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How severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remains largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected Huh7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection in vitro. Western blot validation of the downstream effector molecules of these pathways revealed a dose-dependent activation of Akt, mTOR, S6K1 and 4E-BP1 at 24 hours post infection (hpi). However, we found a significant inhibition of HIF-1α through 24hpi and 48hpi of the infection, suggesting a crosstalk between the SARS-CoV-2 and the Akt/mTOR/HIF-1 signaling pathways. Inhibition of the mTOR signaling pathway using Akt inhibitor MK-2206 showed a significant reduction in virus production. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe coronavirus disease 2019 (COVID-19) patients.

Multiomics technologies improve the biological understanding of health status in people living with HIV on antiretroviral therapy (PWH). Still, a systematic and in-depth characterization of metabolic risk profile during successful long-term treatment is lacking. Here, we used multi-omics (plasma lipidomic, metabolomic, and fecal 16 S microbiome) data-driven stratification and characterization to identify the metabolic at-risk profile within PWH. Through network analysis and similarity network fusion (SNF), we identified three groups of PWH (SNF-1–3): healthy (HC)-like (SNF-1), mild at-risk (SNF-3), and severe at-risk (SNF-2). The PWH in the SNF-2 (45%) had a severe at-risk metabolic profile with increased visceral adipose tissue, BMI, higher incidence of metabolic syndrome (MetS), and increased di- and triglycerides despite having higher CD4 + T-cell counts than the other two clusters. However, the HC-like and the severe at-risk group had a similar metabolic profile differing from HIV-negative controls (HNC), with dysregulation of amino acid metabolism. At the microbiome profile, the HC-like group had a lower α-diversity, a lower proportion of men having sex with men (MSM) and was enriched in Bacteroides. In contrast, in at-risk groups, there was an increase in Prevotella , with a high proportion of MSM, which could potentially lead to higher systemic inflammation and increased cardiometabolic risk profile. The multi-omics integrative analysis also revealed a complex microbial interplay of the microbiome-associated metabolites in PWH. Those severely at-risk clusters may benefit from personalized medicine and lifestyle intervention to improve their dysregulated metabolic traits, aiming to achieve healthier aging.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) has caused a global health emergency. A key feature of COVID-19 is dysregulated interferon-response. Type-I interferon (IFN-I) is one of the earliest antiviral innate immune responses following viral infection and plays a significant role in the pathogenesis of SARS-CoV-2. In this study, using a proteomics-based approach, we identified that SARS-CoV-2 infection induces delayed and dysregulated IFN-I signaling in Huh7 cells. We demonstrate that SARS-CoV-2 is able to inhibit RIG-I mediated IFN-β production. Our results also confirm the recent findings that IFN-I pretreatment is able to reduce the susceptibility of Huh7 cells to SARS-CoV-2, but not post-treatment. Moreover, senescent Huh7 cells, in spite of showing accentuated IFN-I response were more susceptible to SARS-CoV-2 infection, and the virus effectively inhibited IFIT1 in these cells. Finally, proteomic comparison between SARS-CoV-2, SARS-CoV, and MERS-CoV revealed a distinct differential regulatory signature of interferon-related proteins emphasizing that therapeutic strategies based on observations in SARS-CoV and MERS-CoV should be used with caution. Our findings provide a better understanding of SARS-CoV-2 regulation of cellular interferon response and a perspective on its use as a treatment. Investigation of different interferon-stimulated genes and their role in the inhibition of SARS-CoV-2 pathogenesis may direct novel antiviral strategies.

Background Interferons play a central role in antiviral defense, but their dysregulation contributes to inflammation and immune dysfunction in respiratory viral infections, including COVID-19. While interferon-stimulated genes (ISGs) are essential effectors of this response, their expression patterns in patients are heterogeneous and not always predictive of disease severity. The immunometabolic consequences of this heterogeneity remain poorly understood. Methods We analyzed hospitalized COVID-19 patients ( n = 37) and uninfected controls ( n = 31) using whole-blood transcriptomics, immune cell deconvolution, plasma proteomics, and standardized plasma metabolomics from a previously generated dataset within this cohort. Patients were stratified into low (LIS), moderate (MIS), and high (HIS) ISG score clusters. Plasma innate immune activation markers were measured by ELISA. Interferon-directed antibody reactivity was analyzed by multiplex bead-based assays measuring antigen reactivity. Functional immune responses were assessed via ex vivo stimulation of healthy donor immune cells with patient plasma, and correlations were performed between metabolites and immune activation markers. Results HIS patients exhibited increased inflammatory mediators and innate immune cell expansion compared with LIS and MIS groups. However, within the HIS group, severe cases displayed distinct metabolic and immune dysregulation. Specifically, severe HIS cases showed reductions in phospholipids, sphingolipids, and tricarboxylic acid cycle intermediates, suggestive of disrupted mitochondrial and lipid metabolism. Plasma from severe HIS patients tended to impair neutrophil and monocyte activation, indicating functional attenuation of innate immune activation within a shared high-ISG background. Correlation analysis revealed that branched-chain lipids, tryptophan-derived metabolites, and a branched-chain dicarboxylic acid were positively associated with immune activation markers. Although type-I interferon neutralization was detected in a subset of patients with IFN antigen reactivity, these samples did not fully account for the observed ISG heterogeneity or disease severity. Conclusions High ISG expression in COVID-19 defines a transcriptional endotype associated with systemic inflammation and innate immune activation. However, severe cases within this group exhibit metabolic constraints and reduced innate immune responsiveness, supporting an immune-metabolic axis in which inflammatory mediators and altered lipid/energy metabolism intersect with innate immune function. These findings motivate future studies to determine whether interferon-associated immune-metabolic states during acute infection relate to persistent inflammation or post-acute sequelae in selected patient subsets.

Untreated HIV-1 infection leads to a slow decrease in CD4 + T cell lymphocytes over time resulting in increased susceptibility to opportunistic infections (acquired immunodeficiency syndrome, AIDS) and ultimately death of the infected individual. Initially, the host’s immune response controls the infection, but cannot eliminate the HIV-1 from the host. Cytotoxic lymphocytes are the key effector cells in this response and can mediate crucial antiviral responses through the release of a set of proteases called granzymes towards HIV-1-infected cells. However, little is known about the immunological molecular mechanisms by which granzymes could control HIV-1. Since we noted that HIV-1 subtype C (HIV-1C) Gag with the tetrapeptide insertion PYKE contains a putative granzyme M (GrM) cleavage site (KEPL) that overlaps with the PYKE insertion, we analyzed the proteolytic activity of GrM towards Gag. Immunoblot analysis showed that GrM could cleave Gag proteins from HIV-1B and variants from HIV-1C of which the Gag-PYKE variant was cleaved with extremely high efficiency. The main cleavage site was directly after the insertion after leucine residue 483. GrM-mediated cleavage of Gag was also observed in co-cultures using cytotoxic lymphocytes as effector cells and this cleavage could be inhibited by a GrM inhibitor peptide. Altogether, our data indicate towards a noncytotoxic immunological mechanism by which GrM-positive cytotoxic lymphocytes target the HIV-1 Gag protein within infected cells to potentially control HIV-1 infection. This mechanism could be exploited in new therapeutic strategies to treat HIV-1-infected patients to improve immunological control of the infection.

Despite successful combination antiretroviral therapy (cART), persistent low-grade immune activation together with inflammation and toxic antiretroviral drugs can lead to long-lasting metabolic flexibility and adaptation in people living with HIV (PLWH). Our study investigated alterations in the plasma metabolic profiles by comparing PLWH on long-term cART(>5 years) and matched HIV-negative controls (HC) in two cohorts from low- and middle-income countries (LMIC), Cameroon, and India, respectively, to understand the system-level dysregulation in HIV-infection. Using untargeted and targeted LC-MS/MS-based metabolic profiling and applying advanced system biology methods, an altered amino acid metabolism, more specifically to glutaminolysis in PLWH than HC were reported. A significantly lower level of neurosteroids was observed in both cohorts and could potentiate neurological impairments in PLWH. Further, modulation of cellular glutaminolysis promoted increased cell death and latency reversal in pre-monocytic HIV-1 latent cell model U1, which may be essential for the clearance of the inducible reservoir in HIV-integrated cells. Mikaeloff et al. use untargeted and targeted LC-MS/MS-based plasma metabolic profiling to discover dysregulated metabolism including that of glutaminolysis in individuals living with HIV. Furthermore, decreased levels of neurosteroids were detected suggesting a potential connection between HIV and neurological impairment.

Pre-existing SARS-CoV-2-reactive T cells have been identified in SARS-CoV-2-unexposed individuals, potentially modulating COVID-19 and vaccination outcomes. Here, we provide evidence that functional cross-reactive memory CD4⁺ T cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is established in early childhood, mirroring early seroconversion with seasonal human coronavirus OC43. Humoral and cellular immune responses against OC43 and SARS-CoV-2 were assessed in SARS-CoV-2-unexposed children (paired samples at age two and six) and adults (age 26 to 83). Pre-existing SARS-CoV-2-reactive CD4⁺ T cell responses targeting spike, nucleocapsid, and membrane were closely linked to the frequency of OC43-specific memory CD4⁺ T cells in childhood. The functional quality of the cross-reactive memory CD4⁺ T cell responses targeting SARS-CoV-2 spike, but not nucleocapsid, paralleled OC43-specific T cell responses. OC43-specific antibodies were prevalent already at age two. However, they did not increase further with age, contrasting with the antibody magnitudes against HKU1 (β-coronavirus), 229E and NL63 (α-coronaviruses), rhinovirus, Epstein–Barr virus (EBV), and influenza virus, which increased after age two. The quality of the memory CD4⁺ T cell responses peaked at age six and subsequently declined with age, with diminished expression of interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF), and CD38 in late adulthood. Age-dependent qualitative differences in the pre-existing SARS-CoV-2-reactive T cell responses may reflect the ability of the host to control coronavirus infections and respond to vaccination.

Antiretroviral therapy (ART) reduces viral replication, restores T helper cells and improves the survival of people living with HIV (PWH), transforming a life-threatening disease into a manageable chronic infection. Nevertheless, PWH under ART shows aging-related diseases such as bone abnormalities, non-HIV-associated cancers, and cardiovascular and neurocognitive diseases. The complex immune metabolic dysregulation leading to these comorbidities is called immune aging. The main question raised by my thesis was, what are the complex mechanisms responsible for immune aging in HIV? Using advanced system biology and machine learning tools, I used multi-omics-based patient stratification to identify biologic perturbations associated with immune aging in PWH.First, we investigated PWH with Metabolic Syndrome (MetS), a relatively common agingrelated disease in HIV-1. In paper I, we identified the dysregulation of glutamate metabolism in PWH with MetS using plasma metabolomics and measure of cell transporters by flow cytrometry. Then, we investigated the mechanisms of differing PWH on long-term successful ART from HIV-negative controls (HC). In paper II, we identified the dysregulation of amino acids and, more specifically, glutaminolysis (i.e., lysis of glutamine to glutamate) in PWH compared to HC using metabolomics in two independent cohorts to avoid the potential cohort biases. We identified five neurosteroids to be lower in PWH and potentially create neurological impairments in PWH. The glutaminolysis inhibition in chronically infected HIV-1 promonocytic (U1) cells induced apoptosis and latency reversal which could clear HIV reservoirs.The first two papers universally clarified our knowledge about dysregulated metabolic traits following a prolonged ART in PWH. However, we observed heterogeneity among the clinically defined PWH. Therefore, we focused more on the multi-omics data-driven approaches to stratify the at-risk group who were either dysregulated metabolically atrisk PWH (paper III) or immunometabolic at-risk group (paper IV) and clarified the biological aging process by measuring transcriptomics age (paper V).In paper III, we found three groups of PWH based on multi-omics integration of lipidomics, metabolomics, and microbiome. The severe at-risk metabolic complications showed increased weight-related comorbidities and di- and triglycerides compared to the other clusters. At-risk and HC-like groups displayed similar metabolic profiles but were different from HC. An increase in Prevotella was linked to the overrepresentation of men having sex with men (MSM) in the at-risk group. The microbiome-associated metabolites (MAM) appeared dysregulated in all HIV groups compared to controls. We improved this clustering by adding transcriptomics and proteomics data for a refined immunometabolic at-risk-related clustering in PWH. In paper IV, immune-driven HC-like and at-risk groups were clustered based on metabolomics, transcriptomics, and proteomics. Several biomarkers from central carbon metabolism (CCM) and senescence-associated proteins were linked to the at-risk phenotype based on random forest, structural causal modeling, and co-expression networks. Senescent protein changes were associated with a deficiency in macrophage function based on single-cell data, cell profiling, flow cytometry, and proteomics from macrophage data and in vitro validation. We also developed personalized and group-level genome-scale metabolic models (GSMM) and confirmed the implication of metabolites from CCM and polyamides in at-risk phenotypes. Finally, we investigated the accelerated aging process (AAP) in PWH.

Biological aging in people with HIV (PWH) with prolonged successful antiretroviral therapy (ART) is convoluted and poorly defined. Here, we aimed to investigate the transcriptomics age estimator (TAE) in a cohort of 178 PWH on prolonged successful ART with immune reconstitution and viral suppression from the Copenhagen Comorbidity (COCOMO) cohort. We also used 143 clinical, demographical, and lifestyle factors to identify the confounders potentially responsible or associated with age acceleration. Among the PWH, 43% had an accelerated aging process (AAP), and 21% had decelerated aging process (DAP). DAP is linked with older age, European ancestry, and higher use of tenofovir disoproxil/alafenamide fumarate. A directionally class‐based gene set enrichment analysis identified the upregulation of inflammatory pathways (e.g., cytokine and Retinoic acid‐inducible gene I (RIG‐I)‐like receptor signaling pathways) and immune response like T‐cell receptor signaling, antigen processing, and presentation in AAP and the downregulation of metabolic processes like oxidative phosphorylation, pyruvate metabolism.

Clinical implementation of patient cell-based drug testing is hampered by inconsistencies and irreproducible results. Using linear mixed models and drug response data from four cohorts of patients with acute myeloid leukemia (n = 993 samples), we identified sources of variability in ex vivo drug sensitivity testing across the studies. We identified several experimental factors, including the time until sample was tested, the type of culture media and viability assay, which led to systematic differences in drug sensitivity profiles. We further investigated the effects of sample types (fresh or frozen) and disease status (diagnosis, relapse, or refractory), and observed that peripheral blood samples resulted in higher drug sensitivity levels, compared to those derived from bone marrow. Technical factors, such as the type of plate reader and centrifugation procedure, further contributed to the response variability depending on the chosen drug response metric. These factors need to be standardized for improved consistency in functional precision medicine studies.