Sendai Virus Research Articles

Sendai Virus-Vectored Vaccines That Express Envelope Glycoproteins of Respiratory Viruses.

Human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), and human parainfluenza viruses (HPIVs) are leading causes of respiratory disease in young children, the elderly, and individuals of all ages with immunosuppression. Vaccination strategies against these pneumoviruses and paramyxoviruses are vast in number, yet no licensed vaccines are available. Here, we review development of Sendai virus (SeV), a versatile pediatric vaccine that can (a) serve as a Jennerian vaccine against HPIV1, (b) serve as a recombinant vaccine against HRSV, HPIV2, HPIV3, and HMPV, (c) accommodate foreign genes for viral glycoproteins in multiple intergenic positions, (d) induce durable, mucosal, B-cell, and T-cell immune responses without enhanced immunopathology, (e) protect cotton rats, African green monkeys, and chimpanzees from infection, and (f) be formulated into a vaccine cocktail. Clinical phase I safety trials of SeV have been completed in adults and 3–6-year-old children. Clinical testing of SeVRSV, an HRSV fusion (F) glycoprotein gene recombinant, has also been completed in adults. Positive results from these studies, and collaborative efforts with the National Institutes of Health and the Serum Institute of India assist advanced development of SeV-based vaccines. Prospects are now good for vaccine successes in infants and consequent protection against serious viral disease.

Modeling leukemia with pediatric acute leukemia patient-derived iPSCs

Objectiveediatric acute leukemia (AL) is the most common hematological malignancy in childhood. However, the limitation of clinical specimens hindered the progress of research. Therefore, new research platforms are urgently needed to establish and clarify the pathogenesis of pediatric AL, and it is necessary to try to find novel targeted therapies for the clinical use. Here, the induced pluripotent stem cells (iPSCs) derived from AL provide a reliable model for basic research. Methodseukemia cells were sorted by flow cytometry and then reprogrammed into iPSCs by Sendai virus. Cell cycle assay was used to analyze cell proliferation. ResultsiPS cell lines from T cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) cells were successfully established. The reprogramming efficiency of AML cells was much higher than that of ALL cells. Disease iPS cells switched off the expression of the disease marker genes at iPS and HPC stage. When different subtypes of AML-iPSCs were differentiated into hematopoietic progenitor cells, iPS derived from acute megakaryocytic leukemia was more readily differentiated into megakaryocyte-erythroid progenitors. Whereas, the differentiation of multipotent lymphoid progenitor (MLP) and granulocyte macrophage progenitor (GMP) were blocked. The iPS derived from acute monocyte leukemia (AMCL) also showed the differentiation of common myeloid progenitors (CMP), GMP and monocytes significantly increased but MLP differentiation was inhibited. The AML-iPSC could form teratomas and we could obverse three germ layers in vivo, indicating that the AML-iPSCs have full pluripotency. However, there were not enough blood cells in teratoma to identify the leukemia. ConclusionsOur results provide a novel platform for AL research and critical insight into the difference of hematopoietic differentiation between ALL and AML.

Generation of 20 human induced pluripotent stem cell lines from patients with focal segmental glomerulosclerosis (FSGS)

Focal segmental glomerulosclerosis (FSGS) is a major cause of familial nephrotic syndrome. We generated 20 induced pluripotent stem cell lines from patients diagnosed with FSGS. The iPSC lines include 8 female and 12 male lines and cover a donor age range from 31 to 78. The lines were generated from peripheral blood mononuclear cells by integration-free reprogramming using Sendai virus vectors. Cell lines were fully characterized regarding their pluripotency and differentiation potential, and quality controlled for karyotypic integrity, identity and clearance of reprogramming vectors. The generated cell lines represent a valuable tool for disease modelling and drug development for FSGS.

USP18 positively regulates innate antiviral immunity by promoting K63-linked polyubiquitination of MAVS

Activation of MAVS, an adaptor molecule in Rig-I-like receptor (RLR) signaling, is indispensable for antiviral immunity, yet the molecular mechanisms modulating MAVS activation are not completely understood. Ubiquitination has a central function in regulating the activity of MAVS. Here, we demonstrate that a mitochondria-localized deubiquitinase USP18 specifically interacts with MAVS, promotes K63-linked polyubiquitination and subsequent aggregation of MAVS. USP18 upregulates the expression and production of type I interferon following infection with Sendai virus (SeV) or Encephalomyocarditis virus (EMCV). Mice with a deficiency of USP18 are more susceptible to RNA virus infection. USP18 functions as a scaffold protein to facilitate the re-localization of TRIM31 and enhances the interaction between TRIM31 and MAVS in mitochondria. Our results indicate that USP18 functions as a post-translational modulator of MAVS-mediated antiviral signaling.

Establishment of an arrhythmogenic right ventricular cardiomyopathy derived iPSC cell line (USFi004-A) carrying a heterozygous mutation in PKP2 (c.1799delA)

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an autosomal dominant inherited disease, with variable penetrance and expressivity. Currently, more than 14 different genetic loci have been reported for ARVC, the majority being desmosomal genes like Plakophilin-2 (PKP2). Here, we generated an iPSC cell line bearing a pathogenic heterozygous mutation in PKP2 (c.1799delA) from a patient affected by ARVC. Peripheral blood mononuclear cells were reprogrammed by Sendai virus vectors encoding KOS, KLF4, and c-MYC. Derived iPSCs expressed pluripotent markers, showed intact karyotype and demonstrated the ability to differentiate into three germ layers.

Generation of an iPSC cell line (USFi003-A) from a patient with dilated cardiomyopathy carrying a heterozygous mutation in LMNA (p.R541C)

Mutations in the gene that encodes the nuclear envelope proteins lamin A/C (LMNA) are considered to be a prominent cause of Dilated cardiomyopathy (DCM), a leading cause of heart failure and a prevalent indication for heart transplant. Here we described the generation of induced pluripotent stem cells (iPSCs) from a 53-year-old female with DCM plus progressive conduction disease who carry a heterozygous mutation in LMNA (c.1621C > T, p.R541C). PBMCs isolated from the patient were reprogrammed with Yamanaka factors KOS, KLF4, and c-MYC by the non-integrating sendai virus vector system. The obtained iPSC lines demonstrated normal karyotype and pluripotent identity.

Generation of an induced pluripotent stem cell (iPSC) line (JUCTCi017-A) from a patient with limb-girdle muscular dystrophy (LGMD) due to a homozygous p.Lue287Ser fs14* mutation in the SGCB gene

Limb-girdle muscular dystrophies (LGMDs) are a large group of heterogenous genetic diseases characterized by muscle weakness. In this study, an induced pluripotent stem cell (iPSC) line was generated from LGMD patient’s skin dermal fibroblasts, carrying a homozygous mutation in the Sarcoglycan Beta (SGCB) gene; chr4:52890221, c. 859 delC, p.Lue 287Ser fs14*. The reprogramming process was carried out using Sendai viruses encoding for Yamanaka factors. The resulting iPSCs showed normal morphology and karyotype, expressed pluripotency markers, demonstrated the potential to differentiate in vitro into three germ layers and retained the disease-causing SGCB mutation. This iPSC line represents an ideal source of cells for the investigation of LGMD disease mechanisms.

Direct reprogramming with Sendai virus vectors repaired infarct hearts at the chronic stage

Adult hearts have limited regenerative capacity. Hence, after acute myocardial infarction (MI), dead myocardial tissues are digested by immune cells and replaced by fibrosis, leading to ventricular remodeling and heart failure at the chronic stage. Direct reprogramming of the cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) with cardiac transcription factors, including Gata4, Mef2c, and Tbx5 (GMT), may have significant potential for cardiac repair. Sendai virus (SeV) vectors expressing GMT have been reported to reprogram the mouse cardiac fibroblasts into iCMs without any risk of insertional mutagenesis. In vivo reprogramming improved the cardiac function after acute MI in immunodeficient mice. However, it is unknown whether the newly generated iCMs could exist in infarct hearts for a prolonged period and SeV-GMT can improve cardiac function after MI at the chronic stage in immunocompetent mice. Here, we show that SeV vectors efficiently infect CFs in vivo and reprogram them into iCMs, which existed for at least four weeks after MI, in fibroblast-linage tracing mice. Moreover, SeV-GMT improved cardiac function and reduced fibrosis and collagen I expression at 12 weeks after MI in immunocompetent mice. Thus, direct cardiac reprogramming with SeV vectors could be a promising therapy for MI.

SARS-CoV-2 ORF9b antagonizes type I and III interferons by targeting multiple components of the RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways.

The suppression of types I and III interferon (IFN) responses by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) contributes to the pathogenesis of coronavirus disease 2019 (COVID‐19). The strategy used by SARS‐CoV‐2 to evade antiviral immunity needs further investigation. Here, we reported that SARS‐CoV‐2 ORF9b inhibited types I and III IFN production by targeting multiple molecules of innate antiviral signaling pathways. SARS‐CoV‐2 ORF9b impaired the induction of types I and III IFNs by Sendai virus and poly (I:C). SARS‐CoV‐2 ORF9b inhibited the activation of types I and III IFNs induced by the components of cytosolic dsRNA‐sensing pathways of RIG‐I/MDA5‐MAVS signaling, including RIG‐I, MDA‐5, MAVS, TBK1, and IKKε, rather than IRF3‐5D, which is the active form of IRF3. SARS‐CoV‐2 ORF9b also suppressed the induction of types I and III IFNs by TRIF and STING, which are the adaptor protein of the endosome RNA‐sensing pathway of TLR3‐TRIF signaling and the adaptor protein of the cytosolic DNA‐sensing pathway of cGAS–STING signaling, respectively. A mechanistic analysis revealed that the SARS‐CoV‐2 ORF9b protein interacted with RIG‐I, MDA‐5, MAVS, TRIF, STING, and TBK1 and impeded the phosphorylation and nuclear translocation of IRF3. In addition, SARS‐CoV‐2 ORF9b facilitated the replication of the vesicular stomatitis virus. Therefore, the results showed that SARS‐CoV‐2 ORF9b negatively regulates antiviral immunity and thus facilitates viral replication. This study contributes to our understanding of the molecular mechanism through which SARS‐CoV‐2 impairs antiviral immunity and provides an essential clue to the pathogenesis of COVID‐19.

Therapeutic effects of CXCR4+ subpopulation of transgene‐free induced cardiosphere‐derived cells on experimental myocardial infarction

ObjectivesMyocardial infarction (MI) is the most predominant type of cardiovascular diseases with high mortality and morbidity. Stem cell therapy, especially cardiac progenitor cell therapy, has been proposed as a promising approach for cardiac regeneration and MI treatment. Previously, we have successfully generated cardiac progenitor‐like cells, induced cardiosphere (iCS), via somatic reprogramming. However, the genome integration characteristic of virus‐based reprogramming approach hampered their therapeutic applications due to the risk of tumour formation. In the current study, we aim to establish a safer iCS generation strategy with transgene‐free approaches.Materials and MethodsFour transgene‐free approaches for somatic reprogramming, including episome, minicircle, self‐replicative RNA, and sendai virus, were compared, from the perspective of cardiac progenitor marker expression, iCS formation, and cardiac differentiation. The therapeutic effects were assessed in the mouse model of MI, from the perspective of survival rate, cardiac function, and structural alterations.ResultsThe self‐replicative RNA approach produced more iCS, which had cardiomyocyte differentiation ability and therapeutic effects on the mouse model of MI with comparable levels with endogenous cardiospheres and iCS generated with retrovirus. In addition, the CXCR4 (C‐X‐C chemokine receptor 4) positive subpopulation of iCS derived cells (iCSDC) delivered by intravenous injection was found to have similar therapeutic effects with intramyocardial injection on the mouse model of MI, representing a safer delivery approach.ConclusionThus, the optimized strategy for iCS generation is safer and has more therapeutic potentials.

Resident Memory CD8+ T cells in the respiratory tract prevent transmission of respiratory viruses

Abstract Tissue-resident memory CD8+ T cells (TRM) in the respiratory tract reduce viral replication and limit pathology following respiratory virus infections. In infection with heterosubtypic influenza viruses, where pre-existing antibodies does not proved sterilizing immunity, TRM are critical for protection. However, in addition to protecting the host against disease vaccine programs are also designed to limit viral spread in a population. Due to the lack of an animal model to study the effect of TRM on transmission through longitudinal tracking, and with immunological parameters easily identified and manipulated available, this hasn’t been studied before. To solve these issues, we used a luciferase expressing Sendai virus, a natural mouse parainfluenza virus that readily transmits via the aerosol and contact routes, as well as a recombinant influenza virus expressing the immunodominant Sendai NP324-332/Kb epitope to generate SenNP+ memory CD8+ T cells. By utilizing IVIS imaging to non-invasively measure Sendai virus infection over time, we found that mice with pre-existing SenNP+ TRM in the respiratory system don’t transmit Sendai virus to naïve mice when co-housed. In contrast, mice with pre-existing circulating SenNP+ effector memory CD8+ T cells, but no TRM, failed to prevent transmission of Sendai virus to naïve mice when co-housed. In addition, we found that the prevention of transmission was dependent on the production of IFNγ. These findings demonstrates notably that antigen specific lung TRM contributes to herd immunity by preventing respiratory virus transmission. This underscores the importance, and potential, of TRMs generated by vaccines for protection against respiratory viruses.

Generation and characterization of induced pluripotent stem cells heterozygous for the Portuguese BRCA2 founder mutation

Women who inherit heterozygous mutations in the BRCA2 gene have an increased risk of developing cancer, mainly breast and ovarian tumors. A particular BRCA2 mutation (c.156_157insAlu) is exclusively found in families of Portuguese ancestry and is present in approximately 30% of all Portuguese families with hereditary breast and ovarian cancers. We report the generation and characterization of the first iPSC line from a female donor harboring the Portuguese BRCA2 founder mutation. Skin fibroblasts were reprogrammed using a non-integrative Sendai virus. These iPSCs are a valuable tool to study the origin of BRCA2-associated cancer in its earliest phases.

Structure and assembly of double-headed Sendai virus nucleocapsids

Paramyxoviruses, including the mumps virus, measles virus, Nipah virus and Sendai virus (SeV), have non-segmented single-stranded negative-sense RNA genomes which are encapsidated by nucleoproteins into helical nucleocapsids. Here, we reported a double-headed SeV nucleocapsid assembled in a tail-to-tail manner, and resolved its helical stems and clam-shaped joint at the respective resolutions of 2.9 and 3.9 Å, via cryo-electron microscopy. Our structures offer important insights into the mechanism of the helical polymerization, in particular via an unnoticed exchange of a N-terminal hole formed by three loops of nucleoproteins, and unveil the clam-shaped joint in a hyper-closed state for nucleocapsid dimerization. Direct visualization of the loop from the disordered C-terminal tail provides structural evidence that C-terminal tail is correlated to the curvature of nucleocapsid and links nucleocapsid condensation and genome replication and transcription with different assembly forms.

Establishment of a Vietnamese ethnicity induced pluripotent stem cell line (VRISGi001-A) from umbilical cord blood hematopoietic stem cells under a feeder-free system

We have established the original footprint-free Vietnamese human induced pluripotent stem cell line, VRISGi001-A, from cord-blood derived CD34 + hematopoietic stem cells (HSCs) of a 35 year old healthy woman under cGMP-compliant process. For the hiPSC induction, three Sendai virus vectors carrying four reprogramming factors including c-MYC, SOX2, KLF4, and OCT3/4 were delivered into CD34+ HSCs. The VRISGi001-A cell line expresses the majority of the pluripotent markers and differentiate in vitro into derivatives of three germ layers. The availability of Vietnamese hiPSC line could contribute to the improvement of inadequate genetic diversity in the currently available hiPSC lines.

Generation and characterization of six human induced pluripotent stem cell lines (iPSC) from three families with AP4M1-associated hereditary spastic paraplegia (SPG50)

Biallelic loss-of-function variants in the subunits of the adaptor protein complex 4 lead to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1), and SPG52 (AP4S1). Here, we describe the generation of induced pluripotent stem cells (iPSCs) from three AP-4-HSP patients with biallelic, loss-of-function variants in AP4M1 and their sex-matched parents (asymptomatic, heterozygous carriers). Following reprogramming using non-integrating Sendai virus, iPSCs were characterized following standard protocols including karyotyping, embryoid body formation, pluripotency marker expression and STR profiling. These first iPSC lines for SPG50 provide a valuable resource for studying this rare disease and related forms of hereditary spastic paraplegia.

Sendai virus-based immunoadjuvant in hydrogel vaccine intensity-modulated dendritic cells activation for suppressing tumorigenesis

The conventional immunoadjuvants in vaccine have weak effect on stimulating antigen presentation and activating anti-tumor immunity. Unexpectedly, we discovered that non-pathogenic Sendai virus (SeV) could activate antigen-presenting cells (APCs) represented by dendritic cells (DCs). Here, we designed an injectable SeV-based hydrogel vaccine (SHV) to execute multi-channel recruitment and stimulation of DCs for boosting the specific immune response against tumors. After the release of the NIR-triggered antigens from tumor cells, dendritic cells around the vaccine efficiently transport the antigens to lymph nodes and present them to T lymphocytes, thereby inducing systemic anti-tumor immune memory. Our findings demonstrated that the SHV with excellent universality, convenience and flexibility has achieved better immune protection effects in inhibiting the occurrence of melanoma and breast cancer. In conclusion, the SHV system might serve as the next generation of personalized anti-tumor vaccines with enhanced features over standard vaccination regimens, and represented an alternative way to suppress tumorigenesis.

IPSC screening for drug repurposing identifies anti-RNA virus agents modulating host cell susceptibility.

Human pathogenic RNA viruses are threats to public health because they are prone to escaping the human immune system through mutations of genomic RNA, thereby causing local outbreaks and global pandemics of emerging or re‐emerging viral diseases. While specific therapeutics and vaccines are being developed, a broad‐spectrum therapeutic agent for RNA viruses would be beneficial for targeting newly emerging and mutated RNA viruses. In this study, we conducted a screen of repurposed drugs using Sendai virus (an RNA virus of the family Paramyxoviridae), with human‐induced pluripotent stem cells (iPSCs) to explore existing drugs that may present anti‐RNA viral activity. Selected hit compounds were evaluated for their efficacy against two important human pathogens: Ebola virus (EBOV) using Huh7 cells and severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) using Vero E6 cells. Selective estrogen receptor modulators (SERMs), including raloxifene, exhibited antiviral activities against EBOV and SARS‐CoV‐2. Pioglitazone, a PPARγ agonist, also exhibited antiviral activities against SARS‐CoV‐2, and both raloxifene and pioglitazone presented a synergistic antiviral effect. Finally, we demonstrated that SERMs blocked entry steps of SARS‐CoV‐2 into host cells. These findings suggest that the identified FDA‐approved drugs can modulate host cell susceptibility against RNA viruses.

MODELING LONG QT SYNDROME IN IPS-DERIVED CARDIOMYOCYTES FROM A PATIENT HARBORING THE KCNQ1- I588T MUTATION

<h3>Background</h3> Human induced pluripotent stem (iPS) cell-derived cardiomyocytes (hiPSC‐CMs) technology offers an unprecedented tool to study cardiac diseases. Long QT syndrome type 1 (LQT1) is an inherited cardiac channelopathy caused by mutations in KCNQ1, which encodes the main subunit of the potassium channel Kv 7.1, leading to delayed repolarizations and imposing potentially fatal arrhythmias on patients. Therefore, the in vitro patient-specific modeling of LQT1 is crucial to test the pathogenicity of the KCNQ1- I588T mutation. <h3>Objective</h3> The purpose of this study was to test a cellular model of LQT1 based on a KCNQ1-I588T mutation using hiPSC‐CMs. <h3>Methods</h3> Peripheral blood was collected from a proband (a 15-year-old LQT1 symptomatic patient carrying KCNQ1-I588T mutation), a carrier donor (non-symptomatic familial carrying KCNQ1 mutation), and an unrelated control donor. The erythroblast enriched fraction was reprogrammed into iPS using Sendai virus vector. iPS were characterized for pluripotency markers by RT-PCR and flow cytometry, in vitro spontaneous differentiation, and karyotype stability. iPS were differentiated into cardiomyocytes and electrophysiological studies were performed. <h3>Results</h3> The proband was clinically diagnosed with LQT1 with resting corrected QT interval equal to 516 ms. The KCNQ1 mutation (c.1763T>C; p.I588T) was identified by NGS in the proband DNA sample. By Sanger, the same mutation was identified in the familial carrier while it was absent in the control subject. Erythroblasts were successfully reprogrammed into iPS. All iPS expressed the 13 different pluripotent markers evaluated, differentiated into three germ layers and presented a normal chromosomic profile. The differentiation protocol generated cells positive for cardiac troponin T. As expected, proband hiPSC‐CMs exhibited prolongation of action potential duration (APD90 = 312 ± 79 ms) when compared to carrier donor and control donor hiPS-CMs (APD90 = 177 ± 68 and 271 ± 71 ms, respectively). Since the mutation is the same in both the proband and the carrier, a protective/damaging molecular regulation must be involved. We speculate that a polymorphism identified only in the proband (SCN5A c.1673A>G; p.H558R) may be involved in this mechanism. <h3>Conclusions</h3> We identified an LQT1 causative variant that successfully recapitulated LQT1 proband phenotype by in vitro modeling. We also propose the existence of a second variant with a damaging effect on LQT1 symptom development.

Porcine deltacoronavirus nsp10 antagonizes interferon-β production independently of its zinc finger domains

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes serious vomiting and diarrhea in piglets. Previous work demonstrated that PDCoV infection inhibits type I interferon (IFN) production. Here, we found that ectopic expression of PDCoV nsp10 significantly inhibited Sendai virus (SeV)-induced IFN-β production by impairing the phosphorylation and nuclear translocation of two transcription factors, IRF3 and NF-κB p65 subunit. Interestingly, experiments with truncated mutants and site-directed mutagenesis revealed that PDCoV nsp10 mutants with missing or destroyed zinc fingers (ZFs) domains also impeded SeV-induced IFN-β production, suggesting that nsp10 does not require its ZF domains to antagonize IFN-β production. Further work found that co-expression of nsp10 with nsp14 or nsp16, two replicative enzymes, significantly enhanced the inhibitory effects of nsp10 on IFN-β. Taken together, our results demonstrate that PDCoV nsp10 antagonizes IFN via a ZF-independent mechanism and has a synergistic effect with nsp14 and nsp16 on inhibiting IFN-β production.

Establishment of an induced pluripotent stem cell line from a Noonan syndrome patient with the heterozygote mutation p.S257L (c.770C > T) in RAF1 gene

Noonan Syndrome (NS) is an inherited autosome dominant disorder syndrome, which can be caused by the mutations of serine/threonine kinase rapidly accelerated fibrosarcoma 1 (RAF1) gene. Here, an induced pluripotent stem cell (iPSC) line named WMUi022-A derived from urine cells (UCs) of a 9-year-old male NS patient with the heterozygote RAF1 gene mutation p.S257L (c.770C > T) was established through the commercial Sendai virus reprogramming kit. The pluripotent markers like OCT4 and SOX2 can be expressed positively in WMUi022-A, which can be induced into three germ layers in vitro as well as maintain a normal karyotype (46, XY).