Identification of FasL as a crucial host factor driving COVID-19 pathology and lethality.

The dysregulated immune response and inflammation resulting in severe COVID-19 are still incompletely understood. Having recently determined that aberrant death-ligand-induced cell death can cause lethal inflammation, we hypothesized that this process might also cause or contribute to inflammatory disease and lung failure following SARS-CoV-2 infection. To test this hypothesis, we developed a novel mouse-adapted SARS-CoV-2 model (MA20) that recapitulates key pathological features of COVID-19. Concomitantly with occurrence of cell death and inflammation, FasL expression was significantly increased on inflammatory monocytic macrophages and NK cells in the lungs of MA20-infected mice. Importantly, therapeutic FasL inhibition markedly increased survival of both, young and old MA20-infected mice coincident with substantially reduced cell death and inflammation in their lungs. Intriguingly, FasL was also increased in the bronchoalveolar lavage fluid of critically-ill COVID-19 patients. Together, these results identify FasL as a crucial host factor driving the immuno-pathology that underlies COVID-19 severity and lethality, and imply that patients with severe COVID-19 may significantly benefit from therapeutic inhibition of FasL.

RHBDL4-triggered downregulation of COPII adaptor protein TMED7 suppresses TLR4-mediated inflammatory signaling.

The toll-like receptor 4 (TLR4) is a central regulator of innate immunity that primarily recognizes bacterial lipopolysaccharide cell wall constituents to trigger cytokine secretion. We identify the intramembrane protease RHBDL4 as a negative regulator of TLR4 signaling. We show that RHBDL4 triggers degradation of TLR4's trafficking factor TMED7. This counteracts TLR4 transport to the cell surface. Notably, TLR4 activation mediates transcriptional upregulation of RHBDL4 thereby inducing a negative feedback loop to reduce TLR4 trafficking to the plasma membrane. This secretory cargo tuning mechanism prevents the over-activation of TLR4-dependent signaling in an in vitro Mycobacterium tuberculosis macrophage infection model and consequently alleviates septic shock in a mouse model. A hypomorphic RHBDL4 mutation linked to Kawasaki syndrome, an ill-defined inflammatory disorder in children, further supports the pathophysiological relevance of our findings. In this work, we identify an RHBDL4-mediated axis that acts as a rheostat to prevent over-activation of the TLR4 pathway.

Discovery of dual-active ethionamide boosters inhibiting the Mycobacterium tuberculosis ESX-1 secretion system.

Drug-resistant Mycobacterium tuberculosis (Mtb) remains a major public health concern requiring complementary approaches to standard anti-tuberculous regimens. Anti-virulence molecules or compounds that enhance the activity of antimicrobial prodrugs are promising alternatives to conventional antibiotics. Exploiting host cell-based drug discovery, we identified an oxadiazole compound (S3) that blocks the ESX-1 secretion system, a major virulence factor of Mtb. S3-treated mycobacteria showed impaired intracellular growth and a reduced ability to lyse macrophages. RNA sequencing experiments of drug-exposed bacteria revealed strong upregulation of a distinct set of genes including ethA, encoding a monooxygenase activating the anti-tuberculous prodrug ethionamide. Accordingly, we found a strong ethionamide boosting effect in S3-treated Mtb. Extensive structure-activity relationship experiments revealed that anti-virulence and ethionamide-boosting activity can be uncoupled by chemical modification of the primary hit molecule. To conclude, this series of dual-active oxadiazole compounds targets Mtb via two distinct mechanisms of action.

Discovery of highly neutralizing human antibodies targeting Pseudomonas aeruginosa.

Drug-resistant Pseudomonas aeruginosa (PA) poses an emerging threat to human health with urgent need for alternative therapeutic approaches. Here, we deciphered the B cell and antibody response to the virulence-associated type III secretion system (T3SS) in a cohort of patients chronically infected with PA. Single-cell analytics revealed a diverse B cell receptor repertoire directed against the T3SS needle-tip protein PcrV, enabling the production of monoclonal antibodies (mAbs) abrogating T3SS-mediated cytotoxicity. Mechanistic studies involving cryoelectron microscopy identified a surface-exposed C-terminal PcrV epitope as the target of highly neutralizing mAbs with broad activity against drug-resistant PA isolates. These anti-PcrV mAbs were as effective as treatment with conventional antibiotics in vivo. Our study reveals that chronically infected patients represent a source of neutralizing antibodies, which can be exploited as therapeutics against PA.

The unique ORF8 protein from SARS-CoV-2 binds to human dendritic cells and induces a hyper-inflammatory cytokine storm.

The novel coronavirus pandemic, first reported in December 2019, was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection leads to a strong immune response and activation of antigen-presenting cells, which can elicit acute respiratory distress syndrome (ARDS) characterized by the rapid onset of widespread inflammation, the so-called cytokine storm. In response to viral infections, monocytes are recruited into the lung and subsequently differentiate into dendritic cells (DCs). DCs are critical players in the development of the acute lung inflammation that causes ARDS. Here we focus on the interaction of a specific SARS-CoV-2 open reading frame protein, ORF8, with DCs. We show that ORF8 binds to DCs, causes a pre-maturation of differentiating DCs, and induces the secretion of multiple proinflammatory cytokines by these cells. In addition, we identified DC-SIGN as a possible interaction partner of ORF8 on DCs. Blockade of ORF8 leads to reduced production of IL-1ß, IL-6, IL-12p70, TNF-α, MCP-1 (also named CCL2), and IL-10 by DCs. Therefore, a neutralizing antibody blocking the ORF8-mediated cytokine and chemokine response could be an improved therapeutical strategy against severe SARS-CoV-2.

Immunological fingerprint in coronavirus disease-19 convalescents with and without post-COVID syndrome.

Background: Symptoms lasting longer than 12 weeks after severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection are called post-coronavirus disease (COVID) syndrome (PCS). The identification of new biomarkers that predict the occurrence or course of PCS in terms of a post-viral syndrome is vital. T-cell dysfunction, cytokine imbalance, and impaired autoimmunity have been reported in PCS. Nevertheless, there is still a lack of conclusive information on the underlying mechanisms due to, among other things, a lack of controlled study designs. Methods: Here, we conducted a prospective, controlled study to characterize the humoral and cellular immune response in unvaccinated patients with and without PCS following SARS-CoV-2 infection over 7 months and unexposed donors. Results: Patients with PCS showed as early as 6 weeks and 7 months after symptom onset significantly increased frequencies of SARS-CoV-2-specific CD4+ and CD8+ T-cells secreting IFNγ, TNF, and expressing CD40L, as well as plasmacytoid dendritic cells (pDC) with an activated phenotype. Remarkably, the immunosuppressive counterparts type 1 regulatory T-cells (TR1: CD49b/LAG-3+) and IL-4 were more abundant in PCS+. Conclusion: This work describes immunological alterations between inflammation and immunosuppression in COVID-19 convalescents with and without PCS, which may provide potential directions for future epidemiological investigations and targeted treatments.

Fully Human Herpesvirus-Specific Neutralizing IgG Antibodies Generated by EBV Immortalization of Splenocytes-Derived from Immunized Humanized Mice.

Antiviral neutralizing antibodies (nAbs) are commonly derived from B cells developed in immunized or infected animals and humans. Fully human antibodies are preferred for clinical use as they are potentially less immunogenic. However, the function of B cells varies depending on their homing pattern and an additional hurdle for antibody discovery in humans is the source of human tissues with an immunological microenvironment. Here, we show an efficient method to pharm human antibodies using immortalized B cells recovered from Nod.Rag.Gamma (NRG) mice reconstituting the human immune system (HIS). Humanized HIS mice were immunized either with autologous engineered dendritic cells expressing the human cytomegalovirus gB envelope protein (HCMV-gB) or with Epstein-Barr virus-like particles (EB-VLP). Human B cells recovered from spleen of HIS mice were efficiently immortalized with EBV in vitro. We show that these immortalized B cells secreted human IgGs with neutralization capacities against prototypic HCMV-gB and EBV-gp350. Taken together, we show that HIS mice can be successfully used for the generation and pharming fully human IgGs. This technology can be further explored to generate antibodies against emerging infections for diagnostic or therapeutic purposes.

Monocyte-crosstalk drives interferon-mediated signaling following SARS-CoV-2 exposure.

Cells of the innate immune system represent the first line of defense against SARS-CoV-2 and play an essential role in activating adaptive immunity, which mediates long-term protection. In addition, the same cells are key drivers of tissue damage by causing the hyperinflammatory state and cytokine storm that makes COVID-19 a deadly disease. Thus, careful dissection of the host-pathogen interaction on a cellular level is essential to understanding SARS-CoV-2 pathogenesis and developing new treatment modalities against COVID-19. In their recent work, Goffinet and colleagues (Kazmierski et al, 2022) investigate the cell-intrinsic responses of human primary peripheral blood mononuclear cells (PBMCs) exposed to SARS coronaviruses.

Spleen tyrosine kinase mediates innate and adaptive immune crosstalk in SARS-CoV-2 mRNA vaccination.

Durable cell-mediated immune responses require efficient innate immune signaling and the release of pro-inflammatory cytokines. How precisely mRNA vaccines trigger innate immune cells for shaping antigen specific adaptive immunity remains unknown. Here, we show that SARS-CoV-2 mRNA vaccination primes human monocyte-derived macrophages for activation of the NLRP3 inflammasome. Spike protein exposed macrophages undergo NLRP3-driven pyroptotic cell death and subsequently secrete mature interleukin-1ß. These effects depend on activation of spleen tyrosine kinase (SYK) coupled to C-type lectin receptors. Using autologous cocultures, we show that SYK and NLRP3 orchestrate macrophage-driven activation of effector memory T cells. Furthermore, vaccination-induced macrophage priming can be enhanced with repetitive antigen exposure providing a rationale for prime-boost concepts to augment innate immune signaling in SARS-CoV-2 vaccination. Collectively, these findings identify SYK as a regulatory node capable of differentiating between primed and unprimed macrophages, which modulate spike protein-specific T cell responses.

Gasdermin D mediates host cell death but not interleukin-1ß secretion in Mycobacterium tuberculosis-infected macrophages.

Necrotic cell death represents a major pathogenic mechanism of Mycobacterium tuberculosis (Mtb) infection. It is increasingly evident that Mtb induces several types of regulated necrosis but how these are interconnected and linked to the release of pro-inflammatory cytokines remains unknown. Exploiting a clinical cohort of tuberculosis patients, we show here that the number and size of necrotic lesions correlates with IL-1ß plasma levels as a strong indicator of inflammasome activation. Our mechanistic studies reveal that Mtb triggers mitochondrial permeability transition (mPT) and subsequently extensive macrophage necrosis, which requires activation of the NLRP3 inflammasome. NLRP3-driven mitochondrial damage is dependent on proteolytic activation of the pore-forming effector protein gasdermin D (GSDMD), which links two distinct cell death machineries. Intriguingly, GSDMD, but not the membranolytic mycobacterial ESX-1 secretion system, is dispensable for IL-1ß secretion from Mtb-infected macrophages. Thus, our study dissects a novel mechanism of pathogen-induced regulated necrosis by identifying mitochondria as central regulatory hubs capable of delineating cytokine secretion and lytic cell death.

Viral Glycoproteins Induce NLRP3 Inflammasome Activation and Pyroptosis in Macrophages.

Infections with viral pathogens are widespread and can cause a variety of different diseases. In-depth knowledge about viral triggers initiating an immune response is necessary to decipher viral pathogenesis. Inflammasomes, as part of the innate immune system, can be activated by viral pathogens. However, viral structural components responsible for inflammasome activation remain largely unknown. Here we analyzed glycoproteins derived from SARS-CoV-1/2, HCMV and HCV, required for viral entry and fusion, as potential triggers of NLRP3 inflammasome activation and pyroptosis in THP-1 macrophages. All tested glycoproteins were able to potently induce NLRP3 inflammasome activation, indicated by ASC-SPECK formation and secretion of cleaved IL-1ß. Lytic cell death via gasdermin D (GSDMD), pore formation, and pyroptosis are required for IL-1ß release. As a hallmark of pyroptosis, we were able to detect cleavage of GSDMD and, correspondingly, cell death in THP-1 macrophages. CRISPR-Cas9 knockout of NLRP3 and GSDMD in THP-1 macrophages confirmed and strongly support the evidence that viral glycoproteins can act as innate immunity triggers. With our study, we decipher key mechanisms of viral pathogenesis by showing that viral glycoproteins potently induce innate immune responses. These insights could be beneficial in vaccine development and provide new impulses for the investigation of vaccine-induced innate immunity.

In Vivo Lentiviral Gene Delivery of HLA-DR and Vaccination of Humanized Mice for Improving the Human T and B Cell Immune Reconstitution.

Humanized mouse models generated with human hematopoietic stem cells (HSCs) and reconstituting the human immune system (HIS-mice) are invigorating preclinical testing of vaccines and immunotherapies. We have recently shown that human engineered dendritic cells boosted bonafide human T and B cell maturation and antigen-specific responses in HIS-mice. Here, we evaluated a cell-free system based on in vivo co-delivery of lentiviral vectors (LVs) for expression of a human leukocyte antigen (HLA-DRA*01/ HLA-DRB1*0401 functional complex, "DR4"), and a LV vaccine expressing human cytokines (GM-CSF and IFN-α) and a human cytomegalovirus gB antigen (HCMV-gB). Humanized NOD/Rag1null/IL2Rγnull (NRG) mice injected by i.v. with LV-DR4/fLuc showed long-lasting (up to 20 weeks) vector distribution and expression in the spleen and liver. In vivo administration of the LV vaccine after LV-DR4/fLuc delivery boosted the cellularity of lymph nodes, promoted maturation of terminal effector CD4+ T cells, and promoted significantly higher development of IgG+ and IgA+ B cells. This modular lentigenic system opens several perspectives for basic human immunology research and preclinical utilization of LVs to deliver HLAs into HIS-mice.

Long-lived macrophage reprogramming drives spike protein-mediated inflammasome activation in COVID-19.

Innate immunity triggers responsible for viral control or hyperinflammation in COVID-19 are largely unknown. Here we show that the SARS-CoV-2 spike protein (S-protein) primes inflammasome formation and release of mature interleukin-1ß (IL-1ß) in macrophages derived from COVID-19 patients but not in macrophages from healthy SARS-CoV-2 naïve individuals. Furthermore, longitudinal analyses reveal robust S-protein-driven inflammasome activation in macrophages isolated from convalescent COVID-19 patients, which correlates with distinct epigenetic and gene expression signatures suggesting innate immune memory after recovery from COVID-19. Importantly, we show that S-protein-driven IL-1ß secretion from patient-derived macrophages requires non-specific monocyte pre-activation in vivo to trigger NLRP3-inflammasome signaling. Our findings reveal that SARS-CoV-2 infection causes profound and long-lived reprogramming of macrophages resulting in augmented immunogenicity of the SARS-CoV-2 S-protein, a major vaccine antigen and potent driver of adaptive and innate immune signaling.

Induced dendritic cells co-expressing GM-CSF/IFN-α/tWT1 priming T and B cells and automated manufacturing to boost GvL.

Acute myeloid leukemia (AML) patients with minimal residual disease and receiving allogeneic hematopoietic stem cell transplantation (HCT) have poor survival. Adoptive administration of dendritic cells (DCs) presenting the Wilms tumor protein 1 (WT1) leukemia-associated antigen can potentially stimulate de novo T and B cell development to harness the graft-versus-leukemia (GvL) effect after HCT. We established a simple and fast genetic modification of monocytes for simultaneous lentiviral expression of a truncated WT1 antigen (tWT1), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon (IFN)-α, promoting their self-differentiation into potent "induced DCs" (iDCtWT1). A tricistronic integrase-defective lentiviral vector produced under good manufacturing practice (GMP)-like conditions was validated. Transduction of CD14+ monocytes isolated from peripheral blood, cord blood, and leukapheresis material effectively induced their self-differentiation. CD34+ cell-transplanted Nod.Rag.Gamma (NRG)- and Nod.Scid.Gamma (NSG) mice expressing human leukocyte antigen (HLA)-A∗0201 (NSG-A2)-immunodeficient mice were immunized with autologous iDCtWT1. Both humanized mouse models showed improved development and maturation of human T and B cells in the absence of adverse effects. Toward clinical use, manufacturing of iDCtWT1 was up scaled and streamlined using the automated CliniMACS Prodigy system. Proof-of-concept clinical-scale runs were feasible, and the 38-h process enabled standardized production and high recovery of a cryopreserved cell product with the expected identity characteristics. These results advocate for clinical trials testing iDCtWT1 to boost GvL and eradicate leukemia.

Correction: Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV.

[This corrects the article DOI: 10.1371/journal.ppat.1008560.].

A comparative analysis of remdesivir and other repurposed antivirals against SARS-CoV-2.

The ongoing SARS-CoV-2 pandemic stresses the need for effective antiviral drugs that can quickly be applied in order to reduce morbidity, mortality, and ideally viral transmission. By repurposing of broadly active antiviral drugs and compounds that are known to inhibit viral replication of related viruses, several advances could be made in the development of treatment strategies against COVID-19. The nucleoside analog remdesivir, which is known for its potent in vitro activity against Ebolavirus and other RNA viruses, was recently shown to reduce the time to recovery in patients with severe COVID-19. It is to date the only approved antiviral for treating COVID-19. Here, we provide a mechanism and evidence-based comparative review of remdesivir and other repurposed drugs with proven in vitro activity against SARS-CoV-2.

CAR-T Cells Targeting Epstein-Barr Virus gp350 Validated in a Humanized Mouse Model of EBV Infection and Lymphoproliferative Disease.

Epstein-Barr virus (EBV) is a latent and oncogenic human herpesvirus. Lytic viral protein expression plays an important role in EBV-associated malignancies. The EBV envelope glycoprotein 350 (gp350) is expressed abundantly during EBV lytic reactivation and sporadically on the surface of latently infected cells. Here we tested T cells expressing gp350-specific chimeric antigen receptors (CARs) containing scFvs derived from two novel gp350-binding, highly neutralizing monoclonal antibodies. The scFvs were fused to CD28/CD3ζ signaling domains in a retroviral vector. The produced gp350CAR-T cells specifically recognized and killed gp350+ 293T cells in vitro. The best-performing 7A1-gp350CAR-T cells were cytotoxic against the EBV+ B95-8 cell line, showing selectivity against gp350+ cells. Fully humanized Nod.Rag.Gamma mice transplanted with cord blood CD34+ cells and infected with the EBV/M81/fLuc lytic strain were monitored dynamically for viral spread. Infected mice recapitulated EBV-induced lymphoproliferation, tumor development, and systemic inflammation. We tested adoptive transfer of autologous CD8+gp350CAR-T cells administered protectively or therapeutically. After gp350CAR-T cell therapy, 75% of mice controlled or reduced EBV spread and showed lower frequencies of EBER+ B cell malignant lymphoproliferation, lack of tumor development, and reduced inflammation. In summary, CD8+gp350CAR-T cells showed proof-of-concept preclinical efficacy against impending EBV+ lymphoproliferation and lymphomagenesis.

Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV.

Human cytomegalovirus (HCMV) causes serious complications to immune compromised hosts. Dendritic cells (iDCgB) expressing granulocyte-macrophage colony-stimulating factor, interferon-alpha and HCMV-gB were developed to promote de novo antiviral adaptive responses. Mice reconstituted with a human immune system (HIS) were immunized with iDCgB and challenged with HCMV, resulting into 93% protection. Immunization stimulated the expansion of functional effector memory CD8+ and CD4+ T cells recognizing gB. Machine learning analyses confirmed bone marrow T/CD4+, liver B/IgA+ and spleen B/IgG+ cells as predictive biomarkers of immunization (≈87% accuracy). CD8+ and CD4+ T cell responses against gB were validated. Splenic gB-binding IgM-/IgG+ B cells were sorted and analyzed at a single cell level. iDCgB immunizations elicited human-like IgG responses with a broad usage of various IgG heavy chain V gene segments harboring variable levels of somatic hypermutation. From this search, two gB-binding human monoclonal IgGs were generated that neutralized HCMV infection in vitro. Passive immunization with these antibodies provided proof-of-concept evidence of protection against HCMV infection. This HIS/HCMV in vivo model system supported the validation of novel active and passive immune therapies for future clinical translation.

Adult and Cord Blood-Derived High-Affinity gB-CAR-T Cells Effectively React Against Human Cytomegalovirus Infections.

Human cytomegalovirus (HCMV) reactivations are associated with lower overall survival after transplantations. Adoptive transfer of HCMV-reactive expanded or selected T cells can be applied as a compassionate use, but requires that the human leukocyte antigen-matched donor provides memory cells against HCMV. To overcome this, we developed engineered T cells expressing chimeric antigen receptors (CARs) targeted against the HCMV glycoprotein B (gB) expressed upon viral reactivation. Single-chain variable fragments (scFvs) derived from a human high-affinity gB-specific neutralizing monoclonal antibody (SM5-1) were fused to CARs with 4-1BB (BBL) or CD28 (28S) costimulatory domains and subcloned into retroviral vectors. CD4+ and CD8+ T cells obtained from HCMV-seronegative adult blood or cord blood (CB) transduced with the vectors efficiently expressed the gB-CARs. The specificity and potency of gB-CAR-T cells were demonstrated and compared in vitro using the following: 293T cells expressing gB, and with mesenchymal stem cells infected with a HCMV TB40 strain expressing Gaussia luciferase (HCMV/GLuc). BBL-gB-CAR-T cells generated with adult or CB demonstrated significantly higher in vitro activation and cytotoxicity performance than 28-gB-CAR-T cells. Nod.Rag.Gamma (NRG) mice transplanted with human CB CD34+ cells with long-term human immune reconstitution were used to model HCMV/GLuc infection in vivo by optical imaging analyses. One week after administration, response to BBL-gB-CAR-T cell therapy was observed for 5/8 mice, defined by significant reduction of the bioluminescent signal in relation to untreated controls. Response to therapy was sporadically associated with CAR detection in spleen. Thus, exploring scFv derived from the high-affinity gB-antibody SM5-1 and the 4-1BB signaling domain for CAR design enabled an in vitro high on-target effect and cytotoxicity and encouraging results in vivo. Therefore, gB-CAR-T cells can be a future clinical option for treatment of HCMV reactivations, particularly when memory T cells from the donors are not available.

Modeling Human Cytomegalovirus in Humanized Mice for Vaccine Testing.

Human cytomegalovirus (HCMV or HHV-5) is a globally spread pathogen with strictly human tropism that establishes a lifelong persistence. After primary infection, high levels of long-term T and B cell responses are elicited, but the virus is not cleared. HCMV persists mainly in hematopoietic reservoirs, whereby occasional viral reactivation and spread are well controlled in immunocompetent hosts. However, when the immune system cannot control viral infections or reactivations, such as with newborns, patients with immune deficiencies, or immune-compromised patients after transplantations, the lytic outbursts can be severely debilitating or lethal. The development of vaccines for immunization of immune-compromised hosts has been challenging. Several vaccine candidates did not reach the potency expected in clinical trials and were not approved. Before anti-HCMV vaccines can be tested pre-clinically in immune-compromised hosts, reliable in vivo models recapitulating HCMV infection might accelerate their clinical translation. Therefore, immune-deficient mouse strains implanted with human cells and tissues and developing a human immune system (HIS) are being explored to test anti-HCMV vaccines. HIS-mice resemble immune-compromised hosts as they are equipped with antiviral human T and B cells, but the immune reactivity is overall low. Several groups have independently shown that HCMV infections and reactivations can be mirrored in HIS mice. However, these models and the analyses employed varied widely. The path forward is to improve human immune reconstitution and standardize the analyses of adaptive responses so that HIS models can be forthrightly used for testing novel generations of anti-HCMV vaccines in the preclinical pipeline.