression of CX3CR1 along with decreased DNA methylation in the CX3CR1 gene promoter compared with IL-7Rα(high) EM CD8(+) T cells. Altering the DNA methylation status of the CX3CR1 gene promoter changed its activity and gene expression. IL-7Rα(low) EM CD8(+) T cells had an increased migratory capacity to the CX3CR1 ligand fractalkine compared with IL-7Rα(high) EM CD8(+) T cells, suggesting an important biological outcome of the differential expression of CX3CR1. Moreover, IL-7Rα(low) EM CD8(+) T cells induced fractalkine expression on endothelial cells by producing IFN-γ and TNF-α, forming an autocrine amplification loop. Overall, our study shows the role of DNA methylation in generating unique cellular traits in human IL-7Rα(low) and IL-7Rα(high) EM CD8(+) T cells, including differential expression of CX3CR1, as well as potential biological implications of this differential expression.

Prolonged proinflammatory cytokine production in monocytes modulated by interleukin 10 after influenza vaccination in older adults.

We evaluated in vivo innate immune responses in monocyte populations from 67 young (aged 21-30 years) and older (aged ≥65 years) adults before and after influenza vaccination. CD14(+)CD16(+) inflammatory monocytes were induced after vaccination in both young and older adults. In classical CD14(+)CD16(-) and inflammatory monocytes, production of tumor necrosis factor α and interleukin 6, as measured by intracellular staining, was strongly induced after vaccination. Cytokine production was strongly associated with influenza vaccine antibody response; the highest levels were found as late as day 28 after vaccination in young subjects and were substantially diminished in older subjects. Notably, levels of the anti-inflammatory cytokine interleukin 10 (IL-10) were markedly elevated in monocytes from older subjects before and after vaccination. In purified monocytes, we found age-associated elevation in phosphorylated signal transducer and activator of transcription-3, and decreased serine 359 phosphorylation of the negative IL-10 regulator dual-specificity phosphatase 1. These findings for the first time implicate dysregulated IL-10 production in impaired vaccine responses in older adults.

Human monocytes have increased IFN-γ-mediated IL-15 production with age alongside altered IFN-γ receptor signaling.

IL-15 is involved in regulating host defense and inflammation. Monocytes produce the biologically active cell surface IL-15 in response to IFN-γ. Although aging can alter the immune system, little is known about whether and how aging affects IFN-γ-mediated IL-15 production in human monocytes. We showed that monocytes of healthy older adults (age ≥ 65) had increased cell surface IL-15 expression in response to IFN-γ compared to those of healthy young adults (age ≤ 40). This finding stems in part from increased IFN-γ receptor (R)1/2 expression on monocytes in older adults, leading to enhanced STAT1 activation and interferon regulatory factor 1 synthesis with increased IL15 gene expression. Our study suggests that with aging the IFN-γ-mediated IL-15 production pathway in human monocytes is uncompromised, but rather augmented, and could be considered as a therapeutic target point to modulate host defense and inflammation in older adults.

An altered relationship of influenza vaccine-specific IgG responses with T cell immunity occurs with aging in humans.

Alterations in T cell immunity occur with aging. Influenza causes significant morbidity and mortality in the elderly. We investigated the relationship of serum IgG responses with hemagglutinin inhibition (HI) antibody titers and the frequency of distinct T cell subsets in young and elderly people who received the inactivated influenza vaccine. Influenza vaccine-specific IgG responses correlated with the increase of HI antibody titers and the frequency of CD4(+) T cells producing IFN-γ and IL-17 in young, but not elderly, people. Also, only in young people, such IgG responses correlated with the frequency of memory T cells, especially central memory cells, CD45RA(-) effector memory CD8(+) T cells and IL-7 receptor alpha high effector memory CD8(+) T cells with potent survival and proliferative capacity. These findings suggest that aging alters the association of influenza-vaccine specific IgG responses with HI antibody titers, cytokine-producing capacity and proportions of memory T cells in humans.

High-throughput single-cell profiling of B cell responses following inactivated influenza vaccination in young and older adults.

Seasonal influenza contributes to a substantial disease burden, resulting in approximately 10 million hospital visits and 50 thousand deaths in a typical year in the United States. 70 - 85% of the mortality occurs in people over the age of 65. Influenza vaccination is the best protection against the virus, but it is less effective for the elderly, which may be in part due to differences in the quantity or type of B cells induced by vaccination. To investigate this possibility, we sorted pre- and post-vaccination peripheral blood B cells from three young and three older adults with strong antibody responses to the inactivated influenza vaccine and employed single-cell technology to simultaneously profile the gene expression and the B cell receptor (BCR) of the B cells. Prior to vaccination, we observed a higher somatic hypermutation frequency and a higher abundance of activated B cells in older adults than in young adults. Following vaccination, young adults mounted a more clonal response than older adults. The expanded clones included a mix of plasmablasts, activated B cells, and resting memory B cells in both age groups, with a decreased proportion of plasmablasts in older adults. Differential abundance analysis identified additional vaccine-responsive cells that were not part of expanded clones, especially in older adults. We observed broadly consistent gene expression changes in vaccine-responsive plasmablasts and greater heterogeneity among activated B cells between age groups. These quantitative and qualitative differences in the B cells provide insights into age-related changes in influenza vaccination response.

The Plasma Cell Infiltrate Populating the Muscle Tissue of Patients with Inclusion Body Myositis Features Distinct B Cell Receptor Repertoire Properties.

Inclusion body myositis (IBM) is an autoimmune and degenerative disorder of skeletal muscle. The B cell infiltrates in IBM muscle tissue are predominantly fully differentiated Ab-secreting plasma cells, with scarce naive or memory B cells. The role of this infiltrate in the disease pathology is not well understood. To better define the humoral response in IBM, we used adaptive immune receptor repertoire sequencing, of human-derived specimens, to generate large BCR repertoire libraries from IBM muscle biopsies and compared them to those generated from dermatomyositis, polymyositis, and circulating CD27+ memory B cells, derived from healthy controls and Ab-secreting cells collected following vaccination. The repertoire properties of the IBM infiltrate included the following: clones that equaled or exceeded the highly clonal vaccine-associated Ab-secreting cell repertoire in size; reduced somatic mutation selection pressure in the CDRs and framework regions; and usage of class-switched IgG and IgA isotypes, with a minor population of IgM-expressing cells. The IBM IgM-expressing population revealed unique features, including an elevated somatic mutation frequency and distinct CDR3 physicochemical properties. These findings demonstrate that some of IBM muscle BCR repertoire characteristics are distinct from dermatomyositis and polymyositis and circulating Ag-experienced subsets, suggesting that it may form through selection by disease-specific Ags.

Dectin-1 stimulation promotes a distinct inflammatory signature in the setting of HIV-infection and aging.

Dectin-1 is an innate immune receptor that recognizes and binds ß-1, 3/1, 6 glucans on fungi. We evaluated Dectin-1 function in myeloid cells in a cohort of HIV-positive and HIV-negative young and older adults. Stimulation of monocytes with ß-D-glucans induced a pro-inflammatory phenotype in monocytes of HIV-infected individuals that was characterized by increased levels of IL-12, TNF-α, and IL-6, with some age-associated cytokine increases also noted. Dendritic cells showed a striking HIV-associated increase in IFN-α production. These increases in cytokine production paralleled increases in Dectin-1 surface expression in both monocytes and dendritic cells that were noted with both HIV and aging. Differential gene expression analysis showed that HIV-positive older adults had a distinct gene signature compared to other cohorts characterized by a robust TNF-α and coagulation response (increased at baseline), a persistent IFN-α and IFN-γ response, and an activated dendritic cell signature/M1 macrophage signature upon Dectin-1 stimulation. Dectin-1 stimulation induced a strong upregulation of MTORC1 signaling in all cohorts, although increased in the HIV-Older cohort (stimulation and baseline). Overall, our study demonstrates that the HIV Aging population has a distinct immune signature in response to Dectin-1 stimulation. This signature may contribute to the pro-inflammatory environment that is associated with HIV and aging.

Increasing sensitivity of antibody-antigen interactions using photo-cross-linking.

Understanding antibody-antigen interactions in a polyclonal immune response in humans and animal models is critical for rational vaccine design. Current approaches typically characterize antibodies that are functionally relevant or highly abundant. Here, we use photo-cross-linking and single-particle electron microscopy to increase antibody detection and unveil epitopes of low-affinity and low-abundance antibodies, leading to a broader structural characterization of polyclonal immune responses. We employed this approach across three different viral glycoproteins and showed increased sensitivity of detection relative to currently used methods. Results were most noticeable in early and late time points of a polyclonal immune response. Additionally, the use of photo-cross-linking revealed intermediate antibody binding states and demonstrated a distinctive way to study antibody binding mechanisms. This technique can be used to structurally characterize the landscape of a polyclonal immune response of patients in vaccination or post-infection studies at early time points, allowing for rapid iterative design of vaccine immunogens.

Platelet response to influenza vaccination reflects effects of aging.

Platelets are uniquely positioned as mediators of not only hemostasis but also innate immunity. However, how age and geriatric conditions such as frailty influence platelet function during an immune response remains unclear. We assessed the platelet transcriptome at baseline and following influenza vaccination in Younger (age 21-35) and Older (age ≥65) adults (including community-dwelling individuals who were largely non-frail and skilled nursing facility (SNF)-resident adults who nearly all met criteria for frailty). Prior to vaccination, we observed an age-associated increase in the expression of platelet activation and mitochondrial RNAs and decrease in RNAs encoding proteins mediating translation. Age-associated differences were also identified in post-vaccination response trajectories over 28 days. Using tensor decomposition analysis, we found increasing RNA expression of genes in platelet activation pathways in young participants, but decreasing levels in (SNF)-resident adults. Translation RNA trajectories were inversely correlated with these activation pathways. Enhanced platelet activation was found in community-dwelling older adults at the protein level, compared to young individuals both prior to and post-vaccination; whereas SNF residents showed decreased platelet activation compared to community-dwelling older adults that could reflect the influence of decreased translation RNA expression. Our results reveal alterations in the platelet transcriptome and activation responses that may contribute to age-associated chronic inflammation and the increased incidence of thrombotic and pro-inflammatory diseases in older adults.

PD-1highCXCR5-CD4+ peripheral helper T cells promote CXCR3+ plasmablasts in human acute viral infection.

T cell-B cell interaction is the key immune response to protect the host from severe viral infection. However, how T cells support B cells to exert protective humoral immunity in humans is not well understood. Here, we use COVID-19 as a model of acute viral infections and analyze CD4+ T cell subsets associated with plasmablast expansion and clinical outcome. Peripheral helper T cells (Tph cells; denoted as PD-1highCXCR5-CD4+ T cells) are significantly increased, as are plasmablasts. Tph cells exhibit "B cell help" signatures and induce plasmablast differentiation in vitro. Interestingly, expanded plasmablasts show increased CXCR3 expression, which is positively correlated with higher frequency of activated Tph cells and better clinical outcome. Mechanistically, Tph cells help B cell differentiation and produce more interferon γ (IFNγ), which induces CXCR3 expression on plasmablasts. These results elucidate a role for Tph cells in regulating protective B cell response during acute viral infection.

Age-associated decrease in TLR function in primary human dendritic cells predicts influenza vaccine response.

We evaluated TLR function in primary human dendritic cells (DCs) from 104 young (age 21-30 y) and older (> or =65 y) individuals. We used multicolor flow cytometry and intracellular cytokine staining of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) and found substantial decreases in older compared with young individuals in TNF-alpha, IL-6, and/or IL-12 (p40) production in mDCs and in TNF-alpha and IFN-alpha production in pDCs in response to TLR1/2, TLR2/6, TLR3, TLR5, and TLR8 engagement in mDCs and TLR7 and TLR9 in pDCs. These differences were highly significant after adjustment for heterogeneity between young and older groups (e.g., gender, race, body mass index, number of comorbid medical conditions) using mixed-effect statistical modeling. Studies of surface and intracellular expression of TLR proteins and of TLR gene expression in purified mDCs and pDCs revealed potential contributions for both transcriptional and posttranscriptional mechanisms in these age-associated effects. Moreover, intracellular cytokine production in the absence of TLR ligand stimulation was elevated in cells from older compared with young individuals, suggesting a dysregulation of cytokine production that may limit further activation by TLR engagement. Our results provide evidence for immunosenescence in DCs; notably, defects in cytokine production were strongly associated with poor Ab response to influenza immunization, a functional consequence of impaired TLR function in the aging innate immune response.

Metabolomic and transcriptomic signatures of influenza vaccine response in healthy young and older adults.

Seasonal influenza causes mild to severe respiratory infections and significant morbidity, especially in older adults. Transcriptomic analysis in populations across multiple flu seasons has provided insights into the molecular determinants of vaccine response. Still, the metabolic changes that underlie the immune response to influenza vaccination remain poorly characterized. We performed untargeted metabolomics to analyze plasma metabolites in a cohort of younger and older subjects before and after influenza vaccination to identify vaccine-induced molecular signatures. Metabolomic and transcriptomic data were combined to define networks of gene and metabolic signatures indicative of high and low antibody response in these individuals. We observed age-related differences in metabolic baselines and signatures of antibody response to influenza vaccination and the abundance of α-linolenic and linoleic acids, sterol esters, fatty-acylcarnitines, and triacylglycerol metabolism. We identified a metabolomic signature associated with age-dependent vaccine response, finding increased tryptophan and decreased polyunsaturated fatty acids (PUFAs) in young high responders (HRs), while fatty acid synthesis and cholesteryl esters accumulated in older HRs. Integrated metabolomic and transcriptomic analysis shows that depletion of PUFAs, which are building blocks for prostaglandins and other lipid immunomodulators, in young HR subjects at Day 28 is related to a robust immune response to influenza vaccination. Increased glycerophospholipid levels were associated with an inflammatory response in older HRs to flu vaccination. This multi-omics approach uncovered age-related molecular markers associated with influenza vaccine response and provides insight into vaccine-induced metabolic responses that may help guide development of more effective influenza vaccines.