DS-5670a, a novel mRNA-encapsulated lipid nanoparticle vaccine against severe acute respiratory syndrome coronavirus 2: Results from a phase 2 clinical study.

BACKGROUND: DS-5670a is a vaccine candidate for coronavirus disease 2019 (COVID-19) harnessing a novel modality composed of messenger ribonucleic acid (mRNA) encoding the receptor-binding domain (RBD) from the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encapsulated in lipid nanoparticles. Here, we report the safety, immunogenicity, and pharmacokinetic profile of DS-5670a from a phase 2 clinical trial in healthy adults who were immunologically naïve to SARS-CoV-2. METHODS: The study consisted of an open-label, uncontrolled, dose-escalation part and a double-blind, randomized, uncontrolled, 2-arm, parallel-group part. A total of 80 Japanese participants were assigned to receive intramuscular DS-5670a, containing either 30 or 60 µg of mRNA, as two injections administered 4 weeks apart. Safety was assessed by characterization of treatment-emergent adverse events (TEAEs). Immunogenicity was assessed by neutralization titers against SARS-CoV-2, anti-RBD immunoglobulin (Ig)G levels, and SARS-CoV-2 spike-specific T cell responses. Plasma pharmacokinetic parameters of DS-5670a were also evaluated. RESULTS: Most solicited TEAEs were mild or moderate with both the 30 and 60 µg mRNA doses. Four participants (10 %) in the 60 µg mRNA group developed severe redness at the injection site, but all cases resolved without treatment. There were no serious TEAEs and no TEAEs leading to discontinuation. Humoral immune responses in both dose groups were greater than those observed in human convalescent serum; the 60 µg mRNA dose produced better responses. Neutralization titers were found to be correlated with anti-RBD IgG levels (specifically IgG1). DS-5670a elicited antigen-specific T helper 1-polarized cellular immune responses. CONCLUSIONS: The novel mRNA-based vaccine candidate DS-5670a provided favorable immune responses against SARS-CoV-2 with a clinically acceptable safety profile. Confirmatory trials are currently ongoing to evaluate the safety and immunogenicity of DS-5670a as the primary vaccine and to assess the immunogenicity when administered as a heterologous or homologous booster. TRIAL REGISTRY: https://jrct.niph.go.jp/latest-detail/jRCT2071210086.

Galectin-9 restricts hepatitis B virus replication via p62/SQSTM1-mediated selective autophagy of viral core proteins.

Autophagy has been linked to a wide range of functions, including a degradative process that defends host cells against pathogens. Although the involvement of autophagy in HBV infection has become apparent, it remains unknown whether selective autophagy plays a critical role in HBV restriction. Here, we report that a member of the galectin family, GAL9, directs the autophagic degradation of HBV HBc. BRET screening revealed that GAL9 interacts with HBc in living cells. Ectopic expression of GAL9 induces the formation of HBc-containing cytoplasmic puncta through interaction with another antiviral factor viperin, which co-localized with the autophagosome marker LC3. Mechanistically, GAL9 associates with HBc via viperin at the cytoplasmic puncta and enhanced the auto-ubiquitination of RNF13, resulting in p62 recruitment to form LC3-positive autophagosomes. Notably, both GAL9 and viperin are type I IFN-stimulated genes that act synergistically for the IFN-dependent proteolysis of HBc in HBV-infected hepatocytes. Collectively, these results reveal a previously undescribed antiviral mechanism against HBV in infected cells and a form of crosstalk between the innate immune system and selective autophagy in viral infection.

Cleavage of TANK-Binding Kinase 1 by HIV-1 Protease Triggers Viral Innate Immune Evasion.

Type-I interferons (IFN-I) are the innate immune system's principal defense against viral infections. Human immunodeficiency virus-1 (HIV-1) has evolved several ways to suppress or evade the host's innate immunity in order to survive and replicate to sustain infection. Suppression of IFN-I is one among the multiple escape strategies used by HIV-1 to prevent its clearance. HIV-1 protease which helps in viral maturation has also been observed to cleave host cellular protein kinases. In this study we performed a comprehensive screening of a human kinase library using AlphaScreen assay and identified that TANK binding kinase-1 (TBK1) was cleaved by HIV-1 protease (PR). We demonstrate that PR cleaved TBK1 fails to phosphorylate IFN regulatory factor 3 (IRF3), thereby reducing the IFN-I promoter activity and further reveal that the PR mediated suppression of IFN-I could be counteracted by protease inhibitors (PI) in vitro. We have also revealed that mutations of HIV-1 PR that confer drug resistance to PIs reduce the enzyme's ability to cleave TBK1. The findings of this study unearth a direct link between HIV-1 PR activity and evasion of innate immunity by the virus, the possible physiological relevance of which warrants to be determined.

Structure Revision of Protoaculeine B, a Post-translationally Modified N-Terminal Residue in the Peptide Toxin Aculeine B.

The structure of protoaculeine B, the N-terminal residue of the marine peptide toxin aculeine B, is revised to the cis-1,3-disubstituted tetrahydro-ß-carboline framework. We prepared two truncated model compounds that lack a long-chain polyamine using the one-step Pictet-Spengler reaction of tryptophan and compared their NMR, mass spectra, and chemical reactivity with those of the natural protoaculeine B. The synthetic models reproduced the profiles of the natural product well, which confirmed the appropriateness of the structure revision.

Zika virus protease induces caspase-independent pyroptotic cell death by directly cleaving gasdermin D.

The association of Zika virus (ZIKV) infection with a congenital malformation in fetuses, neurological, and other systemic complications in adults have brought significant global health emergency. ZIKV targets nerve cells in the brain and causes cell death, such as pyroptosis, leading to neuroinflammation. Here we described a novel mechanism of pyroptosis caused by ZIKV protease. We found that ZIKV protease directly cleaved the GSDMD into N-terminal fragment (1-249) leading to pyroptosis in a caspase-independent manner, suggesting a direct mechanism of ZIKV-induced cell death and subsequent inflammation. Our findings might shed new light to explore the pathogenesis of ZIKV infections where ZIKV protease might be a suitable target for the development of antiviral agents.

Pectenovarin, A New Ovarian Carotenoprotein from Japanese Scallop Mizuhopecten yessoensis.

The scallop Mizuhopecten yessoensis accumulates carotenoids in the ovary during the maturation stage. Its conspicuous pink color implies the presence of carotenoprotein. However, the carotenoprotein from the scallop ovary has never been isolated and characterized, probably due to its instability and complexity. Here, we developed an extraction and isolation procedure for the carotenoprotein by employing a basic buffer containing potassium bromide to facilitate its efficient extraction from the ovary, and we succeeded in obtaining the carotenoprotein, termed pectenovarin. The carotenoid composition of the pectenovarin was similar to that of the ovary. The N-terminal and internal amino acid sequences of pectenovarin showed a high similarity to those of vitellogenin, the precursor of egg yolk protein under analysis.

Streptococcus pneumoniae hijacks host autophagy by deploying CbpC as a decoy for Atg14 depletion.

Pneumococcal cell surface-exposed choline-binding proteins (CBPs) play pivotal roles in multiple infectious processes with pneumococci. Intracellular pneumococci can be recognized at multiple steps during bactericidal autophagy. However, whether CBPs are involved in pneumococci-induced autophagic processes remains unknown. In this study, we demonstrate that CbpC from S. pneumoniae strain TIGR4 activates autophagy through an interaction with Atg14. However, S. pneumoniae also interferes with autophagy by deploying CbpC as a decoy to cause autophagic degradation of Atg14 through an interaction with p62/SQSTM1. Thus, S. pneumoniae suppresses the autophagic degradation of intracellular pneumococci and survives within cells. Domain analysis reveals that the coiled-coil domain of Atg14 and residue Y83 of the dp3 domain in the N-terminal region of CbpC are crucial for both the CbpC-Atg14 interaction and the subsequent autophagic degradation of Atg14. Although homology modeling indicates that CbpC orthologs have similar structures in the dp3 domain, autophagy induction through Atg14 binding is an intrinsic property of CbpC. Our data provide novel insights into the evolutionary hijacking of host-defense systems by intracellular pneumococci.

Engineering Cellular Biosensors with Customizable Antiviral Responses Targeting Hepatitis B Virus.

SynNotch receptor technology is a versatile tool that uses the regulatory notch core portion with an extracellular scFv and an intracellular transcription factor that enables to program customized input and output functions in mammalian cells. In this study, we designed a novel synNotch receptor comprising scFv against HBs antigen linked with an intracellular artificial transcription factor and exploited it for viral sensing and cellular immunotherapy. The synNotch receptor expressing cells sensed HBV particles and membrane-bound HBs antigens and responded by expressing reporter molecules, secNL or GFP. We also programmed these cells to dispense antiviral responses such as type I interferon and anti-HBV neutralizing mouse-human chimeric antibodies. Our data reveal that synNotch receptor signaling works for membrane-bound ligands such as enveloped viral particles and proteins borne on liposomal vesicles. This study establishes the concepts of "engineered immunity" where the synNotch platform is utilized for cellular immunotherapy against viral infections.

Prolyl Isomerase Pin1 Regulates the Stability of Hepatitis B Virus Core Protein.

The dynamic interplay between virus and host proteins is critical for establishing efficient viral replication and virus-induced pathogenesis. Phosphorylation-dependent prolyl isomerization by Pin1 provides a unique mechanism of molecular switching to control both protein function and stability. We demonstrate here that Pin1 binds and stabilizes hepatitis B virus core protein (HBc) in a phosphorylation-dependent manner, and promotes the efficient viral propagation. Phos-tag gel electrophoresis with various site-directed mutants of HBc revealed that Thr160 and Ser162 residues within the C terminal arginine-rich domain are phosphorylated concomitantly. GST pull-down assay and co-immunoprecipitation analysis demonstrated that Pin1 associated with phosphorylated HBc at the Thr160-Pro and Ser162-Pro motifs. Chemical or genetic inhibition of Pin1 significantly accelerated the rapid degradation of HBc via a lysosome-dependent pathway. Furthermore, we found that the pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2) could dephosphorylate HBc at the Pin1-binding sites, thereby suppressing Pin1-mediated HBc stabilization. Our findings reveal an important regulatory mechanism of HBc stability catalyzed by Pin1 and may facilitate the development of new antiviral therapeutics targeting Pin1 function.

Aseptic meningitis caused by torque teno virus in an infant: a case report.

BACKGROUND: Torque teno virus-induced aseptic meningitis has not been documented, although torque teno virus infections still remain under consideration for etiological agents. This study identified a torque teno virus sequence using next generation sequencing and immunoglobulin M response to the torque teno virus antigen, therefore, that would be a comprehensive diagnosis for torque teno virus infection. CASE PRESENTATION: A 2-month-old Japanese boy was brought to our hospital because he was irritable, drowsy, and lethargic. He was admitted based on his test results which indicated the possibility of septic meningitis. He was started on treatment with high-dose antibiotics and steroids. On the third day of hospitalization, he became afebrile with improvement in his general status and was discharged on the sixth day. He had no developmental problems for up to 1 year after discharge. Metagenomic ribonucleic acid-Seq pathogen detection using next generation sequencing of a sample of his cerebrospinal fluid, which was collected at admission, revealed three short reads homologous to those in torque teno virus out of a total of 1,708,516 reads. This finding indicated that our patient was positive compared to the torque teno virus-negative cerebrospinal fluid samples (controls) from 13 other patients. The torque teno virus has been shown to have a whole genome sequence of 2810 nt by polymerase chain reaction. We prepared a recombinant GP2 antigen from torque teno virus and used it to study our patient's anti-torque teno virus immune response. An anti-GP2 serum immunoglobulin M response was detected, providing further supportive evidence of torque teno virus infection. CONCLUSIONS: This case speculates that torque teno virus-induced aseptic meningitis has a good course. New technologies like next generation sequencing can help in the identification of such cases, and an accumulation of future cases is expected.

PIM kinases facilitate lentiviral evasion from SAMHD1 restriction via Vpx phosphorylation.

Lentiviruses have evolved to acquire an auxiliary protein Vpx to counteract the intrinsic host restriction factor SAMHD1. Although Vpx is phosphorylated, it remains unclear whether such phosphorylation indeed regulates its activity toward SAMHD1. Here we identify the PIM family of serine/threonine protein kinases as the factors responsible for the phosphorylation of Vpx and the promotion of Vpx-mediated SAMHD1 counteraction. Integrated proteomics and subsequent functional analysis reveal that PIM family kinases, PIM1 and PIM3, phosphorylate HIV-2 Vpx at Ser13 and stabilize the interaction of Vpx with SAMHD1 thereby promoting ubiquitin-mediated proteolysis of SAMHD1. Inhibition of the PIM kinases promotes the antiviral activity of SAMHD1, ultimately reducing viral replication. Our results highlight a new mode of virus-host cell interaction in which host PIM kinases facilitate promotion of viral infectivity by counteracting the host antiviral system, and suggest a novel therapeutic strategy involving restoration of SAMHD1-mediated antiviral response.

Production and characterization of monoclonal antibodies specific for major capsid VP1 protein of trichodysplasia spinulosa-associated polyomavirus.

Trichodysplasia spinulosa-associated polyomavirus (TSPyV), a newly identified polyomavirus, has been implicated as a causative agent of trychodysplasia spinulosa (TS), a rare proliferative skin disease in severely immunocompromised hosts. Diagnosis using mAbs is a promising tool with high specificity towards the specific antigen. However, thus far, no suitable mAbs for diagnosing TS disease have been identified. In this study, mAbs specific for VP1 of TSPyV were developed and characterized. Wheat germ cell-free synthesized VP1 protein of TSPyV was used to immunize BALB/c mice to generate hybridomas. Screening of the resultant hybridoma clones resulted in selection of five strongly positive clones that produce mAbs that react with the TSPyV-VP1 antigen. Epitope mapping and bioinformatic analysis showed that these mAbs recognized epitopes located within highly conserved C-terminal region of all clinical isolates of TSPyV-VP1. Further, all these mAbs were highly effective for immunofluorescence and immunoprecipitation analysis. Three of the five mAbs exhibited no cross-reactivity with VP1 of other related polyomaviruses. In addition, one of our mAbs (#14) provided immunohistochemical staining of skin tissue of TS disease. It can be concluded that three of the mAbs in this panel of anti-VP1 antibodies may provide a useful set of tools for studying TSPyV infection and making the specific diagnosis.

A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction.

Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors.

Development of a cell-based assay to identify hepatitis B virus entry inhibitors targeting the sodium taurocholate cotransporting polypeptide.

Sodium taurocholate cotransporting polypeptide (NTCP) is a major entry receptor of hepatitis B virus (HBV) and one of the most attractive targets for anti-HBV drugs. We developed a cell-mediated drug screening method to monitor NTCP expression on the cell surface by generating a HepG2 cell line with tetracycline-inducible expression of NTCP and a monoclonal antibody that specifically detects cell-surface NTCP. Using this system, we screened a small molecule library for compounds that protected against HBV infection by targeting NTCP. We found that glabridin, a licorice-derived isoflavane, could suppress viral infection by inducing caveolar endocytosis of cell-surface NTCP with an IC50 of ~40 µM. We also found that glabridin could attenuate the inhibitory effect of taurocholate on type I interferon signaling by depleting the level of cell-surface NTCP. These results demonstrate that our screening system could be a powerful tool for discovering drugs targeting HBV entry.

Chemical array system, a platform to identify novel hepatitis B virus entry inhibitors targeting sodium taurocholate cotransporting polypeptide.

Current anti-hepatitis B virus (HBV) agents including interferons and nucleos(t)ide analogs efficiently suppress HBV infection. However, as it is difficult to eliminate HBV from chronically infected liver, alternative anti-HBV agents targeting a new molecule are urgently needed. In this study, we applied a chemical array to high throughput screening of small molecules that interacted with sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for HBV. From approximately 30,000 compounds, we identified 74 candidates for NTCP interactants, and five out of these were shown to inhibit HBV infection in cell culture. One of such compound, NPD8716, a coumarin derivative, interacted with NTCP and inhibited HBV infection without causing cytotoxicity. Consistent with its NTCP interaction capacity, this compound was shown to block viral attachment to host hepatocytes. NPD8716 also prevented the infection with hepatitis D virus, but not hepatitis C virus, in agreement with NPD8716 specifically inhibiting NTCP-mediated infection. Analysis of derivative compounds showed that the anti-HBV activity of compounds was apparently correlated with the affinity to NTCP and the capacity to impair NTCP-mediated bile acid uptake. These results are the first to show that the chemical array technology represents a powerful platform to identify novel viral entry inhibitors.

Mutations in genes encoding polycomb repressive complex 2 subunits cause Weaver syndrome.

Weaver syndrome (WS) is a rare congenital overgrowth disorder caused by heterozygous mutations in EZH2 (enhancer of zeste homolog 2) or EED (embryonic ectoderm development). EZH2 and EED are core components of the polycomb repressive complex 2 (PRC2), which possesses histone methyltransferase activity and catalyzes trimethylation of histone H3 at lysine 27. Here, we analyzed eight probands with clinically suspected WS by whole-exome sequencing and identified three mutations: a 25.4-kb deletion partially involving EZH2 and CUL1 (individual 1), a missense mutation (c.707G>C, p.Arg236Thr) in EED (individual 2), and a missense mutation (c.1829A>T, p.Glu610Val) in SUZ12 (suppressor of zeste 12 homolog) (individual 3) inherited from her father (individual 4) with a mosaic mutation. SUZ12 is another component of PRC2 and germline mutations in SUZ12 have not been previously reported in humans. In vitro functional analyses demonstrated that the identified EED and SUZ12 missense mutations cause decreased trimethylation of lysine 27 of histone H3. These data indicate that loss-of-function mutations of PRC2 components are an important cause of WS.

The tumour suppressor APC promotes HIV-1 assembly via interaction with Gag precursor protein.

Diverse cellular proteins and RNAs are tightly regulated in their subcellular localization to exert their local function. Here we report that the tumour suppressor adenomatous polyposis coli protein (APC) directs the localization and assembly of human immunodeficiency virus (HIV)-1 Gag polyprotein at distinct membrane components to enable the efficient production and spread of infectious viral particles. A proteomic analysis and subsequent biomolecular interaction assay reveals that the carboxyl terminus of APC interacts with the matrix region of Gag. Ectopic expression of APC, but not its familial adenomatous polyposis-related truncation mutant, prominently enhances HIV-1 production. Conversely, the depletion of APC leads to a significant decrease in membrane targeting of viral components, resulting in the severe loss of production of infectious virions. Furthermore, APC promotes the directional assembly of viral components at virological synapses, thereby facilitating cell-to-cell viral transmission. These findings reveal an unexpected role of APC in the directional spread of HIV-1.

N-Methyl-ß-carbolinium Salts and an N-Methylated 8-Oxoisoguanine as Acetylcholinesterase Inhibitors from a Solitary Ascidian, Cnemidocarpa irene.

New brominated ß-carbolines irenecarbolines A (1) and B (4) along with known ß-carbolines 2 and 3 and a new 8-oxoisoguanine derivative, 5, were isolated from a solitary ascidian, Cnemidocarpa irene. The structures of these compounds were determined on the basis of their spectral data. All, except for 3, inhibited the action of acetylcholinesterase (AchE). The activities of 1 and 5 were comparable to those of galantamine, a clinically used AchE inhibitor. Compounds 1 and 2 were found to be present in high concentrations in blood, and fluorescence was observed in certain types of cells found in the blood of the tunicate.

Lyn Kinase Suppresses the Transcriptional Activity of IRF5 in the TLR-MyD88 Pathway to Restrain the Development of Autoimmunity.

Interferon regulatory factor-5 (IRF5), a transcription factor critical for the induction of innate immune responses, contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE) in humans and mice. Lyn, a Src family kinase, is also implicated in human SLE, and Lyn-deficient mice develop an SLE-like disease. Here, we found that Lyn physically interacted with IRF5 to inhibit ubiquitination and phosphorylation of IRF5 in the TLR-MyD88 pathway, thereby suppressing the transcriptional activity of IRF5 in a manner independent of Lyn's kinase activity. Conversely, Lyn did not inhibit NF-κB signaling, another major branch downstream of MyD88. Monoallelic deletion of Irf5 alleviated the hyperproduction of cytokines in TLR-stimulated Lyn(-/-) dendritic cells and the development of SLE-like symptoms in Lyn(-/-) mice. Our results reveal a role for Lyn as a specific suppressor of the TLR-MyD88-IRF5 pathway and illustrate the importance of fine-tuning IRF5 activity for the maintenance of immune homeostasis.