Human cytomegalovirus exploits STING signaling and counteracts IFN/ISG induction to facilitate infection of dendritic cells.

Human cytomegalovirus (HCMV) is a widespread pathogen that in immunocompromised hosts can cause life-threatening disease. Studying HCMV-exposed monocyte-derived dendritic cells by single-cell RNA sequencing, we observe that most cells are entered by the virus, whereas less than 30% of them initiate viral gene expression. Increased viral gene expression is associated with activation of the stimulator of interferon genes (STING) that usually induces anti-viral interferon responses, and with the induction of several pro- (RHOB, HSP1A1, DNAJB1) and anti-viral (RNF213, TNFSF10, IFI16) genes. Upon progression of infection, interferon-beta but not interferon-lambda transcription is inhibited. Similarly, interferon-stimulated gene expression is initially induced and then shut off, thus further promoting productive infection. Monocyte-derived dendritic cells are composed of 3 subsets, with one being especially susceptible to HCMV. In conclusion, HCMV permissiveness of monocyte-derived dendritic cells depends on complex interactions between virus sensing, regulation of the interferon response, and viral gene expression.

Systems immunology of transcriptional responses to viral infection identifies conserved antiviral pathways across macaques and humans.

Viral pandemics and epidemics pose a significant global threat. While macaque models of viral disease are routinely used, it remains unclear how conserved antiviral responses are between macaques and humans. Therefore, we conducted a cross-species analysis of transcriptomic data from over 6,088 blood samples from macaques and humans infected with one of 31 viruses. Our findings demonstrate that irrespective of primate or viral species, there are conserved antiviral responses that are consistent across infection phase (acute, chronic, or latent) and viral genome type (DNA or RNA viruses). Leveraging longitudinal data from experimental challenges, we identify virus-specific response kinetics such as host responses to Coronaviridae and Orthomyxoviridae infections peaking 1-3 days earlier than responses to Filoviridae and Arenaviridae viral infections. Our results underscore macaque studies as a powerful tool for understanding viral pathogenesis and immune responses that translate to humans, with implications for viral therapeutic development and pandemic preparedness.

Single B cell transcriptomics identifies multiple isotypes of broadly neutralizing antibodies against flaviviruses.

Sequential dengue virus (DENV) infections often generate neutralizing antibodies against all four DENV serotypes and sometimes, Zika virus. Characterizing cross-flavivirus broadly neutralizing antibody (bnAb) responses can inform countermeasures that avoid enhancement of infection associated with non-neutralizing antibodies. Here, we used single cell transcriptomics to mine the bnAb repertoire following repeated DENV infections. We identified several new bnAbs with comparable or superior breadth and potency to known bnAbs, and with distinct recognition determinants. Unlike all known flavivirus bnAbs, which are IgG1, one newly identified cross-flavivirus bnAb (F25.S02) was derived from IgA1. Both IgG1 and IgA1 versions of F25.S02 and known bnAbs displayed neutralizing activity, but only IgG1 enhanced infection in monocytes expressing IgG and IgA Fc receptors. Moreover, IgG-mediated enhancement of infection was inhibited by IgA1 versions of bnAbs. We demonstrate a role for IgA in flavivirus infection and immunity with implications for vaccine and therapeutic strategies.

Global and cell type-specific immunological hallmarks of severe dengue progression identified via a systems immunology approach.

Severe dengue (SD) is a major cause of morbidity and mortality. To define dengue virus (DENV) target cells and immunological hallmarks of SD progression in children's blood, we integrated two single-cell approaches capturing cellular and viral elements: virus-inclusive single-cell RNA sequencing (viscRNA-Seq 2) and targeted proteomics with secretome analysis and functional assays. Beyond myeloid cells, in natural infection, B cells harbor replicating DENV capable of infecting permissive cells. Alterations in cell type abundance, gene and protein expression and secretion as well as cell-cell communications point towards increased immune cell migration and inflammation in SD progressors. Concurrently, antigen-presenting cells from SD progressors demonstrate intact uptake yet impaired interferon response and antigen processing and presentation signatures, which are partly modulated by DENV. Increased activation, regulation and exhaustion of effector responses and expansion of HLA-DR-expressing adaptive-like NK cells also characterize SD progressors. These findings reveal DENV target cells in human blood and provide insight into SD pathogenesis beyond antibody-mediated enhancement.

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family of receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, ErbB2, and ErbB4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, proinflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production, and disruption of blood-brain barrier integrity in microfluidics-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof of principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Dynamic interactome of the MHC I peptide loading complex in human dendritic cells.

Dendritic cells (DCs) orchestrate immune responses by presenting antigenic peptides on major histocompatibility complex (MHC) molecules to T cells. Antigen processing and presentation via MHC I rely on the peptide-loading complex (PLC), a supramolecular machinery assembled around the transporter associated with antigen processing (TAP), which is the peptide transporter in the endoplasmic reticulum (ER) membrane. We studied antigen presentation in human DCs by isolating monocytes from blood and differentiating them into immature and mature DCs. We uncovered that during DC differentiation and maturation, additional proteins are recruited to the PLC, including B-cell receptor-associated protein 31 (BAP31), vesicle-associated membrane protein-associated protein A (VAPA), and extended synaptotagmin-1 (ESYT1). We demonstrated that these ER cargo export and contact site-tethering proteins colocalize with TAP and are within 40 nm proximity of the PLC, suggesting that the antigen processing machinery is located near ER exit- and membrane contact sites. While CRISPR/Cas9-mediated deletion of TAP and tapasin significantly reduced MHC I surface expression, single-gene deletions of the identified PLC interaction partners revealed a redundant role of BAP31, VAPA, and ESYT1 in MHC I antigen processing in DCs. These data highlight the dynamics and plasticity of PLC composition in DCs that previously was not recognized by the analysis of cell lines.

Single B cell transcriptomics identifies multiple isotypes of broadly neutralizing antibodies against flaviviruses.

Sequential dengue virus (DENV) infections often generate neutralizing antibodies against all four DENV serotypes and sometimes, Zika virus. Characterizing cross-flavivirus broadly neutralizing antibody (bnAb) responses can inform countermeasure strategies that avoid infection enhancement associated with non-neutralizing antibodies. Here, we used single cell transcriptomics to mine the bnAb repertoire following secondary DENV infection. We identified several new bnAbs with comparable or superior breadth and potency to known bnAbs, and with distinct recognition determinants. Unlike all known flavivirus bnAbs, which are IgG1, one newly identified cross-flavivirus bnAb (F25.S02) was derived from IgA1. Both IgG1 and IgA1 versions of F25.S02 and known bnAbs displayed neutralizing activity, but only IgG1 enhanced infection in monocytes expressing IgG and IgA Fc receptors. Moreover, IgG-mediated enhancement of infection was inhibited by IgA1 versions of bnAbs. We demonstrate a role for IgA in flavivirus infection and immunity with implications for vaccine and therapeutic strategies.

Magnitude and kinetics of the human immune cell response associated with severe dengue progression by single-cell proteomics.

Approximately 5 million dengue virus-infected patients progress to a potentially life-threatening severe dengue (SD) infection annually. To identify the immune features and temporal dynamics underlying SD progression, we performed deep immune profiling by mass cytometry of PBMCs collected longitudinally from SD progressors (SDp) and uncomplicated dengue (D) patients. While D is characterized by early activation of innate immune responses, in SDp there is rapid expansion and activation of IgG-secreting plasma cells and memory and regulatory T cells. Concurrently, SDp, particularly children, demonstrate increased proinflammatory NK cells, inadequate expansion of CD16+ monocytes, and high expression of the FcγR CD64 on myeloid cells, yet a signature of diminished antigen presentation. Syndrome-specific determinants include suppressed dendritic cell abundance in shock/hemorrhage versus enriched plasma cell expansion in organ impairment. This study reveals uncoordinated immune responses in SDp and provides insights into SD pathogenesis in humans with potential implications for prediction and treatment.

Anticancer pan-ErbB inhibitors reduce inflammation and tissue injury and exert broad-spectrum antiviral effects.

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

Nanoscale organization of the MHC I peptide-loading complex in human dendritic cells.

Dendritic cells (DCs) translate local innate immune responses into long-lasting adaptive immunity by priming antigen-specific T cells. Accordingly, there is an ample interest in exploiting DCs for therapeutic purposes, e.g., in personalized immunotherapies. Despite recent advances in elucidating molecular pathways of antigen processing, in DCs the exact spatial organization of the underlying processes is largely unknown. Here, we unraveled the nanoscale organization of the transporter associated with antigen processing (TAP)-dependent peptide-loading machinery in human monocyte-derived DCs (moDC). We detected an unexpected accumulation of MHC I peptide-loading complexes (PLCs) and TAP-dependent peptide compartmentalization in protrusions of activated DCs. Using single-molecule localization microscopy we revealed that PLCs display homogeneously sized assemblies, independent of the DC activation status or cellular localization. Our data indicate that moDCs show augmentation of subcellular PLC density during DC maturation. We observed a twofold density increase in the cell body, while an even fourfold accumulation was detected in the tips of the protrusions at the mature DC stage in comparison to immature DCs. In these tip regions, PLC assemblies are found along highly compressed tubular ER networks. These findings provide novel insights into nanoscale organization of the antigen presentation machinery, and open new perspectives on the T cell stimulatory capacity of DCs.

Sequential MAVS and MyD88/TRIF signaling triggers anti-viral responses of tick-borne encephalitis virus-infected murine astrocytes.

Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, is typically transmitted upon tick bite and can cause meningitis and encephalitis in humans. In TBEV-infected mice, mitochondrial antiviral-signaling protein (MAVS), the downstream adaptor of retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling, is needed to induce early type I interferon (IFN) responses and to confer protection. To characterize the brain-resident cell subset that produces protective IFN-ß in TBEV-infected mice, we isolated neurons, astrocytes, and microglia from mice and exposed these cell types to TBEV in vitro. Under such conditions, neurons showed the highest percentage of infected cells, whereas astrocytes and microglia were infected to a lesser extent. In the supernatant (SN) of infected neurons, IFN-ß was not detectable, while infected astrocytes showed high and microglia low IFN-ß expression. Transcriptome analyses of astrocytes implied that MAVS signaling was needed early after TBEV infection. Accordingly, MAVS-deficient astrocytes showed enhanced TBEV infection and significantly reduced early IFN-ß responses. Nevertheless, at later time points, moderate amounts of IFN-ß were detected in the SN of infected MAVS-deficient astrocytes. Transcriptome analyses indicated that MAVS deficiency negatively affected the induction of early anti-viral responses, which resulted in significantly increased TBEV replication. Treatment with MyD88 and TRIF inhibiting peptides reduced only late IFN-ß responses of TBEV-infected WT astrocytes and blocked entirely IFN-ß responses of infected MAVS-deficient astrocytes. Thus, upon TBEV exposure of brain-resident cells, astrocytes are important IFN-ß producers showing biphasic IFN-ß induction that initially depends on MAVS and later on MyD88/TRIF signaling.

Fucosylated lipid nanocarriers loaded with antibiotics efficiently inhibit mycobacterial propagation in human myeloid cells.

Antibiotic treatment of tuberculosis (TB) is complex, lengthy, and can be associated with various adverse effects. As a result, patient compliance often is poor, thus further enhancing the risk of selecting multi-drug resistant bacteria. Macrophage mannose receptor (MMR)-positive alveolar macrophages (AM) constitute a niche in which Mycobacterium tuberculosis replicates and survives. Therefore, we encapsulated levofloxacin in lipid nanocarriers functionalized with fucosyl residues that interact with the MMR. Indeed, such nanocarriers preferentially targeted MMR-positive myeloid cells, and in particular, AM. Intracellularly, fucosylated lipid nanocarriers favorably delivered their payload into endosomal compartments, where mycobacteria reside. In an in vitro setting using infected human primary macrophages as well as dendritic cells, the encapsulated antibiotic cleared the pathogen more efficiently than free levofloxacin. In conclusion, our results point towards carbohydrate-functionalized nanocarriers as a promising tool for improving TB treatment by targeted delivery of antibiotics.

Layer-by-layer modified low density cellulose fiber networks: A sustainable and fireproof alternative to petroleum based foams.

Wood-based cellulose fibers were used to prepare porous, low density and wet-stable fiber networks (FN). Multilayer coatings consisting of chitosan (CH), sodium hexametaphosphate (SHMP) and inorganic nanoparticles comprising of either sodium montmorillonite (MMT), sepiolite (SEP) or colloidal silica (SNP) were deposited by the layer-by-layer (LbL) technique onto FNs in an effort to impart flame-retardancy. A simulated fire scenario measured by cone calorimetry showed that five quadlayers (QL) of CH/SHMP/CH/MMT, CH/SHMP/CH/SEP and CH/SHMP/CH/SNP can produce significant reduction in peak heat release rate (pkHRR). In detail, the coating containing SEP showed the largest reduction of the pkHRR by 47% relative to the uncoated FN. MMT and SEP coated FNs were also able to self-extinguish fire and to retain their shapes after direct exposure to a methane flame. This study hence shows that the LbL assembly is a highly effective way to impart flame-retardant properties to this new type of porous FN.

Reprogramming of Small Noncoding RNA Populations in Peripheral Blood Reveals Host Biomarkers for Latent and Active Mycobacterium tuberculosis Infection.

In tuberculosis (TB), as in other infectious diseases, studies of small noncoding RNAs (sncRNA) in peripheral blood have focused on microRNAs (miRNAs) but have neglected the other major sncRNA classes in spite of their potential functions in host gene regulation. Using RNA sequencing of whole blood, we have therefore determined expression of miRNA, PIWI-interacting RNA (piRNA), small nucleolar RNA (snoRNA), and small nuclear RNA (snRNA) in patients with TB (n = 8), latent TB infection (LTBI; n = 21), and treated LTBI (LTBItt; n = 6) and in uninfected exposed controls (ExC; n = 14). As expected, sncRNA reprogramming was greater in TB than in LTBI, with the greatest changes seen in miRNA populations. However, substantial dynamics were also evident in piRNA and snoRNA populations. One miRNA and 2 piRNAs were identified as moderately accurate (area under the curve [AUC] = 0.70 to 0.74) biomarkers for LTBI, as were 1 miRNA, 1 piRNA, and 2 snoRNAs (AUC = 0.79 to 0.91) for accomplished LTBI treatment. Logistic regression identified the combination of 4 sncRNA (let-7a-5p, miR-589-5p, miR-196b-5p, and SNORD104) as a highly sensitive (100%) classifier to discriminate TB from all non-TB groups. Notably, it reclassified 8 presumed LTBI cases as TB cases, 5 of which turned out to have features of Mycobacterium tuberculosis infection on chest radiographs. SNORD104 expression decreased during M. tuberculosis infection of primary human peripheral blood mononuclear cells (PBMC) and M2-like (P = 0.03) but not M1-like (P = 0.31) macrophages, suggesting that its downregulation in peripheral blood in TB is biologically relevant. Taken together, the results demonstrate that snoRNA and piRNA should be considered in addition to miRNA as biomarkers and pathogenesis factors in the various stages of TB.IMPORTANCE Tuberculosis is the infectious disease with the worldwide largest disease burden and there remains a great need for better diagnostic biomarkers to detect latent and active M. tuberculosis infection. RNA molecules hold great promise in this regard, as their levels of expression may differ considerably between infected and uninfected subjects. We have measured expression changes in the four major classes of small noncoding RNAs in blood samples from patients with different stages of TB infection. We found that, in addition to miRNAs (which are known to be highly regulated in blood cells from TB patients), expression of piRNA and snoRNA is greatly altered in both latent and active TB, yielding promising biomarkers. Even though the functions of many sncRNA other than miRNA are still poorly understood, our results strongly suggest that at least piRNA and snoRNA populations may represent hitherto underappreciated players in the different stages of TB infection.

Preferential uptake of chitosan-coated PLGA nanoparticles by primary human antigen presenting cells.

Biodegradable polymeric nanoparticles (NP) made from poly (lactid-co-glycolide) acid (PLGA) and chitosan (CS) hold promise as innovative formulations for targeted delivery. Since interactions of such NP with primary human immune cells have not been characterized, yet, here we assessed the effect of PLGA or CS-PLGA NP treatment on human peripheral blood mononuclear cells (PBMC), as well as on monocyte-derived DC (moDC). Amongst PBMC, antigen presenting cells (APC) showed higher uptake of both NP preparations than lymphocytes. Furthermore, moDC internalized CS-PLGA NP more efficiently than PLGA NP, presumably because of receptor-mediated endocytosis. Consequently, CS-PLGA NP were delivered mostly to endosomal compartments, whereas PLGA NP primarily ended up in lysosomes. Thus, CS-PLGA NP confer enhanced delivery to endosomal compartments of APC, offering new therapeutic options to either induce or modulate APC function and to inhibit pathogens that preferentially infect APC.

Human monocyte-derived macrophages inhibit HCMV spread independent of classical antiviral cytokines.

Infection of healthy individuals with human cytomegalovirus (HCMV) is usually unnoticed and results in life-long latency, whereas HCMV reactivation as well as infection of newborns or immunocompromised patients can cause life-threatening disease. To better understand HCMV pathogenesis we studied mechanisms that restrict HCMV spread. We discovered that HCMV-infected cells can directly trigger plasmacytoid dendritic cells (pDC) to mount antiviral type I interferon (IFN-I) responses, even in the absence of cell-free virus. In contrast, monocyte-derived cells only expressed IFN-I when stimulated by cell-free HCMV, or upon encounter of HCMV-infected cells that already produced cell-free virus. Nevertheless, also in the absence of cell-free virus, i.e., upon co-culture of infected epithelial/endothelial cells and monocyte-derived macrophages (moMΦ) or dendritic cells (moDC), antiviral responses were induced that limited HCMV spread. The induction of this antiviral effect was dependent on cell-cell contact, whereas cell-free supernatants from co-culture experiments also inhibited virus spread, implying that soluble factors were critically needed. Interestingly, the antiviral effect was independent of IFN-γ, TNF-α, and IFN-I as indicated by cytokine inhibition experiments using neutralizing antibodies or the vaccinia virus-derived soluble IFN-I binding protein B18R, which traps human IFN-α and IFN-ß. In conclusion, our results indicate that human macrophages and dendritic cells can limit HCMV spread by IFN-I dependent as well as independent mechanisms, whereas the latter ones might be particularly relevant for the restriction of HCMV transmission via cell-to-cell spread.

Chemical modification of cellulose-rich fibres to clarify the influence of the chemical structure on the physical and mechanical properties of cellulose fibres and thereof made sheets.

Despite the different chemical approaches used earlier to increase the ductility of fibre-based materials, it has not been possible to link the chemical modification to their mechanical performance. In this study, cellulose fibres have been modified by periodate oxidation, alone or followed either by borohydride reduction, reductive amination or chlorite oxidation. In addition, TEMPO oxidation, and TEMPO oxidation in combination with periodate oxidation and further reduction with sodium borohydride have also been studied. The objective was to gain understanding of the influence of different functional groups on the mechanical and structural properties of handsheets made from the modified fibres. It was found that the modifications studied improved the tensile strength of the fibres to different extents, but that only periodate oxidation followed by borohydride reduction provided more ductile fibre materials. Changes in density, water-holding capacity and mechanical performance were also quantified and all are dependent on the functional group introduced.

Type I IFN and not TNF, is Essential for Cyclic Di-nucleotide-elicited CTL by a Cytosolic Cross-presentation Pathway.

Cyclic di-nucleotides (CDN) are potent stimulators of innate and adaptive immune responses. Cyclic di-AMP (CDA) is a promising adjuvant that generates humoral and cellular immunity. The strong STING-dependent stimulation of type I IFN represents a key feature of CDA. However, recent studies suggested that this is dispensable for adjuvanticity. Here we demonstrate that stimulation of IFN-γ-secreting CD8+ cytotoxic T lymphocytes (CTL) is significantly decreased after vaccination in the absence of type I IFN signaling. The biological significance of this CTL response was confirmed by the stimulation of MHC class I-restricted protection against influenza virus challenge. We show here that type I IFN (and not TNF-α) is essential for CDA-mediated cross-presentation by a cathepsin independent, TAP and proteosome dependent cytosolic antigen processing pathway, which promotes effective cross-priming and further CTL induction. Our data clearly demonstrate that type I IFN signaling is critical for CDN-mediated cross-presentation.

Varicella zoster virus glycoprotein C increases chemokine-mediated leukocyte migration.

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans.