Hybrid immunity to SARS-CoV-2 in patients with chronic lymphocytic leukemia.

OBJECTIVE: Preventing severe COVID-19 remains a priority globally, particularly in the immunocompromised population. As shown in healthy individuals, immunity against SARS-CoV-2 can be yielded by previous infection, vaccination, or both (hybrid immunity). The objective of this observation study was to investigate hybrid immunity in patients with chronic lymphocytic leukemia (CLL). METHODS/RESULTS: Blood samples of six patients with CLL were collected 55 days after fourth COVID-19 vaccination. All patients had a SARS-CoV-2 infection within 12 months before the second booster (fourth vaccination). SARS-CoV-2 spike receptor binding domain (RBD)-specific IgG antibodies were detectable in 6/6 (100.0%) CLL patients after four compared to 4/6 (66.7%) after three vaccinations. The median number of SARS-CoV-2 spike-specific T cells after repeated booster vaccination plus infection was 166 spot-forming cells (SFC) per million peripheral blood mononuclear cells. Overall, 5/5 (100%) studied patients showed a detectable increase in T cell activity. CONCLUSION: Our data reveal an increase of cellular and humoral immune response in CLL patients after fourth COVID-19 vaccination combined with SARS-CoV-2 infection, even in those undergoing B cell-depleting treatment. Patients with prior vaccination failure now show a specific IgG response. Future research should explore the duration and effectiveness of hybrid immunity considering various factors like past infection and vaccination rates, types and numbers of doses, and emerging variants.

IFITM3 blocks influenza virus entry by sorting lipids and stabilizing hemifusion.

Interferon-induced transmembrane protein 3 (IFITM3) inhibits the entry of numerous viruses through undefined molecular mechanisms. IFITM3 localizes in the endosomal-lysosomal system and specifically affects virus fusion with target cell membranes. We found that IFITM3 induces local lipid sorting, resulting in an increased concentration of lipids disfavoring viral fusion at the hemifusion site. This increases the energy barrier for fusion pore formation and the hemifusion dwell time, promoting viral degradation in lysosomes. In situ cryo-electron tomography captured IFITM3-mediated arrest of influenza A virus membrane fusion. Observation of hemifusion diaphragms between viral particles and late endosomal membranes confirmed hemifusion stabilization as a molecular mechanism of IFITM3. The presence of the influenza fusion protein hemagglutinin in post-fusion conformation close to hemifusion sites further indicated that IFITM3 does not interfere with the viral fusion machinery. Collectively, these findings show that IFITM3 induces lipid sorting to stabilize hemifusion and prevent virus entry into target cells.

Methanosaeta and "Candidatus Velamenicoccus archaeovorus".

The phylum "Candidatus Omnitrophica" (candidate division OP3) is ubiquitous in anaerobic habitats but is currently characterized only by draft genomes from metagenomes and single cells. We had visualized cells of the phylotype OP3 LiM in methanogenic cultures on limonene as small epibiotic cells. In this study, we enriched OP3 cells by double density gradient centrifugation and obtained the first closed genome of an apparently clonal OP3 cell population by applying metagenomics and PCR for gap closure. Filaments of acetoclastic Methanosaeta, the largest morphotype in the culture community, contained empty cells, cells devoid of rRNA or of both rRNA and DNA, and dead cells according to transmission electron microscopy (TEM), thin-section TEM, scanning electron microscopy (SEM), catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), and LIVE/DEAD imaging. OP3 LiM cells were ultramicrobacteria (200 to 300 nm in diameter) and showed two physiological stages in CARD-FISH fluorescence signals: strong signals of OP3 LiM cells attached to Bacteria and to Archaea indicated many rRNA molecules and an active metabolism, whereas free-living OP3 cells had weak signals. Metaproteomics revealed that OP3 LiM lives with highly expressed secreted proteins involved in depolymerization and uptake of macromolecules and an active glycolysis and energy conservation by the utilization of pyruvate via a pyruvate:ferredoxin oxidoreductase and an Rnf complex (ferredoxin:NAD oxidoreductase). Besides sugar fermentation, a nucleotidyl transferase may contribute to energy conservation by phosphorolysis, the phosphate-dependent depolymerization of nucleic acids. Thin-section TEM showed distinctive structures of predation. Our study demonstrated a predatory metabolism for OP3 LiM cells, and therefore, we propose the name "Candidatus Velamenicoccus archaeovorus" gen. nov., sp. nov., for OP3 LiM. IMPORTANCE Epibiotic bacteria are known to live on and off bacterial cells. Here, we describe the ultramicrobacterial anaerobic epibiont OP3 LiM living on Archaea and Bacteria. We detected sick and dead cells of the filamentous archaeon Methanosaeta in slowly growing methanogenic cultures. OP3 LiM lives as a sugar fermenter, likely on polysaccharides from outer membranes, and has the genomic potential to live as a syntroph. The predatory lifestyle of OP3 LiM was supported by its genome, the first closed genome for the phylum "Candidatus Omnitrophica," and by images of cell-to-cell contact with prey cells. We propose naming OP3 LiM "Candidatus Velamenicoccus archaeovorus." Its metabolic versatility explains the ubiquitous presence of "Candidatus Omnitrophica" 3 in anoxic habitats and gives ultramicrobacterial epibionts an important role in the recycling and remineralization of microbial biomass. The removal of polysaccharides from outer membranes by ultramicrobacteria may also influence biological interactions between pro- and eukaryotes.

Cryo-correlative light and electron microscopy workflow for cryo-focused ion beam milled adherent cells.

In situ cryo-electron tomography of cryo-focused ion beam (cryo-FIB) milled cells enables the study of cellular organelles in unperturbed conditions and close to the molecular resolution. However, due to the crowdedness of the cellular environment, the identification of individual macromolecular complexes either on organelles or inside the cytosol in cryo-electron tomograms is challenging. Cryo-correlative light and electron microscopy (cryo-CLEM) employs a fluorescently labeled feature of interest imaged by cryo-light microscopy that is correlated to cryo-electron microscopy maps of cryo-FIB milled lamellae using correlation markers discernable by both imaging methods. Here, we provide a protocol for a post-correlation on-lamella cryo-CLEM approach for localization of fluorescently labeled organelles of interest in cryo-lamellae after cryo-FIB milling and tomography of adherent plunge frozen cells.

Post-correlation on-lamella cryo-CLEM reveals the membrane architecture of lamellar bodies.

Lamellar bodies (LBs) are surfactant-rich organelles in alveolar cells. LBs disassemble into a lipid-protein network that reduces surface tension and facilitates gas exchange in the alveolar cavity. Current knowledge of LB architecture is predominantly based on electron microscopy studies using disruptive sample preparation methods. We established and validated a post-correlation on-lamella cryo-correlative light and electron microscopy approach for cryo-FIB milled cells to structurally characterize and validate the identity of LBs in their unperturbed state. Using deconvolution and 3D image registration, we were able to identify fluorescently labeled membrane structures analyzed by cryo-electron tomography. In situ cryo-electron tomography of A549 cells as well as primary Human Small Airway Epithelial Cells revealed that LBs are composed of membrane sheets frequently attached to the limiting membrane through "T"-junctions. We report a so far undescribed outer membrane dome protein complex (OMDP) on the limiting membrane of LBs. Our data suggest that LB biogenesis is driven by parallel membrane sheet import and by the curvature of the limiting membrane to maximize lipid storage capacity.

Biopearling of Interconnected Outer Membrane Vesicle Chains by a Marine Flavobacterium.

Large surface-to-volume ratios provide optimal nutrient uptake conditions for small microorganisms in oligotrophic habitats. The surface area can be increased with appendages. Here, we describe chains of interconnecting vesicles protruding from cells of strain Hel3_A1_48, affiliating with Formosa spp. within the Flavobacteriia and originating from coastal free-living bacterioplankton. The chains were up to 10 µm long and had vesicles emanating from the outer membrane with a single membrane and a size of 80 to 100 nm by 50 to 80 nm. Cells extruded membrane tubes in the exponential phase, whereas vesicle chains dominated on cells in the stationary growth phase. This formation is known as pearling, a physical morphogenic process in which membrane tubes protrude from liposomes and transform into chains of interconnected vesicles. Proteomes of whole-cell membranes and of detached vesicles were dominated by outer membrane proteins, including the type IX secretion system and surface-attached peptidases, glycoside hydrolases, and endonucleases. Fluorescein-labeled laminarin stained the cells and the vesicle chains. Thus, the appendages provide binding domains and degradative enzymes on their surfaces and probably storage volume in the vesicle lumen. Both may contribute to the high abundance of these Formosa-affiliated bacteria during laminarin utilization shortly after spring algal blooms.IMPORTANCE Microorganisms produce membrane vesicles. One synthesis pathway seems to be pearling that describes the physical formation of vesicle chains from phospholipid vesicles via extended tubes. Bacteria with vesicle chains had been observed as well as bacteria with tubes, but pearling was so far not observed. Here, we report the observation of, initially, tubes and then vesicle chains during the growth of a flavobacterium, suggesting biopearling of vesicle chains. The flavobacterium is abundant during spring bacterioplankton blooms developing after algal blooms and has a special set of enzymes for laminarin, the major storage polysaccharide of microalgae. We demonstrated with fluorescently labeled laminarin that the vesicle chains bind laminarin or contain laminarin-derived compounds. Proteomic analyses revealed surface-attached degradative enzymes on the outer membrane vesicles. We conclude that the large surface area and the lumen of vesicle chains may contribute to the ecological success of this marine bacterium.

Alpha-synuclein fibrils recruit TBK1 and OPTN to lysosomal damage sites and induce autophagy in microglial cells.

Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not clear and most previous work was done in neurons and not in microglial cells. Here, we report that exogenous fibrillary, but not monomeric, alpha-synuclein (AS, also known as SNCA) induces autophagy in microglial cells. We extensively studied the dynamics of this response using both live-cell imaging and correlative light-electron microscopy (CLEM), and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and optineurin (OPTN) to ubiquitylated lysosomes. In addition, we observed that LC3 (MAP1LC3B) recruitment to damaged lysosomes was dependent on TBK1 activity. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS.This article has an associated First Person interview with the first author of the paper.

A new method for cryo-sectioning cell monolayers using a correlative workflow.

Cryo-electron microscopy (cryo-EM) techniques have made a huge advancement recently, providing close to atomic resolution of the structure of protein complexes. Interestingly, this imaging technique can be performed in cells, giving access to the molecular machines in their natural context, therefore bridging structural and cell biology. However, in situ structural electron microscopy faces one major challenge, which is the ability to focus on specific subcellular regions to capture the objects of interest. Correlative light and electron microscopy (CLEM) is one very efficient solution for this. Here we present a sample preparation technique that enables cryo-sections of vitrified cell monolayers in an orientation that places the cryo-section parallel to the fluorescence imaging plane. The main advantage of this approach is that it exploits the potentials of CLEM for cryo-EM investigation, for selecting specific cells of interest in a heterogeneous population, or for finding identified subcellular regions on sections.

In vivo testing of gold nanoparticles using the Caenorhabditis elegans model organism.

Gold nanoparticles (AuNPs) are present in many man-made products and cosmetics and are also used by the food and medical industries. Tight regulations regarding the use of mammalian animals for product testing can hamper the study of the specific interactions between engineered nanoparticles and biological systems. Invertebrate models, such as the nematode Caenorhabditis elegans (C. elegans), can offer alternative approaches during the early phases of nanoparticle discovery. Here, we thoroughly evaluated the biodistribution of 11-nm and 150-nm citrate-capped AuNPs in the model organism C. elegans at multiple scales, moving from micrometric to nanometric resolution and from the organismal to cellular level. We confirmed that the nanoparticles were not able to cross the intestinal and dermal barriers. We investigated the effect of AuNPs on the survival and reproductive performance of C. elegans, and correlated these effects with the uptake of AuNPs in terms of their number, surface area, and metal mass. In general, exposure to 11-nm AuNPs resulted in a higher toxicity than the larger 150-nm AuNPs. NP aggregation inside C. elegans was determined using absorbance microspectroscopy, which allowed the plasmonic properties of AuNPs to be correlated with their confinement inside the intestinal lumen, where anatomical traits, acidic pH and the presence of biomolecules play an essential role on NP aggregation. Finally, quantitative PCR of selected molecular markers indicated that exposure to AuNPs did not significantly affect endocytosis and intestinal barrier integrity. STATEMENT OF SIGNIFICANCE: This work highlights how the simple, yet information-rich, animal model C. elegans is ideally suited for preliminary screening of nanoparticles or chemicals mitigating most of the difficulties associated with mammalian animal models, namely the ethical issues, the high cost, and time constraints. This is of particular relevance to the cosmetic, food, and pharmaceutical industries, which all have to justify the use of animals, especially during the discovery, development and initial screening phases. This work provides a detailed and thorough analysis of 11-nm and 150-nm AuNPs at multiple levels of organization (the whole organism, organs, tissues, cells and molecules).

Bio-identity and fate of albumin-coated SPIONs evaluated in cells and by the C. elegans model.

UNLABELLED: Nanoparticles which surface adsorb proteins in an uncontrolled and non-reproducible manner will have limited uses as nanomedicinal products. A promising approach to avoid nanoparticle non-specific interactions with proteins is to design bio-hybrids by purposely pre-forming a protein corona around the inorganic cores. Here, we investigate, in vitro and in vivo, the newly acquired bio-identity of superparamagnetic iron oxide nanoparticles (SPIONs) upon their functionalization with a pre-formed and well-defined bovine serum albumin (BSA) corona. Cellular uptake, intracellular particle distribution and cytotoxicity were studied in two cell lines: adherent and non-adherent cells. BSA decreases nanoparticle internalization in both cell lines and protects the iron core once they have been internalized. The physiological response to the nanoparticles is then in vivo evaluated by oral administration to Caenorhabditis elegans, which was selected as a model of a functional intestinal barrier. Nanoparticle biodistribution, at single particle resolution, is studied by transmission electron microscopy. The analysis reveals that the acidic intestinal environment partially digests uncoated SPIONs but does not affect BSA-coated ones. It also discloses that some particles could enter the nematode's enterocytes, likely by endocytosis which is a different pathway than the one described for the worm nutrients. STATEMENT OF SIGNIFICANCE: Unravelling meaningful relationships between the physiological impact of engineered nanoparticles and their synthetic and biological identity is of vital importance when considering nanoparticles biomedical uses and when establishing their nanotoxicological profile. This study contributes to better comprehend the inorganic nanoparticles' behavior in real biological milieus. We synthesized a controlled pre-formed BSA protein corona on SPIONs to lower unspecific cell uptake and decrease nanoparticle fouling with other proteins. Such findings may be of relevance considering clinical translation and regulatory issues of inorganic nanoparticles. Moreover, we have advanced in the validation of C. elegans as a simple animal model for assessing biological responses of engineering nanomaterials. The physiological response of BSA coated SPIONs was evaluated in vivo after their oral administration to C. elegans. Analyzing ultra-thin cross-sections of the worms by TEM with single-particle precision, we could track NP biodistribution along the digestive tract and determine unambiguously their translocation through biological barriers and cell membranes.

NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication.

UNLABELLED: Induction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA viruses. These altered membranes serve as scaffolds for the assembly of viral replication factories (RFs). We have recently shown that hepatitis C virus (HCV) infection induces endoplasmic reticulum-derived double-membrane vesicles (DMVs) representing the major constituent of the RF within the infected cell. RF formation requires the concerted action of nonstructural action of nonstructural protein (NS)3, -4A, protein (NS)3 -4A, -4B, -5A, and -5B. Although the sole expression of NS5A is sufficient to induce DMV formation, its efficiency is very low. In this study, we dissected the determinants within NS5A responsible for DMV formation and found that RNA-binding domain 1 (D1) and the amino-terminal membrane anchor are indispensable for this process. In contrast, deletion of NS5A D2 or D3 did not affect DMV formation but disrupted RNA replication and virus assembly, respectively. To identify cis- and trans-acting factors of DMV formation, we established a trans cleavage assay. We found that induction of DMVs requires full-length NS3, whereas a helicase-lacking mutant was unable to trigger DMV formation in spite of efficient polyprotein cleavage. Importantly, a mutation accelerating cleavage kinetics at the NS4B-5A site diminished DMV formation, while the insertion of an internal ribosome entry site mimicking constitutive cleavage at this boundary completely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible implications for our understanding of how RFs are formed in other positive-strand RNA viruses. IMPORTANCE: Like all positive-strand RNA viruses, hepatitis C virus (HCV) extensively reorganizes intracellular membranes to allow efficient RNA replication. Double-membrane vesicles (DMVs) that putatively represent sites of HCV RNA amplification are induced by the concerted action of viral and cellular factors. However, the contribution of individual proteins to this process remains poorly understood. Here we identify determinants in the HCV replicase that are required for DMV biogenesis. Major contributors to this process are domain 1 of nonstructural protein 5A and the helicase domain of nonstructural protein 3. In addition, efficient DMV induction depends on cis cleavage of the viral polyprotein, as well as tightly regulated cleavage kinetics. These results identify key determinants governing the biogenesis of the HCV replication factory with possible implications for our understanding of how this central compartment is formed in other positive-strand RNA viruses.

African swine fever virus assembles a single membrane derived from rupture of the endoplasmic reticulum.

Collective evidence argues that two members of the nucleocytoplasmic large DNA viruses (NCLDVs) acquire their membrane from open membrane intermediates, postulated to be derived from membrane rupture. We now study membrane acquisition of the NCLDV African swine fever virus. By electron tomography (ET), the virion assembles a single bilayer, derived from open membrane precursors that collect as ribbons in the cytoplasm. Biochemically, lumenal endoplasmic reticulum (ER) proteins are released into the cytosol, arguing that the open intermediates are ruptured ER membranes. ET shows that viral capsid assembles on the convex side of the open viral membrane to shape it into an icosahedron. The viral capsid is composed of tiny spikes with a diameter of ∼5 nm, connected to the membrane by a 6 nm wide structure displaying thin striations, as observed by several complementary electron microscopy imaging methods. Immature particles display an opening that closes after uptake of the viral genome and core proteins, followed by the formation of the mature virion. Together with our previous data, this study shows a common principle of NCLDVs to build a single internal envelope from open membrane intermediates. Our data now provide biochemical evidence that these open intermediates result from rupture of a cellular membrane, the ER.

Open membranes are the precursors for assembly of large DNA viruses.

Nucleo cytoplasmic large DNA viruses (NCLDVs) are a group of double-stranded DNA viruses that replicate their DNA partly or entirely in the cytoplasm in association with viral factories (VFs). They share about 50 genes suggesting that they are derived from a common ancestor. Using transmission electron microscopy (TEM) and electron tomography (ET) we showed that the NCLDV vaccinia virus (VACV) acquires its membrane from open membrane intermediates, derived from the ER. These open membranes contribute to the formation of a single open membrane of the immature virion, shaped into a sphere by the assembly of the viral scaffold protein on its convex side. We now compare VACV with the NCLDV Mimivirus by TEM and ET and show that the latter also acquires its membrane from open membrane intermediates that accumulate at the periphery of the cytoplasmic VF. In analogy to VACV this membrane is shaped by the assembly of a layer on the convexside of its membrane, likely representing the Mimivirus capsid protein. By quantitative ET we show for both viruses that the open membrane intermediates of assembly adopt an 'open-eight' conformation with a characteristic diameter of 90 nm for Mimi- and 50 nm for VACV. We discuss these results with respect to the common ancestry of NCLDVs and propose a hypothesis on the possible origin of this unusual membrane biogenesis.

Vaccinia virus lacking A17 induces complex membrane structures composed of open membrane sheets.

The vaccinia virus (VACV) precursor membrane, the crescent, consists of an open membrane sheet and is formed by rupture of a cellular compartment. Here, we asked whether A17, a viral membrane protein, plays a role in membrane rupture. Without A17 synthesis, crescents are not formed, and instead, tubular and vesicular membranes accumulate (Rodriguez et al. in J Virol 69:4640-4648, 1). We used electron tomography (ET) to analyze whether the viral membranes lacking A17 consist of open membrane sheets. Tubular, vesicular and so far not described onion-shaped membranes, which consisted of open membrane sheets, were seen. Thus, the data show that membrane rupture occurs independently of the A17 protein.