An inhaled ACE2 decoy confers protection against SARS-CoV-2 infection in preclinical models.

The Omicron variant continuously evolves under the humoral immune pressure exerted by vaccination and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and the resulting Omicron subvariants display further immune evasion and antibody escape. An engineered angiotensin-converting enzyme 2 (ACE2) decoy composed of high-affinity ACE2 and an IgG1 Fc domain could offer an alternative modality to neutralize SARS-CoV-2. We previously reported its broad spectrum and therapeutic potential in rodent models. Here, we demonstrate that the engineered ACE2 decoy retains neutralization activity against Omicron subvariants, including the currently emerging XBB and BQ.1 strains, which completely evade antibodies currently in clinical use. SARS-CoV-2, under the suboptimal concentration of neutralizing drugs, generated SARS-CoV-2 mutants escaping wild-type ACE2 decoy and monoclonal antibodies, whereas no escape mutant emerged against the engineered ACE2 decoy. Furthermore, inhalation of aerosolized decoys improved the outcomes of rodents infected with SARS-CoV-2 at a 20-fold lower dose than that of intravenous administration. Last, the engineered ACE2 decoy exhibited therapeutic efficacy for cynomolgus macaques infected with SARS-CoV-2. These results indicate that this engineered ACE2 decoy represents a promising therapeutic strategy to overcome immune-evading SARS-CoV-2 variants and that liquid aerosol inhalation could be considered as a noninvasive approach to enhance the efficacy of COVID-19 treatments.

Nsp14 of SARS-CoV-2 inhibits mRNA processing and nuclear export by targeting the nuclear cap-binding complex.

To facilitate selfish replication, viruses halt host gene expression in various ways. The nuclear export of mRNA is one such process targeted by many viruses. SARS-CoV-2, the etiological agent of severe acute respiratory syndrome, also prevents mRNA nuclear export. In this study, Nsp14, a bifunctional viral replicase subunit, was identified as a novel inhibitor of mRNA nuclear export. Nsp14 induces poly(A)+ RNA nuclear accumulation and the dissolution/coalescence of nuclear speckles. Genome-wide gene expression analysis revealed the global dysregulation of splicing and 3'-end processing defects of replication-dependent histone mRNAs by Nsp14. These abnormalities were also observed in SARS-CoV-2-infected cells. A mutation introduced at the guanine-N7-methyltransferase active site of Nsp14 diminished these inhibitory activities. Targeted capillary electrophoresis-mass spectrometry analysis (CE-MS) unveiled the production of N7-methyl-GTP in Nsp14-expressing cells. Association of the nuclear cap-binding complex (NCBC) with the mRNA cap and subsequent recruitment of U1 snRNP and the stem-loop binding protein (SLBP) were impaired by Nsp14. These data suggest that the defects in mRNA processing and export arise from the compromise of NCBC function by N7-methyl-GTP, thus exemplifying a novel viral strategy to block host gene expression.

A computationally designed ACE2 decoy has broad efficacy against SARS-CoV-2 omicron variants and related viruses in vitro and in vivo.

SARS-CoV-2, especially B.1.1.529/omicron and its sublineages, continues to mutate to evade monoclonal antibodies and antibodies elicited by vaccination. Affinity-enhanced soluble ACE2 (sACE2) is an alternative strategy that works by binding the SARS-CoV-2 S protein, acting as a 'decoy' to block the interaction between the S and human ACE2. Using a computational design strategy, we designed an affinity-enhanced ACE2 decoy, FLIF, that exhibited tight binding to SARS-CoV-2 delta and omicron variants. Our computationally calculated absolute binding free energies (ABFE) between sACE2:SARS-CoV-2 S proteins and their variants showed excellent agreement to binding experiments. FLIF displayed robust therapeutic utility against a broad range of SARS-CoV-2 variants and sarbecoviruses, and neutralized omicron BA.5 in vitro and in vivo. Furthermore, we directly compared the in vivo therapeutic efficacy of wild-type ACE2 (non-affinity enhanced ACE2) against FLIF. A few wild-type sACE2 decoys have shown to be effective against early circulating variants such as Wuhan in vivo. Our data suggest that moving forward, affinity-enhanced ACE2 decoys like FLIF may be required to combat evolving SARS-CoV-2 variants. The approach described herein emphasizes how computational methods have become sufficiently accurate for the design of therapeutics against viral protein targets. Affinity-enhanced ACE2 decoys remain highly effective at neutralizing omicron subvariants.

Assessing prognostic factors correlating with response to nintedanib for connective tissue disease-associated interstitial lung disease: A real-world single-center study.

OBJECTIVE: For patients with connective tissue disease-associated interstitial lung disease (CTD-ILD), early medical intervention would be desirable. This study analyzed the real-world, single-center use of nintedanib for CTD-ILD patients. METHODS: Patients with CTD who received nintedanib from January 2020 to July 2022 were enrolled. Medical records review and stratified analyses of the collected data were conducted. RESULTS: Reduction in the percentage of predicted forced vital capacity (%FVC) was seen in the elderly group (>70 years; P = .210), males (P = .027), the late group who started nintedanib >80 months after confirmation of an ILD disease activity (P = .03), the severe %DLco (diffusing capacity for carbon monoxide as a percentage of predicted) group (<40%; P = .20), the group who had extensive pulmonary fibrosis at the beginning of nintedanib (pulmonary fibrosis score >35%), and the low-dose group (nintedanib 50-100 mg/d; P = .40). %FVC did not decrease by >5% in the young group (<55 years), the early group who started nintedanib within 10 months after confirmation of an ILD disease activity, and the group whose pulmonary fibrosis score at the beginning of nintedanib was <35%. CONCLUSION: It is important to diagnose ILD early and start antifibrotic drugs with proper timing for cases in need. It is better to start nintedanib early, especially for patients at risk (>70 years old, male, <40% DLco, and >35% areas of pulmonary fibrosis).

Antibody feedback contributes to facilitating the development of Omicron-reactive memory B cells in SARS-CoV-2 mRNA vaccinees.

In contrast to a second dose of the SARS-CoV-2 mRNA vaccine, a third dose elicits potent neutralizing activity against the Omicron variant. To address the underlying mechanism for this differential antibody response, we examined spike receptor-binding domain (RBD)-specific memory B cells in vaccinated individuals. Frequency of Omicron-reactive memory B cells increased ∼9 mo after the second vaccine dose. These memory B cells show an altered distribution of epitopes from pre-second memory B cells, presumably due to an antibody feedback mechanism. This hypothesis was tested using mouse models, showing that an addition or a depletion of RBD-induced serum antibodies results in a concomitant increase or decrease, respectively, of Omicron-reactive germinal center (GC) and memory B cells. Our data suggest that pre-generated antibodies modulate the selection of GC and subsequent memory B cells after the second vaccine dose, accumulating more Omicron-reactive memory B cells over time, which contributes to the generation of Omicron-neutralizing antibodies elicited by the third vaccine dose.

SARS-CoV-2 ORF6 disrupts nucleocytoplasmic trafficking to advance viral replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF6 is an antagonist of interferon (IFN)-mediated antiviral signaling, achieved through the prevention of STAT1 nuclear localization. However, the exact mechanism through which ORF6 prevents STAT1 nuclear trafficking remains unclear. Herein, we demonstrate that ORF6 directly binds to STAT1 with or without IFN stimulation, resulting in the nuclear exclusion of STAT1. ORF6 also recognizes importin α subtypes with different modes, in particular, high affinity to importin α1 but a low affinity to importin α5. Although ORF6 potentially disrupts the importin α/importin ß1-mediated nuclear transport, thereby suppressing the nuclear translocation of the other classical nuclear localization signal-containing cargo proteins, the inhibitory effect of ORF6 is modest when compared with that of STAT1. The results indicate that the drastic nuclear exclusion of STAT1 is attributed to the specific binding with ORF6, which is a distinct strategy for the importin α1-mediated pathway. Combined with the results from a newly-produced replicon system and a hamster model, we conclude that SARS-CoV-2 ORF6 acts as a virulence factor via regulation of nucleocytoplasmic trafficking to accelerate viral replication, resulting in disease progression.

Cell response analysis in SARS-CoV-2 infected bronchial organoids.

The development of an in vitro cell model that can be used to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is expected. Here we conducted infection experiments in bronchial organoids (BO) and an BO-derived air-liquid interface model (BO-ALI) using 8 SARS-CoV-2 variants. The infection efficiency in BO-ALI was more than 1,000 times higher than that in BO. Among the bronchial epithelial cells, we found that ciliated cells were infected with the virus, but basal cells were not. Ciliated cells died 7 days after the viral infection, but basal cells survived after the viral infection and differentiated into ciliated cells. Fibroblast growth factor 10 signaling was essential for this differentiation. These results indicate that BO and BO-ALI may be used not only to evaluate the cell response to SARS-CoV-2 and coronavirus disease 2019 (COVID-19) therapeutic agents, but also for airway regeneration studies.

An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo.

The Omicron (B.1.1.529) SARS-CoV-2 variant contains an unusually high number of mutations in the spike protein, raising concerns of escape from vaccines, convalescent serum, and therapeutic drugs. Here, we analyzed the degree to which Omicron pseudo-virus evades neutralization by serum or therapeutic antibodies. Serum samples obtained 3 months after two doses of BNT162b2 vaccination exhibited 18-fold lower neutralization titers against Omicron than parental virus. Convalescent serum samples from individuals infected with the Alpha and Delta variants allowed similar frequencies of Omicron breakthrough infections. Domain-wise analysis using chimeric spike proteins revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor binding domain but that multiple mutations in the N-terminal domain contributed as well. Omicron escaped a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. Angiotensin-converting enzyme 2 (ACE2) decoys are another virus-neutralizing drug modality that are free, at least in theory, from complete escape. Deep mutational analysis demonstrated that an engineered ACE2 molecule prevented escape for each single-residue mutation in the receptor binding domain, similar to immunized serum. Engineered ACE2 neutralized Omicron comparably to the Wuhan strain and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Similar to previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.

Establishment of monoclonal antibodies broadly neutralize infection of hepatitis B virus.

Antibodies against hepatitis B virus S protein can protect against hepatitis B virus (HBV) infection. Therefore, hepatitis B immunoglobulin (HBIG), which contains HBsAb, is used clinically as a therapy for HBV infection. In this study, a series of monoclonal antibodies that recognize multiple HBV genotypes was obtained. All the antibodies recognized conformational epitopes of S protein, but not linear epitopes. Several antibodies neutralized HBV infection and exhibited strong affinities and neutralizing activities. Antigenic epitope analysis demonstrated that they recognized residue Ile152 of S protein, which is localized outside the "a" determinant. Ile152 is highly conserved, and a mutation in this residue resulted in reduced expression of large hepatitis B surface proteins (L protein), suggesting that the amino acid at this position is involved in the expression of L protein. In addition, the antibodies neutralized the infection of hepatitis D virus possessing a Gly145 mutation to Arg in S protein, which is a well-known escape mutation against HBIG treatment. Using mouse monoclonal antibodies, a humanized antibody possessing affinities and neutralizing activities similar to those of the original mouse antibody was successfully established. The antibodies generated in this study may have the potential for use in alternative antibody therapies for HBV infection.

Engineered ACE2 receptor therapy overcomes mutational escape of SARS-CoV-2.

SARS-CoV-2 has mutated during the global pandemic leading to viral adaptation to medications and vaccinations. Here we describe an engineered human virus receptor, ACE2, by mutagenesis and screening for binding to the receptor binding domain (RBD). Three cycles of random mutagenesis and cell sorting achieved sub-nanomolar affinity to RBD. Our structural data show that the enhanced affinity comes from better hydrophobic packing and hydrogen-bonding geometry at the interface. Additional disulfide mutations caused the fixing of a closed ACE2 conformation to avoid off-target effects of protease activity, and also improved structural stability. Our engineered ACE2 neutralized SARS-CoV-2 at a 100-fold lower concentration than wild type; we also report that no escape mutants emerged in the co-incubation after 15 passages. Therapeutic administration of engineered ACE2 protected hamsters from SARS-CoV-2 infection, decreased lung virus titers and pathology. Our results provide evidence of a therapeutic potential of engineered ACE2.

Hepatitis C virus modulates signal peptide peptidase to alter host protein processing.

Immunoevasins are viral proteins that prevent antigen presentation on major histocompatibility complex (MHC) class I, thus evading host immune recognition. Hepatitis C virus (HCV) evades immune surveillance to induce chronic infection; however, how HCV-infected hepatocytes affect immune cells and evade immune recognition remains unclear. Herein, we demonstrate that HCV core protein functions as an immunoevasin. Its expression interfered with the maturation of MHC class I molecules catalyzed by the signal peptide peptidase (SPP) and induced their degradation via HMG-CoA reductase degradation 1 homolog, thereby impairing antigen presentation to CD8+ T cells. The expression of MHC class I in the livers of HCV core transgenic mice and chronic hepatitis C patients was impaired but was restored in patients achieving sustained virological response. Finally, we show that the human cytomegalovirus US2 protein, possessing a transmembrane region structurally similar to the HCV core protein, targets SPP to impair MHC class I molecule expression. Thus, SPP represents a potential target for the impairment of MHC class I molecules by DNA and RNA viruses.

Deneddylation by SENP8 restricts hepatitis B virus propagation.

Proteins newly synthesized from messenger RNA undergo Posttranslational modifications (PTMs) such as phosphorylation, glycosylation, methylation, and ubiquitination. These PTMs have important roles in protein stability, localization, and conformation and have been reported to be involved in hepatitis B virus (HBV) propagation. Although ubiquitination plays an essential role in HBV life cycles, the involvement of ubiquitin-like proteins (UBLs) in HBV life cycles has been understudied. Through comprehensive gain- and loss-of-function screening of UBLs, we observed that neddylation, a PTM in which neural precursor cell, expressed developmentally downregulated 8 (NEDD8) is conjugated to substrate proteins, was required for efficient HBV propagation. We also found that overexpression of sentrin-specific protease 8 (SENP8), which cleaves conjugated NEDD8, suppressed HBV propagation. Further, the catalytic activity of SENP8 was required for the suppression of HBV propagation. These results indicated that the reduction of neddylation negatively regulated HBV propagation. In addition, we demonstrated that suppression of HBV propagation via SENP8 overexpression was independent of hepatitis B protein X (HBx) and HBV promoter activity. Therefore, our data suggested that neddylation plays an important role in the late stages of HBV life cycles.

Monitoring of glutamate-induced excitotoxicity by mitochondrial oxygen consumption.

Dysfunction of mitochondrial activity is often associated with the onset and progress of neurodegenerative diseases. Membrane depolarization induced by Na+ influx increases intracellular Ca2+ levels in neurons, which upregulates mitochondrial activity. However, overlimit of Na+ influx and its prolonged retention ultimately cause excitotoxicity leading to neuronal cell death. To return the membrane potential to the normal level, Na+ /K+ -ATPase exchanges intracellular Na+ with extracellular K+ by consuming a large amount of ATP. This is a reason why mitochondria are important for maintaining neurons. In addition, astrocytes are thought to be important for supporting neighboring neurons by acting as energy providers and eliminators of excessive neurotransmitters. In this study, we examined the meaning of changes in the mitochondrial oxygen consumption rate (OCR) in primary mouse neuronal populations. By varying the medium constituents and using channel modulators, we found that pyruvate rather than lactate supported OCR levels and conferred on neurons resistance to glutamate-mediated excitotoxicity. Under a pyruvate-restricted condition, our OCR monitoring could detect excitotoxicity induced by glutamate at only 10 µM. The OCR monitoring also revealed the contribution of the N-methyl-D-aspartate receptor and Na+ /K+ -ATPase to the toxicity, which allowed evaluating spontaneous excitation. In addition, the OCR monitoring showed that astrocytes preferentially used glutamate, not glutamine, for a substrate of the tricarboxylic acid cycle. This mechanism may be coupled with astrocyte-dependent protection of neurons from glutamate-mediated excitotoxicity. These results suggest that OCR monitoring would provide a new powerful tool to analyze the mechanisms underlying neurotoxicity and protection against it.

Carnosol suppresses interleukin-6 production in mouse lungs injured by ischemia-reperfusion operation and in RAW264.7 macrophages treated with lipopolysaccharide.

Carnosol is a naturally occurring herbal compound, known for its antioxidative properties. We previously found that carnosol protected mouse lungs from ischemia-reperfusion injury in ex vivo cultures. To elucidate the molecular mechanisms underpinning carnosol-mediated lung protection, we analyzed modes of interleukin-6 (IL-6) gene expression, which is associated with lung ischemia-reperfusion injury. Microarray analysis of mouse lungs suggested that IL-6 mRNA levels were elevated in the mouse lungs subjected to clamp-reperfusion, which was associated with elevated levels of other inflammatory modulators, such as activating transcription factor 3 (ATF3). Carnosol pretreatment lowered the IL-6 protein levels in mouse lung homogenates prepared after the clamp-reperfusion. On the other hand, the ATF3 gene expression was negatively correlated with that of IL-6 in RAW264.7 cells. IL-6 mRNA levels and gene promoter activities were suppressed by carnosol in RAW264.7 cells, but rescued by ATF3 knockdown. When RAW264.7 cells were subjected to hypoxia-reoxygenation, carnosol treatment lowered oxygen consumption after reoxygenation, which was coupled with a correlation with a transient production of mitochondrial reactive oxygen species and following ATF3 gene expression. These results suggest that carnosol treatment could be a new strategy for protecting lungs from ischemia-reperfusion injury by modulating the ATF3-IL-6 axis.

Simple chronic colitis model using hypopigmented mice with a Hermansky-Pudlak syndrome 5 gene mutation.

Pigmentation in mammals is important for protection of skin and eyes from ultraviolet radiation. Dysregulation of pigmentation is often associated with other conditions that are not directly linked to pigmentation. Here, we isolated spontaneously occurring hypopigmented mice that occasionally experienced severe diarrhea during lactation. Treatment of these mice with dextran sulfate sodium salt, a conventional method to induce acute colitis, caused chronic diarrhea with granulomatous colitis. Gene mapping and sequencing revealed that the mice had a nonsense mutation in the Hermansky-Pudlak syndrome (Hps)5 gene. As some HPS patients can develop granulomatous colitis, the simple induction of chronic colitis in spontaneously mutated Hps5-deficient mice may become an invaluable model for exploring treatment options in patients with HPS as well as other patients with inflammatory bowel disease.

Pterosin B prevents chondrocyte hypertrophy and osteoarthritis in mice by inhibiting Sik3.

Osteoarthritis is a common debilitating joint disorder. Risk factors for osteoarthritis include age, which is associated with thinning of articular cartilage. Here we generate chondrocyte-specific salt-inducible kinase 3 (Sik3) conditional knockout mice that are resistant to osteoarthritis with thickened articular cartilage owing to a larger chondrocyte population. We also identify an edible Pteridium aquilinum compound, pterosin B, as a Sik3 pathway inhibitor. We show that either Sik3 deletion or intraarticular injection of mice with pterosin B inhibits chondrocyte hypertrophy and protects cartilage from osteoarthritis. Collectively, our results suggest Sik3 regulates the homeostasis of articular cartilage and is a target for the treatment of osteoarthritis, with pterosin B as a candidate therapeutic.

Pterosin B has multiple targets in gluconeogenic programs, including coenzyme Q in RORα-SRC2 signaling.

Hepatic gluconeogenic programs are regulated by a variety of signaling cascades. Glucagon-cAMP signaling is the main initiator of the gluconeogenic programs, including glucose-6-phosphatase catalytic subunit (G6pc) gene expression. Pterosin B, an ingredient in Pteridium aquilinum, inhibits salt-inducible kinase 3 signaling that represses cAMP-response element-binding protein regulated transcription coactivator 2, an inducer of gluconeogenic programs. As the results, pterosin B promotes G6pc expression even in the absence of cAMP. In this work, however, we noticed that once cAMP signaling was initiated, pterosin B became a strong repressor of G6pc expression. The search for associated transcription factors for pterosin B actions revealed that retinoic acid receptor-related orphan receptor alpha-steroid receptor coactivator 2 (RORα-SRC2) complex on the G6pc promoter was the target. Meanwhile, pterosin B impaired the oxidation-reduction cycle of coenzyme Q in mitochondrial oxidative phosphorylation (OXPHOS); and antimycin A, an inhibitor of coenzyme Q: cytochrome c-oxidoreductase (termed mitochondrial complex III), also mimicked pterosin B actions on RORα-SRC2 signaling. Although other respiratory toxins (rotenone and oligomycin) also suppressed G6pc expression accompanied by lowered ATP levels following the activation of AMP-activated kinase, minimal or no effect of these other toxins on RORα-SRC2 activity was observed. These results suggested that individual components in OXPHOS differentially linked to different transcriptional machineries for hepatic gluconeogenic programs, and the RORα-SRC2 complex acted as a sensor for oxidation-reduction cycle of coenzyme Q and regulated G6Pc expression. This was a site disrupted by pterosin B in gluconeogenic programs.

A Simple Method for Labeling Human Embryonic Stem Cells Destined to Lose Undifferentiated Potency.

Mitochondrial oxidative phosphorylation is a major source of cellular ATP. Its usage as an energy source varies, not only according to the extracellular environment, but also during development and differentiation, as indicated by the reported changes in the flux ratio of glycolysis to oxidative phosphorylation during embryonic stem (ES) cell differentiation. The fluorescent probe JC-1 allows visualization of changes in the mitochondrial membrane potential produced by oxidative phosphorylation. Strong JC-1 signals were localized in the differentiated cells located at the edge of H9 ES colonies that expressed vimentin, an early differentiation maker. The JC-1 signals were further intensified when individual adjacent colonies were in contact with each other. Time-lapse analyses revealed that JC-1-labeled H9 cells under an overconfluent condition were highly differentiated after subculture, suggesting that monitoring oxidative phosphorylation in live cells might facilitate the prediction of induced pluripotent stem cells, as well as ES cells, that are destined to lose their undifferentiated potency.