Seasonal human coronavirus antibodies are boosted upon SARS-CoV-2 infection but not associated with protection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread within the human population. Although SARS-CoV-2 is a novel coronavirus, most humans had been previously exposed to other antigenically distinct common seasonal human coronaviruses (hCoVs) before the coronavirus disease 2019 (COVID-19) pandemic. Here, we quantified levels of SARS-CoV-2-reactive antibodies and hCoV-reactive antibodies in serum samples collected from 431 humans before the COVID-19 pandemic. We then quantified pre-pandemic antibody levels in serum from a separate cohort of 251 individuals who became PCR-confirmed infected with SARS-CoV-2. Finally, we longitudinally measured hCoV and SARS-CoV-2 antibodies in the serum of hospitalized COVID-19 patients. Our studies indicate that most individuals possessed hCoV-reactive antibodies before the COVID-19 pandemic. We determined that ∼20% of these individuals possessed non-neutralizing antibodies that cross-reacted with SARS-CoV-2 spike and nucleocapsid proteins. These antibodies were not associated with protection against SARS-CoV-2 infections or hospitalizations, but they were boosted upon SARS-CoV-2 infection.

The Crystal Structure of a Biological Insulated Transmembrane Molecular Wire.

A growing number of bacteria are recognized to conduct electrons across their cell envelope, and yet molecular details of the mechanisms supporting this process remain unknown. Here, we report the atomic structure of an outer membrane spanning protein complex, MtrAB, that is representative of a protein family known to transport electrons between the interior and exterior environments of phylogenetically and metabolically diverse microorganisms. The structure is revealed as a naturally insulated biomolecular wire possessing a 10-heme cytochrome, MtrA, insulated from the membrane lipidic environment by embedding within a 26 strand ß-barrel formed by MtrB. MtrAB forms an intimate connection with an extracellular 10-heme cytochrome, MtrC, which presents its hemes across a large surface area for electrical contact with extracellular redox partners, including transition metals and electrodes.

Predicting plasma cell retention and loss over a lifetime.

Plasma cells (PCs) rely on external survival cues for persistence, which limits the size of the PC pool. How, then, are new specificities incorporated into a saturated system? In this issue of Immunity, Simons and Karin put forward a mathematical framework to explain PC retention that makes testable predictions about steady-state lifespan structure, withstands tests based on accrual and displaceability, and accounts for lifespan stratification with specificity.

Conversion of vaccines from low to high immunogenicity by antibodies with epitope complementarity.

Existing antibodies (Abs) have varied effects on humoral immunity during subsequent infections. Here, we leveraged in vivo systems that allow precise control of antigen-specific Abs and B cells to examine the impact of Ab dose, affinity, and specificity in directing B cell activation and differentiation. Abs competing with the B cell receptor (BCR) epitope showed affinity-dependent suppression. By contrast, Abs targeting a complementary epitope, not overlapping with the BCR, shifted B cell differentiation toward Ab-secreting cells. Such Abs allowed for potent germinal center (GC) responses to otherwise poorly immunogenic sites by promoting antigen capture and presentation by low-affinity B cells. These mechanisms jointly diversified the B cell repertoire by facilitating the recruitment of high- and low-affinity B cells into Ab-secreting cell, GC, and memory B cell fates. Incorporation of small amounts of monoclonal Abs into protein- or mRNA-based vaccines enhanced immunogenicity and facilitated sustained immune responses, with implications for vaccine design and our understanding of protective immunity.

A DNA-Based T Cell Receptor Reveals a Role for Receptor Clustering in Ligand Discrimination.

A T cell mounts an immune response by measuring the binding strength of its T cell receptor (TCR) for peptide-loaded MHCs (pMHC) on an antigen-presenting cell. How T cells convert the lifetime of the extracellular TCR-pMHC interaction into an intracellular signal remains unknown. Here, we developed a synthetic signaling system in which the extracellular domains of the TCR and pMHC were replaced with short hybridizing strands of DNA. Remarkably, T cells can discriminate between DNA ligands differing by a single base pair. Single-molecule imaging reveals that signaling is initiated when single ligand-bound receptors are converted into clusters, a time-dependent process requiring ligands with longer bound times. A computation model reveals that receptor clustering serves a kinetic proofreading function, enabling ligands with longer bound times to have disproportionally greater signaling outputs. These results suggest that spatial reorganization of receptors plays an important role in ligand discrimination in T cell signaling.

Interleukin-21, acting beyond the immunological synapse, independently controls T follicular helper and germinal center B cells.

Germinal centers (GCs), transient structures within B cell follicles and central to affinity maturation, require the coordinated behavior of T and B cells. IL-21, a pleiotropic T cell-derived cytokine, is key to GC biology through incompletely understood mechanisms. By genetically restricting production and receipt of IL-21 in vivo, we reveal how its independent actions on T and B cells combine to regulate the GC. IL-21 established the magnitude of the GC B cell response by promoting CD4+ T cell expansion and differentiation in a dose-dependent manner and with paracrine activity. Within GC, IL-21 specifically promoted B cell centroblast identity and, when bioavailability was high, plasma cell differentiation. Critically, these actions may occur irrespective of cognate T-B interactions, making IL-21 a general promoter of growth as distinct to a mediator of affinity-driven selection via synaptic delivery. This promiscuous activity of IL-21 explains the consequences of IL-21 deficiency on antibody-based immunity.

A Dangerous Liaison with a Conscience.

The contribution of the immunoglobulin E (IgE)-mast cell response to allergy portrays the axis as a villain with malicious intent. A new study from Starkl et al. tells a different story, highlighting a more worthwhile purpose of protecting us against bacterial toxins.

CLSTN3ß enforces adipocyte multilocularity to facilitate lipid utilization.

Multilocular adipocytes are a hallmark of thermogenic adipose tissue1,2, but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3ß) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3ß is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER-LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3ß have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3ß is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3ß associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3ß-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes.

Broad defects in the energy metabolism of leukocytes underlie immunoparalysis in sepsis.

The acute phase of sepsis is characterized by a strong inflammatory reaction. At later stages in some patients, immunoparalysis may be encountered, which is associated with a poor outcome. By transcriptional and metabolic profiling of human patients with sepsis, we found that a shift from oxidative phosphorylation to aerobic glycolysis was an important component of initial activation of host defense. Blocking metabolic pathways with metformin diminished cytokine production and increased mortality in systemic fungal infection in mice. In contrast, in leukocytes rendered tolerant by exposure to lipopolysaccharide or after isolation from patients with sepsis and immunoparalysis, a generalized metabolic defect at the level of both glycolysis and oxidative metabolism was apparent, which was restored after recovery of the patients. Finally, the immunometabolic defects in humans were partially restored by therapy with recombinant interferon-γ, which suggested that metabolic processes might represent a therapeutic target in sepsis.

Formaldehyde regulates one-carbon metabolism and epigenetics.

Recently, Pham et al. used an array of model systems to uncover a role for the enzyme methionine adenosyltransferase (MAT)-1A, which is mainly expressed in liver, in both sensing formaldehyde and regulating transcriptional responses that protect against it. This provides a new lens for understanding the effects of formaldehyde on gene regulation.

An optogenetic method for the controlled release of single molecules.

We developed a system for optogenetic release of single molecules in cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for a light dose-dependent delivery of functional proteins to the cytosol and plasma membrane in amounts compatible with single-molecule imaging, greatly simplifying access to single-molecule microscopy of any protein in live cells. We were able to reconstitute ion conductance by delivering BK and LRRC8/volume-regulated anion channels to the plasma membrane. Finally we were able to induce NF-kB signaling in T lymphoblasts stimulated by interleukin-1 by controlled release of a signaling protein that had been knocked out. We observed light-induced formation of functional inflammatory signaling complexes that triggered phosphorylation of the inhibitor of nuclear factor kappa-B kinase only in activated cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single-molecule level.

Sod1-deficient cells are impaired in formation of the modified nucleosides mcm5s2U and yW in tRNA.

Uridine residues present at the wobble position of eukaryotic cytosolic tRNAs often carry a 5-carbamoylmethyl (ncm5), 5-methoxycarbonylmethyl (mcm5), or 5-methoxycarbonylhydroxymethyl (mchm5) side-chain. The presence of these side-chains allows proper pairing with cognate codons and they are particularly important in tRNA species where the U34 residue is also modified with a 2-thio (s2) group. The first step in synthesis of the ncm5, mcm5, and mchm5 side-chains is dependent on the six-subunit Elongator complex, whereas the thiolation of the 2-position is catalyzed by the Ncs6/Ncs2 complex. In both yeast and metazoans, allelic variants of Elongator subunit genes show genetic interactions with mutant alleles of SOD1, which encodes the cytosolic Cu,Zn-superoxide dismutase. However, the cause of these genetic interactions remains unclear. Here, we show that yeast sod1 null mutants are impaired in the formation of 2-thio-modified U34 residues. In addition, the lack of Sod1 induces a defect in the biosynthesis of wybutosine, which is a modified nucleoside found at position 37 of tRNAPhe Our results suggest that these tRNA modification defects are caused by superoxide-induced inhibition of the iron-sulfur cluster-containing Ncs6/Ncs2 and Tyw1 enzymes. Since mutations in Elongator subunit genes generate strong negative genetic interactions with mutant ncs6 and ncs2 alleles, our findings at least partially explain why the activity of Elongator can modulate the phenotypic consequences of SOD1/sod1 alleles. Collectively, our results imply that tRNA hypomodification may contribute to impaired proteostasis in Sod1-deficient cells.

High-Resolution Reconstruction of a C. elegans Ribosome Sheds Light on Evolutionary Dynamics and Tissue Specificity.

Caenorhabditis elegans is an important model organism for human health and disease, with foundational contributions to the understanding of gene expression and tissue patterning in animals. An invaluable tool in modern gene expression research is the presence of a high-resolution ribosome structure, though no such structure exists for C. elegans. Here we present a high-resolution single-particle cryogenic electron microscopy (cryoEM) reconstruction and molecular model of a C. elegans ribosome, revealing a significantly streamlined animal ribosome. Many facets of ribosome structure are conserved in C. elegans, including overall ribosomal architecture and the mechanism of cycloheximide, while other facets such as expansion segments and eL28 are rapidly evolving. We identify uL5 and uL23 as two instances of tissue-specific ribosomal protein paralog expression conserved in Caenorhabditis, suggesting that C. elegans ribosomes vary across tissues. The C. elegans ribosome structure will provide a basis for future structural, biochemical, and genetic studies of translation in this important animal system.

Structure-functional characterization of Lactococcus AbiA phage defense system.

Bacterial reverse transcriptases (RTs) are a large and diverse enzyme family. AbiA, AbiK and Abi-P2 are abortive infection system (Abi) RTs that mediate defense against bacteriophages. What sets Abi RTs apart from other RT enzymes is their ability to synthesize long DNA products of random sequences in a template- and primer-independent manner. Structures of AbiK and Abi-P2 representatives have recently been determined, but there are no structural data available for AbiA. Here, we report the crystal structure of Lactococcus AbiA polymerase in complex with a single-stranded polymerization product. AbiA comprises three domains: an RT-like domain, a helical domain that is typical for Abi polymerases, and a higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain that is common for many antiviral proteins. AbiA forms a dimer that distinguishes it from AbiK and Abi-P2, which form trimers/hexamers. We show the DNA polymerase activity of AbiA in an in vitro assay and demonstrate that it requires the presence of the HEPN domain which is enzymatically inactive. We validate our biochemical and structural results in vivo through bacteriophage infection assays. Finally, our in vivo results suggest that AbiA-mediated phage defense may not rely on AbiA-mediated cell death.

The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArg and tRNALys isoacceptors in the P site.

The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.

IgG3 subclass antibodies recognize antigenically drifted influenza viruses and SARS-CoV-2 variants through efficient bivalent binding.

The constant domains of antibodies are important for effector functions, but less is known about how they can affect binding and neutralization of viruses. Here, we evaluated a panel of human influenza virus monoclonal antibodies (mAbs) expressed as IgG1, IgG2, or IgG3. We found that many influenza virus-specific mAbs have altered binding and neutralization capacity depending on the IgG subclass encoded and that these differences result from unique bivalency capacities of the subclasses. Importantly, subclass differences in antibody binding and neutralization were greatest when the affinity for the target antigen was reduced through antigenic mismatch. We found that antibodies expressed as IgG3 bound and neutralized antigenically drifted influenza viruses more effectively. We obtained similar results using a panel of SARS-CoV-2-specific mAbs and the antigenically advanced B.1.351 and BA.1 strains of SARS-CoV-2. We found that a licensed therapeutic mAb retained neutralization breadth against SARS-CoV-2 variants when expressed as IgG3, but not IgG1. These data highlight that IgG subclasses are not only important for fine-tuning effector functionality but also for binding and neutralization of antigenically drifted viruses.

The structural basis of hyperpromiscuity in a core combinatorial network of type II toxin-antitoxin and related phage defense systems.

Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralizing antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins), we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralization by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralizing mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralization, while in multidomain antitoxins the neutralization is mediated by other domains, such as PAD1, Phd-C, and ZFD. We hypothesize that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 NetFlax TA systems and used domain annotations and metabolic labeling assays to predict their potential mechanisms of toxicity (such as membrane disruption, and inhibition of cell division or protein synthesis) as well as biological functions (such as antiphage defense). We have validated the antiphage activity of a RosmerTA system encoded by Gordonia phage Kita, and used fluorescence microscopy to confirm its predicted membrane-depolarizing activity. The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/.

Myddosome clustering in IL-1 receptor signaling regulates the formation of an NF-kB activating signalosome.

IL-1 receptor (IL-1R) signaling can activate thresholded invariant outputs and proportional outputs that scale with the amount of stimulation. Both responses require the Myddosome, a multiprotein complex. The Myddosome is required for polyubiquitin chain formation and NF-kB signaling. However, how these signals are spatially and temporally regulated to drive switch-like and proportional outcomes is not understood. During IL-1R signaling, Myddosomes dynamically reorganize into multi-Myddosome clusters at the cell membrane. Blockade of clustering using nanoscale extracellular barriers reduces NF-kB activation. Myddosomes function as scaffolds that assemble an NF-kB signalosome consisting of E3-ubiquitin ligases TRAF6 and LUBAC, K63/M1-linked polyubiquitin chains, phospho-IKK, and phospho-p65. This signalosome preferentially assembles at regions of high Myddosome density, which enhances the recruitment of TRAF6 and LUBAC. Extracellular barriers that restrict Myddosome clustering perturbed the recruitment of both ligases. We find that LUBAC was especially sensitive to clustering with 10-fold lower recruitment to single Myddosomes than clustered Myddosomes. These data reveal that the clustering behavior of Myddosomes provides a basis for digital and analog IL-1R signaling.

Antimicrobial Drug Penetration Is Enhanced by Lung Tissue Inflammation and Injury.

Rationale: Pneumonia is a frequent and feared complication in intubated critically ill patients. Tissue concentrations of antimicrobial drugs need to be sufficiently high to treat the infection and also prevent development of bacterial resistance. It is uncertain whether pulmonary inflammation and injury affect antimicrobial drug penetration into lung tissue.Objectives: To determine and compare tissue and BAL fluid concentrations of ceftaroline fosamil and linezolid in a model of unilateral acute lung injury in pigs and to evaluate whether dose adjustment is necessary to reach sufficient antimicrobial concentrations in injured lung tissue.Methods: After induction of unilateral acute lung injury, ceftaroline fosamil and linezolid were administered intravenously. Drug concentrations were measured in lung tissue through microdialysis and in blood and BAL fluid samples during the following 8 hours. The primary endpoint was the tissue concentration area under the concentration curve in the first 8 hours (AUC0-8 h) of the two antimicrobial drugs.Measurements and Main Results: In 10 pigs, antimicrobial drug concentrations were higher in inflamed and injured lung tissue compared with those in uninflamed and uninjured lung tissue (median ceftaroline fosamil AUC0-8 h [and interquartile range] = 26.7 mg ⋅ h ⋅ L-1 [19.7-39.0] vs. 16.0 mg ⋅ h ⋅ L-1 [13.6-19.9], P = 0.02; median linezolid AUC0-8 h 76.0 mg ⋅ h ⋅ L-1 [68.1-96.0] vs. 54.6 mg ⋅ h ⋅ L-1 [42.7-60.9], P = 0.01), resulting in a longer time above the minimal inhibitory concentration and in higher peak concentrations and dialysate/plasma ratios. Penetration into BAL fluid was excellent for both antimicrobials, but without left-to-right differences (ceftaroline fosamil, P = 0.78; linezolid, P = 1.00).Conclusions: Tissue penetration of two commonly used antimicrobial drugs for pneumonia is enhanced by early lung tissue inflammation and injury, resulting in longer times above the minimal inhibitory concentration. Thus, lung tissue inflammation ameliorates antimicrobial drug penetration during the acute phase.

The Plasma Lipidomic Landscape in Patients with Sepsis due to Community-acquired Pneumonia.

Rationale: The plasma lipidome has the potential to reflect many facets of the host status during severe infection. Previous work is limited to specific lipid groups or was focused on lipids as prognosticators.Objectives: To map the plasma lipidome during sepsis due to community-acquired pneumonia (CAP) and determine the disease specificity and associations with clinical features.Methods: We analyzed 1,833 lipid species across 33 classes in 169 patients admitted to the ICU with sepsis due to CAP, 51 noninfected ICU patients, and 48 outpatient controls. In a paired analysis, we reanalyzed patients still in the ICU 4 days after admission (n = 82).Measurements and Main Results: A total of 58% of plasma lipids were significantly lower in patients with CAP-attributable sepsis compared with outpatient controls (6% higher, 36% not different). We found strong lipid class-specific associations with disease severity, validated across two external cohorts, and inflammatory biomarkers, in which triacylglycerols, cholesterol esters, and lysophospholipids exhibited the strongest associations. A total of 36% of lipids increased over time, and stratification by survival revealed diverging lipid recovery, which was confirmed in an external cohort; specifically, a 10% increase in cholesterol ester levels was related to a lower odds ratio (0.84; P = 0.006) for 30-day mortality (absolute mortality, 18 of 82). Comparison with noninfected ICU patients delineated a substantial common illness response (57.5%) and a distinct lipidomic signal for patients with CAP-attributable sepsis (37%).Conclusions: Patients with sepsis due to CAP exhibit a time-dependent and partially disease-specific shift in their plasma lipidome that correlates with disease severity and systemic inflammation and is associated with higher mortality.