Biology subjects

Torii, S.

Publications and source records attributed to Torii, S..

5 recordsLinked to original sources

bioRxiv2021

Dynamic expression of smRNA from fecal exosome in disease progression of an inflammatory bowel disorder mouse model.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract affecting over 3 million adults in the United States. Despite being widespread, reliable early diagnostic tests are not available. We examined exosomal small RNA (smRNA), specifically targeting microRNA (miRNA) and piRNA from the stool samples of IBD model mice, interleukin 10 knockout mice (IL-10 KO), as a potential diagnostic marker. Stool samples were specifically chosen because they are readily available, and collection is noninvasive. At the end of the experimental period, the gastrointestinal (GI) tract was collected, and disease severity was scored. Histopathology showed a significant increase in inflammation and proliferation within the proximal and distal large intestines. smRNA profiles were examined upon conventional housing (start-point) which is a determinant factor of spontaneous IBD progression in the IL-10 KO mice, terminal illness (end-point), and 6 weeks before the end-point (mid-point), when the mice were still phenotypically healthy. We found 504 smRNA that were significantly differentially expressed between before symptom onset and terminal sedation. These changes were not detected in wild-type samples. Moreover, clustering analysis of expression changes over the disease progression identified a unique set of smRNAs that primarily target pro-inflammatory or anti-inflammatory genes. The expression of smRNAs that suppresses pro-inflammatory genes was higher at 6 weeks before terminal sedation, suggesting the downregulation of the pro-inflammatory genes advances the terminal illness of the IBD. In summary, our study proposes that fecal exosomal smRNA profiling offers a new opportunity to monitor the inflammatory status of the gut with a capability of detecting its pro-inflammatory (asymptomatic) status. Our next step is to understand the spatiotemporal interplay of these exosomes and the host cells in the gut as well as the complete biochemical makeup of fecal exosomes, such as mRNA, DNA, protein, and lipids. This will lead to an exciting development of reengineered exosomes that can be utilized to treat or even prevent the pro-inflammatory colonic lesion while the host is still clinically asymptomatic.

genomics↗

bioRxiv2021

Anti-CoVid19 plasmid DNA vaccine induces a potent immune response in rodents by Pyro-drive Jet Injector intradermal inoculation

There is an urgent need to limit and stop the worldwide coronavirus disease 2019 (COVID-19) pandemic via quick development of efficient and safe vaccination methods. Plasmid DNA vaccines are one of the most remarkable vaccines that can be developed in a short term. pVAX1-SARS-CoV2-co, which is a plasmid DNA vaccine, was designed to express severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein. The produced antibodies lead to Immunoreactions against S protein, anti-receptor-binding-domain, and neutralizing action of pVAX1-SARS-CoV2-co, as confirmed in a previous study. To promote the efficacy of the pVAX1-SARS-CoV2-co vaccine, a pyro-drive jet injector (PJI) was employed. PJI is an injection device that can adjust the injection pressure depending on various target tissues. Intradermally-adjusted PJI demonstrated that pVAX1-SARS-CoV2-co vaccine injection caused a strong production of anti-S protein antibodies, triggered immunoreactions and neutralizing actions against SARS-CoV-2. Moreover, a high dose of pVAX1-SARS-CoV2-co intradermal injection via PJI did not cause any serious disorders in the rat model. Finally, virus infection challenge in mice, confirmed that intradermally immunized (via PJI) mice were potently protected from COVID-19 infection. Thus, pVAX1-SARS-CoV2-co intradermal injection via PJI is a safe and promising vaccination method to overcome the COVID-19 pandemic.

immunology↗

bioRxiv2020

An infectivity-enhancing site on the SARS-CoV-2 spike protein is targeted by COVID-19 patient antibodies

SARS-CoV-2 infection causes severe symptoms in a subset of patients, suggesting the presence of certain unknown risk factors. Although antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike have been shown prevent SARS-CoV-2 infection, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from COVID-19 patients, and found that some of antibodies against the N-terminal domain (NTD) dramatically enhanced the binding capacity of the spike protein to ACE2, and thus increased SARS-CoV2 infectivity. Surprisingly, mutational analysis revealed that all the infectivity-enhancing antibodies recognized a specific site on the surface of the NTD. The antibodies against this infectivity-enhancing site were detected in all samples of hospitalized COVID-19 patients in the study. However, the ratio of infectivity-enhancing antibodies to neutralizing antibodies differed among patients. Furthermore, the antibodies against the infectivity-enhancing site were detected in 3 out of 48 uninfected donors, albeit at low levels. These findings suggest that the production of antibodies against SARS-CoV-2 infectivity-enhancing site could be considered as a possible exacerbating factors for COVID-19 and that a spike protein lacking such antibody epitopes may be required for safe vaccine development, especially for individuals with pre-existing enhancing antibodies.

microbiology↗

bioRxiv2020

The N-terminal domain of spike glycoprotein mediates SARS-CoV-2 infection by associating with L-SIGN and DC-SIGN

The widespread occurrence of SARS-CoV-2 has had a profound effect on society and a vaccine is currently being developed. Angiotensin-converting enzyme 2 (ACE2) is the primary host cell receptor that interacts with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Although pneumonia is the main symptom in severe cases of SARS-CoV-2 infection, the expression levels of ACE2 in the lung is low, suggesting the presence of another receptor for the spike protein. In order to identify the additional receptors for the spike protein, we screened a receptor for the SARS-CoV-2 spike protein from the lung cDNA library. We cloned L-SIGN as a specific receptor for the N-terminal domain (NTD) of the SARS-CoV-2 spike protein. The RBD of the spike protein did not bind to L-SIGN. In addition, not only L-SIGN but also DC-SIGN, a closely related C-type lectin receptor to L-SIGN, bound to the NTD of the SARS-CoV-2 spike protein. Importantly, cells expressing L-SIGN and DC-SIGN were both infected by SARS-CoV-2. Furthermore, L-SIGN and DC-SIGN induced membrane fusion by associating with the SARS-CoV-2 spike protein. Serum antibodies from infected patients and a patient-derived monoclonal antibody against NTD inhibited SARS-CoV-2 infection of L-SIGN or DC-SIGN expressing cells. Our results highlight the important role of NTD in SARS-CoV-2 dissemination through L-SIGN and DC-SIGN and the significance of having anti-NTD neutralizing antibodies in antibody-based therapeutics.

microbiology↗

bioRxiv2020

Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). While the development of specific treatments and a vaccine is urgently needed, functional analyses of SARS-CoV-2 have been limited by the lack of convenient mutagenesis methods. In this study, we established a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. Notably, the construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (~5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. We hope that our reverse genetics system will contribute to the further understanding of SARS-CoV-2.

microbiology↗