Stability of gut microbiome after COVID-19 vaccination in healthy and immuno-compromised individuals.

Bidirectional interactions between the immune system and the gut microbiota are key contributors to various physiological functions. Immune-associated diseases such as cancer and autoimmunity, and efficacy of immunomodulatory therapies, have been linked to microbiome variation. Although COVID-19 infection has been shown to cause microbial dysbiosis, it remains understudied whether the inflammatory response associated with vaccination also impacts the microbiota. Here, we investigate the temporal impact of COVID-19 vaccination on the gut microbiome in healthy and immuno-compromised individuals; the latter included patients with primary immunodeficiency and cancer patients on immunomodulating therapies. We find that the gut microbiome remained remarkably stable post-vaccination irrespective of diverse immune status, vaccine response, and microbial composition spanned by the cohort. The stability is evident at all evaluated levels including diversity, phylum, species, and functional capacity. Our results indicate the resilience of the gut microbiome to host immune changes triggered by COVID-19 vaccination and suggest minimal, if any, impact on microbiome-mediated processes. These findings encourage vaccine acceptance, particularly when contrasted with the significant microbiome shifts observed during COVID-19 infection.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Age-associated B cells predict impaired humoral immunity after COVID-19 vaccination in patients receiving immune checkpoint blockade.

Age-associated B cells (ABC) accumulate with age and in individuals with different immunological disorders, including cancer patients treated with immune checkpoint blockade and those with inborn errors of immunity. Here, we investigate whether ABCs from different conditions are similar and how they impact the longitudinal level of the COVID-19 vaccine response. Single-cell RNA sequencing indicates that ABCs with distinct aetiologies have common transcriptional profiles and can be categorised according to their expression of immune genes, such as the autoimmune regulator (AIRE). Furthermore, higher baseline ABC frequency correlates with decreased levels of antigen-specific memory B cells and reduced neutralising capacity against SARS-CoV-2. ABCs express high levels of the inhibitory FcγRIIB receptor and are distinctive in their ability to bind immune complexes, which could contribute to diminish vaccine responses either directly, or indirectly via enhanced clearance of immune complexed-antigen. Expansion of ABCs may, therefore, serve as a biomarker identifying individuals at risk of suboptimal responses to vaccination.

A histone deacetylase 3 and mitochondrial complex I axis regulates toxic formaldehyde production.

Cells produce considerable genotoxic formaldehyde from an unknown source. We carry out a genome-wide CRISPR-Cas9 genetic screen in metabolically engineered HAP1 cells that are auxotrophic for formaldehyde to find this cellular source. We identify histone deacetylase 3 (HDAC3) as a regulator of cellular formaldehyde production. HDAC3 regulation requires deacetylase activity, and a secondary genetic screen identifies several components of mitochondrial complex I as mediators of this regulation. Metabolic profiling indicates that this unexpected mitochondrial requirement for formaldehyde detoxification is separate from energy generation. HDAC3 and complex I therefore control the abundance of a ubiquitous genotoxic metabolite.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism.

A challenge for screening new candidate drugs to treat cancer is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels to propagate cells. Which nutrients are available can influence how cancer cells use metabolism to proliferate and impact sensitivity to some drugs, but a general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To enable screening of compounds to determine how the nutrient environment impacts drug efficacy, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We used this system to screen several small molecule libraries and found that compounds targeting metabolic enzymes were enriched as having differential efficacy in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

B cell receptor repertoire kinetics after SARS-CoV-2 infection and vaccination.

B cells are important in immunity to both severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and vaccination, but B cell receptor (BCR) repertoire development in these contexts has not been compared. We analyze serial samples from 171 SARS-CoV-2-infected individuals and 63 vaccine recipients and find the global BCR repertoire differs between them. Following infection, immunoglobulin (Ig)G1/3 and IgA1 BCRs increase, somatic hypermutation (SHM) decreases, and, in severe disease, IgM and IgA clones are expanded. In contrast, after vaccination, the proportion of IgD/M BCRs increase, SHM is unchanged, and expansion of IgG clones is prominent. VH1-24, which targets the N-terminal domain (NTD) and contributes to neutralization, is expanded post infection except in the most severe disease. Infection generates a broad distribution of SARS-CoV-2-specific clones predicted to target the spike protein, while a more focused response after vaccination mainly targets the spike's receptor-binding domain. Thus, the nature of SARS-CoV-2 exposure differentially affects BCR repertoire development, potentially informing vaccine strategies.

Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease.

The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.

Neural stem cells traffic functional mitochondria via extracellular vesicles.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

Increased demand for NAD+ relative to ATP drives aerobic glycolysis.

Aerobic glycolysis, or preferential fermentation of glucose-derived pyruvate to lactate despite available oxygen, is associated with proliferation across many organisms and conditions. To better understand that association, we examined the metabolic consequence of activating the pyruvate dehydrogenase complex (PDH) to increase pyruvate oxidation at the expense of fermentation. We find that increasing PDH activity impairs cell proliferation by reducing the NAD+/NADH ratio. This change in NAD+/NADH is caused by increased mitochondrial membrane potential that impairs mitochondrial electron transport and NAD+ regeneration. Uncoupling respiration from ATP synthesis or increasing ATP hydrolysis restores NAD+/NADH homeostasis and proliferation even when glucose oxidation is increased. These data suggest that when demand for NAD+ to support oxidation reactions exceeds the rate of ATP turnover in cells, NAD+ regeneration by mitochondrial respiration becomes constrained, promoting fermentation, despite available oxygen. This argues that cells engage in aerobic glycolysis when the demand for NAD+ is in excess of the demand for ATP.

Treatment of COVID-19 with remdesivir in the absence of humoral immunity: a case report.

The response to the coronavirus disease 2019 (COVID-19) pandemic has been hampered by lack of an effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antiviral therapy. Here we report the use of remdesivir in a patient with COVID-19 and the prototypic genetic antibody deficiency X-linked agammaglobulinaemia (XLA). Despite evidence of complement activation and a robust T cell response, the patient developed persistent SARS-CoV-2 pneumonitis, without progressing to multi-organ involvement. This unusual clinical course is consistent with a contribution of antibodies to both viral clearance and progression to severe disease. In the absence of these confounders, we take an experimental medicine approach to examine the in vivo utility of remdesivir. Over two independent courses of treatment, we observe a temporally correlated clinical and virological response, leading to clinical resolution and viral clearance, with no evidence of acquired drug resistance. We therefore provide evidence for the antiviral efficacy of remdesivir in vivo, and its potential benefit in selected patients.

Dissecting cell-type-specific metabolism in pancreatic ductal adenocarcinoma.

Tumors are composed of many different cell types including cancer cells, fibroblasts, and immune cells. Dissecting functional metabolic differences between cell types within a mixed population can be challenging due to the rapid turnover of metabolites relative to the time needed to isolate cells. To overcome this challenge, we traced isotope-labeled nutrients into macromolecules that turn over more slowly than metabolites. This approach was used to assess differences between cancer cell and fibroblast metabolism in murine pancreatic cancer organoid-fibroblast co-cultures and tumors. Pancreatic cancer cells exhibited increased pyruvate carboxylation relative to fibroblasts, and this flux depended on both pyruvate carboxylase and malic enzyme 1 activity. Consequently, expression of both enzymes in cancer cells was necessary for organoid and tumor growth, demonstrating that dissecting the metabolism of specific cell populations within heterogeneous systems can identify dependencies that may not be evident from studying isolated cells in culture or bulk tissue.

Tumors contain a mixture of many different types of cells, including cancer cells and non-cancer cells. The interactions between these two groups of cells affect how the cancer cells use nutrients, which, in turn, affects how fast these cells grow and divide. Furthermore, different cell types may use nutrients in diverse ways to make other molecules ­ known as metabolites ­ that the cell needs to survive. Fibroblasts are a subset of non-cancer cells that are typically found in tumors and can help them form. Separating fibroblasts from cancer cells in a tumor takes a lot longer than the chemical reactions in each cell of the tumor that produce and use up nutrients, also known as the cell's metabolism. Therefore, measuring the levels of glucose (the sugar that is the main energy source for cells) and other metabolites in each tumor cell after separating them does not necessarily provide accurate information about the tumor cell's metabolism. This makes it difficult to study how cancer cells and fibroblasts use nutrients differently. Lau et al. have developed a strategy to study the metabolism of cancer cells and fibroblasts in tumors. Mice with tumors in their pancreas were provided glucose that had been labelled using biochemical techniques. As expected, when the cell processed the glucose, the label was transferred into metabolites that got used up very quickly. But the label also became incorporated into larger, more stable molecules, such as proteins. Unlike the small metabolites, these larger molecules do not change in the time it takes to separate the cancer cells from the fibroblasts. Lau et al. sorted cells from whole pancreatic tumors and analyzed large, stable molecules that can incorporate the label from glucose in cancer cells and fibroblasts. The experiments showed that, in cancer cells, these molecules were more likely to have labeling patterns that are characteristic of two specific enzymes called pyruvate carboxylase and malic enzyme 1. This suggests that these enzymes are more active in cancer cells. Lau et al. also found that pancreatic cancer cells needed these two enzymes to metabolize glucose and to grow into large tumors. Pancreatic cancer is one of the most lethal cancers and current therapies offer limited benefit to many patients. Therefore, it is important to develop new drugs to treat this disease. Understanding how cancer cells and non-cancer cells in pancreatic tumors use nutrients differently is important for developing drugs that only target cancer cells.

Effective control of SARS-CoV-2 transmission between healthcare workers during a period of diminished community prevalence of COVID-19.

Previously, we showed that 3% (31/1032)of asymptomatic healthcare workers (HCWs) from a large teaching hospital in Cambridge, UK, tested positive for SARS-CoV-2 in April 2020. About 15% (26/169) HCWs with symptoms of coronavirus disease 2019 (COVID-19) also tested positive for SARS-CoV-2 (Rivett et al., 2020). Here, we show that the proportion of both asymptomatic and symptomatic HCWs testing positive for SARS-CoV-2 rapidly declined to near-zero between 25th April and 24th May 2020, corresponding to a decline in patient admissions with COVID-19 during the ongoing UK 'lockdown'. These data demonstrate how infection prevention and control measures including staff testing may help prevent hospitals from becoming independent 'hubs' of SARS-CoV-2 transmission, and illustrate how, with appropriate precautions, organizations in other sectors may be able to resume on-site work safely.

Integrative functional genomics decodes herpes simplex virus 1.

The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution.

Promiscuous Targeting of Cellular Proteins by Vpr Drives Systems-Level Proteomic Remodeling in HIV-1 Infection.

HIV-1 encodes four "accessory proteins" (Vif, Vpr, Vpu, and Nef), dispensable for viral replication in vitro but essential for viral pathogenesis in vivo. Well characterized cellular targets have been associated with Vif, Vpu, and Nef, which counteract host restriction and promote viral replication. Conversely, although several substrates of Vpr have been described, their biological significance remains unclear. Here, we use complementary unbiased mass spectrometry-based approaches to demonstrate that Vpr is both necessary and sufficient for the DCAF1/DDB1/CUL4 E3 ubiquitin ligase-mediated degradation of at least 38 cellular proteins, causing systems-level changes to the cellular proteome. We therefore propose that promiscuous targeting of multiple host factors underpins complex Vpr-dependent cellular phenotypes and validate this in the case of G2/M cell cycle arrest. Our model explains how Vpr modulates so many cell biological processes and why the functional consequences of previously described Vpr targets, identified and studied in isolation, have proved elusive.

An Interferon-Driven Oxysterol-Based Defense against Tumor-Derived Extracellular Vesicles.

Tumor-derived extracellular vesicles (TEV) "educate" healthy cells to promote metastases. We found that melanoma TEV downregulated type I interferon (IFN) receptor and expression of IFN-inducible cholesterol 25-hydroxylase (CH25H). CH25H produces 25-hydroxycholesterol, which inhibited TEV uptake. Low CH25H levels in leukocytes from melanoma patients correlated with poor prognosis. Mice incapable of downregulating the IFN receptor and Ch25h were resistant to TEV uptake, TEV-induced pre-metastatic niche, and melanoma lung metastases; however, ablation of Ch25h reversed these phenotypes. An anti-hypertensive drug, reserpine, suppressed TEV uptake and disrupted TEV-induced formation of the pre-metastatic niche and melanoma lung metastases. These results suggest the importance of CH25H in defense against education of normal cells by TEV and argue for the use of reserpine in adjuvant melanoma therapy.

Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants.

Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function.

NOTCH1 mediates a switch between two distinct secretomes during senescence.

Senescence, a persistent form of cell-cycle arrest, is often associated with a diverse secretome, which provides complex functionality for senescent cells within the tissue microenvironment. We show that oncogene-induced senescence is accompanied by a dynamic fluctuation of NOTCH1 activity, which drives a TGF-ß-rich secretome, while suppressing the senescence-associated pro-inflammatory secretome through inhibition of C/EBPß. NOTCH1 and NOTCH1-driven TGF-ß contribute to 'lateral induction of senescence' through a juxtacrine NOTCH-JAG1 pathway. In addition, NOTCH1 inhibition during senescence facilitates upregulation of pro-inflammatory cytokines, promoting lymphocyte recruitment and senescence surveillance in vivo. As enforced activation of NOTCH1 signalling confers a near mutually exclusive secretory profile compared with typical senescence, our data collectively indicate that the dynamic alteration of NOTCH1 activity during senescence dictates a functional balance between these two distinct secretomes: one representing TGF-ß and the other pro-inflammatory cytokines, highlighting that NOTCH1 is a temporospatial controller of secretome composition.

GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells.

Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing.