RIG-I and Other RNA Sensors in Antiviral Immunity.

Pattern recognition receptors (PRRs) survey intra- and extracellular spaces for pathogen-associated molecular patterns (PAMPs) within microbial products of infection. Recognition and binding to cognate PAMP ligand by specific PRRs initiates signaling cascades that culminate in a coordinated intracellular innate immune response designed to control infection. In particular, our immune system has evolved specialized PRRs to discriminate viral nucleic acid from host. These are critical sensors of viral RNA to trigger innate immunity in the vertebrate host. Different families of PRRs of virus infection have been defined and reveal a diversity of PAMP specificity for wide viral pathogen coverage to recognize and extinguish virus infection. In this review, we discuss recent insights in pathogen recognition by the RIG-I-like receptors, related RNA helicases, Toll-like receptors, and other RNA sensor PRRs, to present emerging themes in innate immune signaling during virus infection.

RIPK3 Restricts Viral Pathogenesis via Cell Death-Independent Neuroinflammation.

Receptor-interacting protein kinase-3 (RIPK3) is an activator of necroptotic cell death, but recent work has implicated additional roles for RIPK3 in inflammatory signaling independent of cell death. However, while necroptosis has been shown to contribute to antiviral immunity, death-independent roles for RIPK3 in host defense have not been demonstrated. Using a mouse model of West Nile virus (WNV) encephalitis, we show that RIPK3 restricts WNV pathogenesis independently of cell death. Ripk3-/- mice exhibited enhanced mortality compared to wild-type (WT) controls, while mice lacking the necroptotic effector MLKL, or both MLKL and caspase-8, were unaffected. The enhanced susceptibility of Ripk3-/- mice arose from suppressed neuronal chemokine expression and decreased central nervous system (CNS) recruitment of T lymphocytes and inflammatory myeloid cells, while peripheral immunity remained intact. These data identify pleiotropic functions for RIPK3 in the restriction of viral pathogenesis and implicate RIPK3 as a key coordinator of immune responses within the CNS.

The Nucleotide Sensor ZBP1 and Kinase RIPK3 Induce the Enzyme IRG1 to Promote an Antiviral Metabolic State in Neurons.

As long-lived post-mitotic cells, neurons employ unique strategies to resist pathogen infection while preserving cellular function. Here, using a murine model of Zika virus (ZIKV) infection, we identified an innate immune pathway that restricts ZIKV replication in neurons and is required for survival upon ZIKV infection of the central nervous system (CNS). We found that neuronal ZIKV infection activated the nucleotide sensor ZBP1 and the kinases RIPK1 and RIPK3, core components of virus-induced necroptotic cell death signaling. However, activation of this pathway in ZIKV-infected neurons did not induce cell death. Rather, RIPK signaling restricted viral replication by altering cellular metabolism via upregulation of the enzyme IRG1 and production of the metabolite itaconate. Itaconate inhibited the activity of succinate dehydrogenase, generating a metabolic state in neurons that suppresses replication of viral genomes. These findings demonstrate an immunometabolic mechanism of viral restriction during neuroinvasive infection.

Potently neutralizing and protective human antibodies against SARS-CoV-2.

The ongoing pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health1 and the medical countermeasures available so far are limited2,3. Moreover, we currently lack a thorough understanding of the mechanisms of humoral immunity to SARS-CoV-24. Here we analyse a large panel of human monoclonal antibodies that target the spike (S) glycoprotein5, and identify several that exhibit potent neutralizing activity and fully block the receptor-binding domain of the S protein (SRBD) from interacting with human angiotensin-converting enzyme 2 (ACE2). Using competition-binding, structural and functional studies, we show that the monoclonal antibodies can be clustered into classes that recognize distinct epitopes on the SRBD, as well as distinct conformational states of the S trimer. Two potently neutralizing monoclonal antibodies, COV2-2196 and COV2-2130, which recognize non-overlapping sites, bound simultaneously to the S protein and neutralized wild-type SARS-CoV-2 virus in a synergistic manner. In two mouse models of SARS-CoV-2 infection, passive transfer of COV2-2196, COV2-2130 or a combination of both of these antibodies protected mice from weight loss and reduced the viral burden and levels of inflammation in the lungs. In addition, passive transfer of either of two of the most potent ACE2-blocking monoclonal antibodies (COV2-2196 or COV2-2381) as monotherapy protected rhesus macaques from SARS-CoV-2 infection. These results identify protective epitopes on the SRBD and provide a structure-based framework for rational vaccine design and the selection of robust immunotherapeutic agents.

A Small Molecule RIG-I Agonist Serves as an Adjuvant to Induce Broad Multifaceted Influenza Virus Vaccine Immunity.

Retinoic acid-inducible gene I (RIG-I) is essential for activating host cell innate immunity to regulate the immune response against many RNA viruses. We previously identified that a small molecule compound, KIN1148, led to the activation of IFN regulatory factor 3 (IRF3) and served to enhance protection against influenza A virus (IAV) A/California/04/2009 infection. We have now determined direct binding of KIN1148 to RIG-I to drive expression of IFN regulatory factor 3 and NF-κB target genes, including specific immunomodulatory cytokines and chemokines. Intriguingly, KIN1148 does not lead to ATPase activity or compete with ATP for binding but activates RIG-I to induce antiviral gene expression programs distinct from type I IFN treatment. When administered in combination with a vaccine against IAV, KIN1148 induces both neutralizing Ab and IAV-specific T cell responses compared with vaccination alone, which induces comparatively poor responses. This robust KIN1148-adjuvanted immune response protects mice from lethal A/California/04/2009 and H5N1 IAV challenge. Importantly, KIN1148 also augments human CD8+ T cell activation. Thus, we have identified a small molecule RIG-I agonist that serves as an effective adjuvant in inducing noncanonical RIG-I activation for induction of innate immune programs that enhance adaptive immune protection of antiviral vaccination.

Autoimmunity initiates in nonhematopoietic cells and progresses via lymphocytes in an interferon-dependent autoimmune disease.

The type I interferon (IFN) response initiated by detection of nucleic acids is important for antiviral defense but is also associated with specific autoimmune diseases. Mutations in the human 3' repair exonuclease 1 (Trex1) gene cause Aicardi-Goutières syndrome (AGS), an IFN-associated autoimmune disease. However, the source of the type I IFN response and the precise mechanisms of disease in AGS remain unknown. Here, we demonstrate that Trex1 is an essential negative regulator of the STING-dependent antiviral response. We used an in vivo reporter of IFN activity in Trex1-deficient mice to localize the initiation of disease to nonhematopoietic cells. These IFNs drove T cell-mediated inflammation and an autoantibody response that targeted abundant, tissue-restricted autoantigens. However, B cells contributed to mortality independently of T cell-mediated tissue damage. These findings reveal a stepwise progression of autoimmune disease in Trex1-deficient mice, with implications for the treatment of AGS and related disorders.

Immune signaling by RIG-I-like receptors.

The RIG-I-like receptors (RLRs) RIG-I, MDA5, and LGP2 play a major role in pathogen sensing of RNA virus infection to initiate and modulate antiviral immunity. The RLRs detect viral RNA ligands or processed self RNA in the cytoplasm to trigger innate immunity and inflammation and to impart gene expression that serves to control infection. Importantly, RLRs cooperate in signaling crosstalk networks with Toll-like receptors and other factors to impart innate immunity and to modulate the adaptive immune response. RLR regulation occurs at a variety of levels ranging from autoregulation to ligand and cofactor interactions and posttranslational modifications. Abberant RLR signaling or dysregulation of RLR expression is now implicated in the development of autoimmune diseases. Understanding the processes of RLR signaling and response will provide insights to guide RLR-targeted therapeutics for antiviral and immune-modifying applications.

Differential and Overlapping Immune Programs Regulated by IRF3 and IRF5 in Plasmacytoid Dendritic Cells.

We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5, an immune-regulatory transcription factor. We show that the protein kinases IKKα, IKKß, IKKε, and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization, thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets, we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs, linking IRF5 with immune regulatory and proinflammatory gene expression. Thus, TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome.

Class A Scavenger Receptor-Mediated Double-Stranded RNA Internalization Is Independent of Innate Antiviral Signaling and Does Not Require Phosphatidylinositol 3-Kinase Activity.

dsRNA is a potent trigger of innate immune signaling, eliciting effects within virally infected cells and after release from dying cells. Given its inherent stability, extracellular dsRNA induces both local and systemic effects. Although the class A scavenger receptors (SR-As) mediate dsRNA entry, it is unknown whether they contribute to signaling beyond ligand internalization. In this study, we investigated whether SR-As contribute to innate immune signaling independent of the classic TLR and retinoic acid-inducible gene-I-like receptor (RLR) pathways. We generated a stable A549 human epithelial cell line with inducible expression of the hepatitis C virus protease NS3/4A, which efficiently cleaves TRIF and IFN-ß promoter stimulator 1, adaptors for TLR3 and the RLRs, respectively. Cells expressing NS3/4A and TLR3/MyD88/IFN-ß promoter stimulator 1(-/-) mouse embryonic fibroblasts completely lacked antiviral activity to extracellular dsRNA relative to control cells, suggesting that SR-As do not possess signaling capacity independent of TLR3 or the RLRs. Previous studies implicated PI3K signaling in SR-A-mediated activities and in downstream production of type I IFN. We found that SR-A-mediated dsRNA internalization occurs independent of PI3K activation, whereas downstream signaling leading to IFN production was partially dependent on PI3K activity. Overall, these findings suggest that SR-A-mediated dsRNA internalization is independent of innate antiviral signaling.

Membrane Perturbation-Associated Ca2+ Signaling and Incoming Genome Sensing Are Required for the Host Response to Low-Level Enveloped Virus Particle Entry.

UNLABELLED: The type I interferon (IFN) response is an important aspect of innate antiviral defense, and the transcription factor IRF3 plays an important role in its induction. Membrane perturbation during fusion, a necessary step for enveloped virus particle entry, appears sufficient to induce transcription of a subset of IFN-stimulated genes (ISGs) in an IRF3-dependent, IFN-independent fashion. IRF3 is emerging as a central node in host cell stress responses, although it remains unclear how different forms of stress activate IRF3. Here, we investigated the minimum number of Sendai virus (SeV) and human cytomegalovirus (HCMV) particles required to activate IRF3 and trigger an antiviral response. We found that Ca(2+) signaling associated with membrane perturbation and recognition of incoming viral genomes by cytosolic nucleic acid receptors are required to activate IRF3 in response to fewer than 13 particles of SeV and 84 particles of HCMV per cell. Moreover, it appears that Ca(2+) signaling is important for activation of STING and IRF3 following HCMV particle entry, suggesting that Ca(2+) signaling sensitizes cells to recognize genomes within incoming virus particles. To our knowledge, this is the first evidence that cytosolic nucleic acid sensors recognize genomes within incoming virus particles prior to virus replication. These studies highlight the exquisite sensitivity of the cellular response to low-level stimuli and suggest that virus particle entry is sensed as a stress signal. IMPORTANCE: The mechanism by which replicating viruses trigger IRF3 activation and type I IFN induction through the generation and accumulation of viral pathogen-associated molecular patterns has been well characterized. However, the mechanism by which enveloped virus particle entry mediates a stress response, leading to IRF3 activation and the IFN-independent response, remained elusive. Here, we find that Ca(2+) signaling associated with membrane perturbation appears to sensitize cells to recognize genomes within incoming virus particles. To our knowledge, this is the first study to show that cytosolic receptors recognize genomes within incoming virus particles prior to virus replication. These findings not only highlight the sensitivity of cellular responses to low-level virus particle stimulation, but provide important insights into how nonreplicating virus vectors or synthetic lipid-based carriers used as clinical delivery vehicles activate innate immune responses.

Targeting Innate Immunity for Antiviral Therapy through Small Molecule Agonists of the RLR Pathway.

UNLABELLED: The cellular response to virus infection is initiated when pathogen recognition receptors (PRR) engage viral pathogen-associated molecular patterns (PAMPs). This process results in induction of downstream signaling pathways that activate the transcription factor interferon regulatory factor 3 (IRF3). IRF3 plays a critical role in antiviral immunity to drive the expression of innate immune response genes, including those encoding antiviral factors, type 1 interferon, and immune modulatory cytokines, that act in concert to restrict virus replication. Thus, small molecule agonists that can promote IRF3 activation and induce innate immune gene expression could serve as antivirals to induce tissue-wide innate immunity for effective control of virus infection. We identified small molecule compounds that activate IRF3 to differentially induce discrete subsets of antiviral genes. We tested a lead compound and derivatives for the ability to suppress infections caused by a broad range of RNA viruses. Compound administration significantly decreased the viral RNA load in cultured cells that were infected with viruses of the family Flaviviridae, including West Nile virus, dengue virus, and hepatitis C virus, as well as viruses of the families Filoviridae (Ebola virus), Orthomyxoviridae (influenza A virus), Arenaviridae (Lassa virus), and Paramyxoviridae (respiratory syncytial virus, Nipah virus) to suppress infectious virus production. Knockdown studies mapped this response to the RIG-I-like receptor pathway. This work identifies a novel class of host-directed immune modulatory molecules that activate IRF3 to promote host antiviral responses to broadly suppress infections caused by RNA viruses of distinct genera. IMPORTANCE: Incidences of emerging and reemerging RNA viruses highlight a desperate need for broad-spectrum antiviral agents that can effectively control infections caused by viruses of distinct genera. We identified small molecule compounds that can selectively activate IRF3 for the purpose of identifying drug-like molecules that can be developed for the treatment of viral infections. Here, we report the discovery of a hydroxyquinoline family of small molecules that can activate IRF3 to promote cellular antiviral responses. These molecules can prophylactically or therapeutically control infection in cell culture by pathogenic RNA viruses, including West Nile virus, dengue virus, hepatitis C virus, influenza A virus, respiratory syncytial virus, Nipah virus, Lassa virus, and Ebola virus. Our study thus identifies a class of small molecules with a novel mechanism to enhance host immune responses for antiviral activity against a variety of RNA viruses that pose a significant health care burden and/or that are known to cause infections with high case fatality rates.

Direct, interferon-independent activation of the CXCL10 promoter by NF-κB and interferon regulatory factor 3 during hepatitis C virus infection.

Hepatitis C virus (HCV) infection of hepatocytes leads to transcriptional induction of the chemokine CXCL10, which is considered an interferon (IFN)-stimulated gene. However, we have recently shown that IFNs are not required for CXCL10 induction in hepatocytes during acute HCV infection. Since the CXCL10 promoter contains binding sites for several proinflammatory transcription factors, we investigated the contribution of these factors to CXCL10 transcriptional induction during HCV infection in vitro. Wild-type and mutant CXCL10 promoter-luciferase reporter constructs were used to identify critical sites of transcriptional regulation. The proximal IFN-stimulated response element (ISRE) and NF-κB binding sites positively regulated CXCL10 transcription during HCV infection as well as following exposure to poly(I·C) (a Toll-like receptor 3 [TLR3] stimulus) and 5' poly(U) HCV RNA (a retinoic acid-inducible gene I [RIG-I] stimulus) from two viral genotypes. Conversely, binding sites for AP-1 and CCAAT/enhancer-binding protein ß (C/EBP-ß) negatively regulated CXCL10 induction in response to TLR3 and RIG-I stimuli, while only C/EBP-ß negatively regulated CXCL10 during HCV infection. We also demonstrated that interferon-regulatory factor 3 (IRF3) is transiently recruited to the proximal ISRE during HCV infection and localizes to the nucleus in HCV-infected primary human hepatocytes. Furthermore, IRF3 activated the CXCL10 promoter independently of type I or type III IFN signaling. The data indicate that sensing of HCV infection by RIG-I and TLR3 leads to direct recruitment of NF-κB and IRF3 to the CXCL10 promoter. Our study expands upon current knowledge regarding the mechanisms of CXCL10 induction in hepatocytes and lays the foundation for additional mechanistic studies that further elucidate the combinatorial and synergistic aspects of immune signaling pathways.

Convergent evolution of escape from hepaciviral antagonism in primates.

The ability to mount an interferon response on sensing viral infection is a critical component of mammalian innate immunity. Several viruses directly antagonize viral sensing pathways to block activation of the host immune response. Here, we show that recurrent viral antagonism has shaped the evolution of the host protein MAVS--a crucial component of the viral-sensing pathway in primates. From sequencing and phylogenetic analyses of MAVS from 21 simian primates, we found that MAVS has evolved under strong positive selection. We focused on how this positive selection has shaped MAVS' susceptibility to Hepatitis C virus (HCV). We functionally tested MAVS proteins from diverse primate species for their ability to resist antagonism by HCV, which uses its protease NS3/4A to cleave human MAVS. We found that MAVS from multiple primates are resistant to inhibition by the HCV protease. This resistance maps to single changes within the protease cleavage site in MAVS, which protect MAVS from getting cleaved by the HCV protease. Remarkably, most of these changes have been independently acquired at a single residue 506 that evolved under positive selection. We show that "escape" mutations lower affinity of the NS3 protease for MAVS and allow it to better restrict HCV replication. We further show that NS3 proteases from all other primate hepaciviruses, including the highly divergent GBV-A and GBV-C viruses, are functionally similar to HCV. We conclude that convergent evolution at residue 506 in multiple primates has resulted in escape from antagonism by hepaciviruses. Our study provides a model whereby insights into the ancient history of viral infections in primates can be gained using extant host and virus genes. Our analyses also provide a means by which primates might clear infections by extant hepaciviruses like HCV.

Uridine composition of the poly-U/UC tract of HCV RNA defines non-self recognition by RIG-I.

Viral infection of mammalian cells triggers the innate immune response through non-self recognition of pathogen associated molecular patterns (PAMPs) in viral nucleic acid. Accurate PAMP discrimination is essential to avoid self recognition that can generate autoimmunity, and therefore should be facilitated by the presence of multiple motifs in a PAMP that mark it as non-self. Hepatitis C virus (HCV) RNA is recognized as non-self by RIG-I through the presence of a 5'-triphosphate (5'-ppp) on the viral RNA in association with a 3' poly-U/UC tract. Here we define the HCV PAMP and the criteria for RIG-I non-self discrimination of HCV by examining the RNA structure-function attributes that impart PAMP function to the poly-U/UC tract. We found that the 34 nucleotide poly-uridine "core" of this sequence tract was essential for RIG-I activation, and that interspersed ribocytosine nucleotides between poly-U sequences in the RNA were required to achieve optimal RIG-I signal induction. 5'-ppp poly-U/UC RNA variants that stimulated strong RIG-I activation efficiently bound purified RIG-I protein in vitro, and RNA interaction with both the repressor domain and helicase domain of RIG-I was required to activate signaling. When appended to 5'-ppp RNA that lacks PAMP activity, the poly-U/UC U-core sequence conferred non-self recognition of the RNA and innate immune signaling by RIG-I. Importantly, HCV poly-U/UC RNA variants that strongly activated RIG-I signaling triggered potent anti-HCV responses in vitro and hepatic innate immune responses in vivo using a mouse model of PAMP signaling. These studies define a multi-motif PAMP signature of non-self recognition by RIG-I that incorporates a 5'-ppp with poly-uridine sequence composition and length. This HCV PAMP motif drives potent RIG-I signaling to induce the innate immune response to infection. Our studies define a basis of non-self discrimination by RIG-I and offer insights into the antiviral therapeutic potential of targeted RIG-I signaling activation.

AZD3152 neutralizes SARS-CoV-2 historical and contemporary variants and is protective in hamsters and well tolerated in adults.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in variants that can escape neutralization by therapeutic antibodies. Here, we describe AZD3152, a SARS-CoV-2-neutralizing monoclonal antibody designed to provide improved potency and coverage against emerging variants. AZD3152 binds to the back left shoulder of the SARS-CoV-2 spike protein receptor binding domain and prevents interaction with the human angiotensin-converting enzyme 2 receptor. AZD3152 potently neutralized a broad panel of pseudovirus variants, including the currently dominant Omicron variant JN.1 but has reduced potency against XBB subvariants containing F456L. In vitro studies confirmed F456L resistance and additionally identified T415I and K458E as escape mutations. In a Syrian hamster challenge model, prophylactic administration of AZD3152 protected hamsters from weight loss and inflammation-related lung pathologies and reduced lung viral load. In the phase 1 sentinel safety cohort of the ongoing SUPERNOVA study (ClinicalTrials.gov: NCT05648110), a single 600-mg intramuscular injection of AZD5156 (containing 300 mg each of AZD3152 and cilgavimab) was well tolerated in adults through day 91. Observed serum concentrations of AZD3152 through day 91 were similar to those observed with cilgavimab and consistent with predictions for AZD7442, a SARS-CoV-2-neutralizing antibody combination of cilgavimab and tixagevimab, in a population pharmacokinetic model. On the basis of its pharmacokinetic characteristics, AZD3152 is predicted to provide durable protection against symptomatic coronavirus disease 2019 caused by susceptible SARS-CoV-2 variants, such as JN.1, in humans.

Antibody targeting of conserved sites of vulnerability on the SARS-CoV-2 spike receptor-binding domain.

Given the continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VoCs), immunotherapeutics that target conserved epitopes on the spike (S) glycoprotein have therapeutic advantages. Here, we report the crystal structure of the SARS-CoV-2 S receptor-binding domain (RBD) at 1.95 Å and describe flexibility and distinct conformations of the angiotensin-converting enzyme 2 (ACE2)-binding site. We identify a set of SARS-CoV-2-reactive monoclonal antibodies (mAbs) with broad RBD cross-reactivity including SARS-CoV-2 Omicron subvariants, SARS-CoV-1, and other sarbecoviruses and determine the crystal structures of mAb-RBD complexes with Ab246 and CR3022 mAbs targeting the class IV site, WRAIR-2134, which binds the recently designated class V epitope, and WRAIR-2123, the class I ACE2-binding site. The broad reactivity of class IV and V mAbs to conserved regions of SARS-CoV-2 VoCs and other sarbecovirus provides a framework for long-term immunotherapeutic development strategies.

Isoflavone agonists of IRF-3 dependent signaling have antiviral activity against RNA viruses.

There is a growing need for novel antiviral therapies that are broad spectrum, effective, and not subject to resistance due to viral mutations. Using high-throughput screening methods, including computational docking studies and an interferon-stimulated gene 54 (ISG54)-luciferase reporter assay, we identified a class of isoflavone compounds that act as specific agonists of innate immune signaling pathways and cause activation of the interferon regulatory factor (IRF-3) transcription factor. The isoflavone compounds activated the ISG54 promoter, mediated nuclear translocation of IRF-3, and displayed highly potent activity against hepatitis C virus (HCV) and influenza virus. Additionally, these agonists efficiently activated IRF-3 in the presence of the HCV protease NS3-4A, which is known to blunt the host immune response. Furthermore, genomic studies showed that discrete innate immune pathways centered on IRF signaling were regulated following agonist treatment without causing global changes in host gene expression. Following treatment, the expression of only 64 cellular genes was significantly induced. This report provides the first evidence that innate immune pathways dependent on IRF-3 can be successfully targeted by small-molecule drugs for the development of novel broad-spectrum antiviral compounds.

Interferon response factors 3 and 7 protect against Chikungunya virus hemorrhagic fever and shock.

Chikungunya virus (CHIKV) infections can produce severe disease and mortality. Here we show that CHIKV infection of adult mice deficient in interferon response factors 3 and 7 (IRF3/7(-/-)) is lethal. Mortality was associated with undetectable levels of alpha/beta interferon (IFN-α/ß) in serum, ∼50- and ∼10-fold increases in levels of IFN-γ and tumor necrosis factor (TNF), respectively, increased virus replication, edema, vasculitis, hemorrhage, fever followed by hypothermia, oliguria, thrombocytopenia, and raised hematocrits. These features are consistent with hemorrhagic shock and were also evident in infected IFN-α/ß receptor-deficient mice. In situ hybridization suggested CHIKV infection of endothelium, fibroblasts, skeletal muscle, mononuclear cells, chondrocytes, and keratinocytes in IRF3/7(-/-) mice; all but the latter two stained positive in wild-type mice. Vaccination protected IRF3/7(-/-) mice, suggesting that defective antibody responses were not responsible for mortality. IPS-1- and TRIF-dependent pathways were primarily responsible for IFN-α/ß induction, with IRF7 being upregulated >100-fold in infected wild-type mice. These studies suggest that inadequate IFN-α/ß responses following virus infection can be sufficient to induce hemorrhagic fever and shock, a finding with implications for understanding severe CHIKV disease and dengue hemorrhagic fever/dengue shock syndrome.

Thymic stromal lymphopoietin is induced by respiratory syncytial virus-infected airway epithelial cells and promotes a type 2 response to infection.

BACKGROUND: Respiratory viral infection, including respiratory syncytial virus (RSV) and rhinovirus, has been linked to respiratory disease in pediatric patients, including severe acute bronchiolitis and asthma exacerbation. OBJECTIVE: The study examined the role of the epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) in the response to RSV infection. METHODS: Infection of human airway epithelial cells was used to examine TSLP induction after RSV infection. Air-liquid interface cultures from healthy children and children with asthma were also tested for TSLP production after infection. Finally, a mouse model was used to directly test the role of TSLP signaling in the response to RSV infection. RESULTS: Infection of airway epithelial cells with RSV led to the production of TSLP via activation of an innate signaling pathway that involved retinoic acid induced gene I, interferon promoter-stimulating factor 1, and nuclear factor-κB. Consistent with this observation, airway epithelial cells from asthmatic children a produced significantly greater levels of TSLP after RSV infection than cells from healthy children. In mouse models, RSV-induced TSLP expression was found to be critical for the development of immunopathology. CONCLUSION: These findings suggest that RSV can use an innate antiviral signaling pathway to drive a potentially nonproductive immune response and has important implications for the role of TSLP in viral immune responses in general.

Developability profiling of a panel of Fc engineered SARS-CoV-2 neutralizing antibodies.

To combat the COVID-19 pandemic, potential therapies have been developed and moved into clinical trials at an unprecedented pace. Some of the most promising therapies are neutralizing antibodies against SARS-CoV-2. In order to maximize the therapeutic effectiveness of such neutralizing antibodies, Fc engineering to modulate effector functions and to extend half-life is desirable. However, it is critical that Fc engineering does not negatively impact the developability properties of the antibodies, as these properties play a key role in ensuring rapid development, successful manufacturing, and improved overall chances of clinical success. In this study, we describe the biophysical characterization of a panel of Fc engineered ("TM-YTE") SARS-CoV-2 neutralizing antibodies, the same Fc modifications as those found in AstraZeneca's Evusheld (AZD7442; tixagevimab and cilgavimab), in which the TM modification (L234F/L235E/P331S) reduce binding to FcγR and C1q and the YTE modification (M252Y/S254T/T256E) extends serum half-life. We have previously shown that combining both the TM and YTE Fc modifications can reduce the thermal stability of the CH2 domain and possibly lead to developability challenges. Here we show, using a diverse panel of TM-YTE SARS-CoV-2 neutralizing antibodies, that despite lowering the thermal stability of the Fc CH2 domain, the TM-YTE platform does not have any inherent developability liabilities and shows an in vivo pharmacokinetic profile in human FcRn transgenic mice similar to the well-characterized YTE platform. The TM-YTE is therefore a developable, effector function reduced, half-life extended antibody platform.