Inhibition of lung microbiota-derived proapoptotic peptides ameliorates acute exacerbation of pulmonary fibrosis.

Idiopathic pulmonary fibrosis is an incurable disease of unknown etiology. Acute exacerbation of idiopathic pulmonary fibrosis is associated with high mortality. Excessive apoptosis of lung epithelial cells occurs in pulmonary fibrosis acute exacerbation. We recently identified corisin, a proapoptotic peptide that triggers acute exacerbation of pulmonary fibrosis. Here, we provide insights into the mechanism underlying the processing and release of corisin. Furthermore, we demonstrate that an anticorisin monoclonal antibody ameliorates lung fibrosis by significantly inhibiting acute exacerbation in the human transforming growth factorß1 model and acute lung injury in the bleomycin model. By investigating the impact of the anticorisin monoclonal antibody in a general model of acute lung injury, we further unravel the potential of corisin to impact such diseases. These results underscore the role of corisin in the pathogenesis of acute exacerbation of pulmonary fibrosis and acute lung injury and provide a novel approach to treating this incurable disease.

Non-propagative human parainfluenza virus type 2 nasal vaccine robustly protects the upper and lower airways against SARS-CoV-2.

We developed an intranasal vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using the replication-incompetent human parainfluenza virus type 2 (hPIV2) vector BC-PIV, which can deliver ectopic gene as stable RNA and ectopic protein on the envelope. BC-PIV expressing the full-length prefusion-stabilized spike gene (K986P/V987P) of SARS-CoV-2, S-2PM, possessed a corona-like viral envelope. Intranasal vaccination of mice with BC-PIV/S-2PM induced high levels of neutralizing immunoglobulin G (IgG) and mucosal IgA antibodies against the spike protein. Although BC-PIV showed hemagglutinating activity, BC-PIV/S-2PM lacked such activity, in accordance with the presence of the massive spike protein on the viral surface. Furthermore, single-dose intranasal vaccination of hamsters with BC-PIV/S-2PM completely protected the lungs from SARS-CoV-2 at 11-week post-immunization, and boost vaccination two weeks before the challenge conferred virtually complete protection of the nasal turbinates against SARS-CoV-2. Thus, this chimeric hPIV2/spike intranasal vaccine is a promising vaccine candidate for SARS-CoV-2 to curtail virus transmission.

A Staphylococcus pro-apoptotic peptide induces acute exacerbation of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal disease of unknown etiology; however, apoptosis of lung alveolar epithelial cells plays a role in disease progression. This intractable disease is associated with increased abundance of Staphylococcus and Streptococcus in the lungs, yet their roles in disease pathogenesis remain elusive. Here, we report that Staphylococcus nepalensis releases corisin, a peptide conserved in diverse staphylococci, to induce apoptosis of lung epithelial cells. The disease in mice exhibits acute exacerbation after intrapulmonary instillation of corisin or after lung infection with corisin-harboring S. nepalensis compared to untreated mice or mice infected with bacteria lacking corisin. Correspondingly, the lung corisin levels are significantly increased in human IPF patients with acute exacerbation compared to patients without disease exacerbation. Our results suggest that bacteria shedding corisin are involved in acute exacerbation of IPF, yielding insights to the molecular basis for the elevation of staphylococci in pulmonary fibrosis.

A versatile platform technology for recombinant vaccines using non-propagative human parainfluenza virus type 2 vector.

Ectopic protein with proper steric structure was efficiently loaded onto the envelope of the F gene-defective BC-PIV vector derived from human parainfluenza virus type 2 (hPIV2) by a reverse genetics method of recombinant virus production. Further, ectopic antigenic peptide was successfully loaded either outside, inside, or at both sides of the envelope of the vector. The BC-PIV vector harboring the Ebola virus GP gene was able to elicit neutralizing antibodies in mice. In addition, BC-PIV with antigenic epitopes of both melanoma gp100 and WT1 tumor antigen induced a CD8+ T-cell-mediated response in tumor-transplanted syngeneic mice. Considering the low pathogenicity and recurrent infections of parental hPIV2, BC-PIV can be used as a versatile vector with high safety for recombinant vaccine development, addressing unmet medical needs.

Nucleocytoplasmic shuttling of the human parainfluenza virus type 2 phosphoprotein.

Human parainfluenza virus type 2 phosphoprotein (P) is an essential component of viral polymerase. The P gene encodes both P and accessory V proteins by a specific gene editing mechanism. Therefore, the N-terminal 164 amino acids of P protein are common to V protein. Interestingly, while P protein is located in the cytoplasm, V protein is found mainly in the nucleus. Using deletion mutants, we show the presence of a nuclear localization signal (NLS) in the P/V common domain, and a nuclear export signal (NES) in the C-terminal P specific region. The NLS region makes a complex with importin α5 or 7. In the presence of leptomycin B, P protein is retained in the nucleus, indicating that it contains a CRM1-dependent NES. We identified the NLS (65PVKPRRKK72) and the NES (225IIELLKGLDL234) using ß-galactosidase fusion proteins. Moreover, nucleocytoplasmic shuttling of P protein appears to be important for efficient viral polymerase activity.

Human parainfluenza virus type 2 vector induces dendritic cell maturation without viral RNA replication/transcription.

The dendritic cell (DC), a most potent antigen-presenting cell, plays a key role in vaccine therapy against infectious diseases and malignant tumors. Although advantages of viral vectors for vaccine therapy have been reported, potential risks for adverse effects prevent them from being licensed for clinical use. Human parainfluenza virus type 2 (hPIV2), one of the members of the Paramyxoviridae family, is a nonsegmented and negative-stranded RNA virus. We have developed a reverse genetics system for the production of infectious hPIV2 lacking the F gene (hPIV2ΔF), wherein various advantages for vaccine therapy exist, such as cytoplasmic replication/transcription, nontransmissible infectivity, and extremely high transduction efficacy in various types of target cells. Here we demonstrate that hPIV2ΔF shows high transduction efficiency in human DCs, while not so high in mouse DCs. In addition, hPIV2ΔF sufficiently induces maturation of both human and murine DCs, and the maturation state of both human and murine DCs is almost equivalent to that induced by lipopolysaccharide. Moreover, alkylating agent ß-propiolactone-inactivated hPIV2ΔF (BPL-hPIV2ΔF) elicits DC maturation without viral replication/transcription. These results suggest that hPIV2ΔF may be a useful tool for vaccine therapy as a novel type of paramyxoviral vector, which is single-round infectious vector and has potential adjuvant activity.

Neuroblastoma oligo-capping cDNA project: toward the understanding of the genesis and biology of neuroblastoma.

Neuroblastoma (NBL) is a common pediatric cancer originated from the neuronal precursor cells of sympathoadrenal lineage. NBLs show a variety of clinical phenotypes from spontaneous regression to malignant progression with acquirement of resistance to therapy. To understand the molecular mechanism of the genesis, progression, and regression of NBL, we need to identify key molecules determining the neuronal development of sympathoadrenal lineage. To this end, we have performed the NBL cDNA project. It includes (1) mass-cloning of the expressed genes from oligo-capping cDNA libraries derived from primary NBLs with different clinical and biological features; (2) mass-identification of differentially expressed genes between favorable and unfavorable subsets; and (3) molecular and functional analyses of the novel genes, which could be useful prognostic indicators. To date, 10,000 cDNA clones in total, approximately 40% of which contained novel sequences, were randomly picked up and DNA sequenced. We have identified approximately 500 differentially expressed genes between favorable and unfavorable subsets of NBL, among which more than 250 were the genes with unknown function.

Sendai virus vector-mediated gene transfer of glial cell line-derived neurotrophic factor prevents delayed neuronal death after transient global ischemia in gerbils.

We have developed a cytoplasmic replicating virus vector of Sendai virus (SeV) that infects and replicates in most mammalian cells, including neurons, and directs high-level gene expression. To investigate the protective effect of SeV vector-mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF) on the delayed neuronal death caused by transient global ischemia in gerbils, SeV vectors carrying either GDNF (SeV/GDNF) or enhanced green fluorescent protein gene (SeV/GFP) were stereotaxically microinjected into the lateral ventricle. Four days after injection, occlusion of the bilateral common carotid arteries for 5 min produced transient global forebrain ischemia. Treatment with SeV/GDNF significantly decreased the delayed neuronal death of the hippocampal CA1 pyramidal neurons observed 6 days after the operation. TUNEL staining demonstrated that SeV/GDNF treatment markedly reduced the number of apoptotic cells in the hippocampal CA1 neurons, indicating that SeV/GDNF treatment prevented apoptosis. Furthermore, delayed neuronal death on the contralateral side of the hippocampal CA1 was also prevented to a similar extent as that on the ipsilateral side. These results suggest that SeV/GDNF prevents the delayed neuronal death induced by ischemia and is potentially useful for gene therapy for stroke.

Recombinant Sendai virus-mediated gene transfer into adult rat retinal tissue: efficient gene transfer by brief exposure.

To determine the usefulness of recombinant Sendai virus (SeV) for ocular gene transfer, the authors characterized SeV-mediated gene transfer to the retinal tissue of adult rats via subretinal injection. Recombinant SeV encoding the lacZ gene achieved frequent transgene expression in the retinal pigment epithelium (RPE) (mean=38.76%), while gene transfer to other retinal cells was rare. These findings are similar to those of previous reports using adenoviruses. Peak reporter gene expression of SeV in cultured RPE cells was similar to that of adenovirus at the same titer; however, SeV achieved high levels of expression after a brief vector-cell contact time, while adenovirus required over 3hr for efficient gene transfer. This finding was also observed in vivo following a brief SeV filling in the subretinal space, and may therefore provide a clinical advantage in avoiding retinal damage due to prolonged detachment. The observed SeV-mediated gene expression in the rat retina was transient. The initial phase of the decrease in luciferase activity could be prevented by daily eye drops of dexamethasone, suggesting that the corticosteroid-sensitive host reaction may affect early clearance of the virus. The late decline of transgene expression (2 weeks) was inhibited by the immunosuppressant, cyclosporin A, in a dose-dependent manner, suggesting that the cytotoxic T-lymphocyte response may be important in this phase. This work represents the first report of SeV-mediated gene transfer to ocular tissue, and identifies recombinant SeV as a new tool for studies of retinal gene transfer and gene therapy.

The nasal epithelium as a factory for systemic protein delivery.

We have previously shown that recombinant Sendai virus (SeV) produces efficient in vivo airway epithelial gene transfer. The ability to produce therapeutic levels of circulating proteins following noninvasive gene transfer would have widespread clinical application. Here, we compared nose, lung, and skeletal muscle for the ability to produce circulating levels of the secreted mouse antiinflammatory cytokine interleukin-10 (IL10) following SeV-mediated gene transfer. High levels of serum IL10 were obtained from each site with a potency order of lung > nose > muscle for a given viral titer. Serum levels from each site were within the likely required range for anti-inflammatory effects. The combination of a high-efficiency gene transfer agent (SeV) and sites that can be assessed noninvasively (nose or lung) may circumvent several current challenges to gene therapy.