A previously unknown SARS-CoV-2 vaccine protecting nonhuman primates from SARS-CoV-2 challenge was developed using RBD-P2 and N.

Adeno-associated virus (AAV)-mediated gene delivery holds great promise for gene therapy. However, the non-invasive delivery of AAV for lung tissues has not been adequately established. Here, we revealed that the intratracheal administration of an appropriate amount of AAV2/8 predominantly targets lung tissue. AAV-mediated gene delivery that we used in this study induced the expression of the desired protein in lung parenchymal cells, including alveolar type II cells. We harnessed the technique to develop severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-susceptible mice. Three kinds of immune function-relevant gene knockout (KO) mice were transduced with AAV encoding human angiotensin-converting enzyme 2 (hACE2) and then injected with SARS-CoV-2. Among these mice, type I interferon receptor (IFNAR) KO mice showed increased viral titer in the lungs compared to that in the other KO mice. Moreover, nucleocapsid protein of SARS-CoV-2 and multiple lesions in the trachea and lung were observed in AAV-hACE2-transduced, SARS-CoV-2-infected IFNAR KO mice, indicating the involvement of type I interferon signaling in the protection of SARS-CoV-2. In this study, we demonstrate the ease and rapidness of the intratracheal administration of AAV for targeting lung tissue in mice, and this can be used to study diverse pulmonary diseases.

The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) pandemic is causing a global crisis. It is still unresolved. Although many therapies and vaccines are being studied, they are still in their infancy. As this pandemic continues, rapid and accurate research for the development of therapies and vaccines is needed. Therefore, it is necessary to understand characteristics of diseases caused by SARS-CoV-2 through animal models. Syrian hamsters are known to be susceptible to SARS-CoV-2. They were intranasally inoculated with SARS-CoV-2. At 2, 4, 8, 12, and 16 days post-infection (dpi), these hamsters were euthanized, and tissues were collected for ultrastructural and microstructural examinations. Microscopic lesions were prominent in the upper and lower respiratory tracts from 2 and 4 dpi groups, respectively. The respiratory epithelium in the trachea, bronchiole, and alveolar showed pathological changes. Inflammatory cells including neutrophils, lymphocytes, macrophages, and eosinophils were infiltrated in/around tracheal lamina propria, pulmonary vessels, alveoli, and bronchiole. In pulmonary lesions, alveolar wall was thickened with infiltrated inflammatory cells, mainly neutrophils and macrophages. In the trachea, epithelial damages started from 2 dpi and recovered from 8 dpi, consistent with microscopic results, High levels of SARS-CoV-2 nucleoprotein were detected at 2 dpi and 4 dpi. In the lung, lesions were most severe at 8 dpi. Meanwhile, high levels of SARS-CoV-2 were detected at 4 dpi. Electron microscopic examinations revealed cellular changes in the trachea epithelium and alveolar epithelium such as vacuolation, sparse micro-organelle, and poor cellular margin. In the trachea epithelium, the number of cytoplasmic organelles was diminished, and small vesicles were prominent from 2 dpi. Some of these electron-lucent vesicles were filled with virion particles. From 8 dpi, the trachea epithelium started to recover. Because of shrunken nucleus and swollen cytoplasm, the N/C ratio of type 2 pneumocyte decreased at 8 and 12 dpi. From 8 dpi, lamellar bodies on type 2 pneumocyte cytoplasm were increasingly observed. Their number then decreased from 16 dpi. However, there was no significant change in type 1 pneumocyte. Viral vesicles were only observed in the cytoplasm of type 2 pneumocyte. In conclusion, ultra- and micro-structural changes presented in this study may provide useful information for SARS-CoV-2 studies in various fields.

The effects of pentylenetetrazol (PTZ), a GABA receptor antagonist, were studied on passive avoidance learning and expression of heat shock protein 70 (hsp70), neuroglobin, and fatty acid binding protein‐7 (fabp‐7) genes. Zebrafish were trained to stay in a dark compartment to avoid a weight dropping in an acryl shuttle box with a central sliding door. In two training sessions of 2 h interval, each consisting of 3 trials, the crossing time was significantly increased from 43.2±14.4s to 149.3±38.5s in the first training session and remained 116.1 ±36.0 s in the first trial of the second training session in the control. In zebrafish treated with PTZ before the first training session, the crossing time was significantly increased neither in the first nor in the second training session. However, the increased crossing time was maintained in the second training session when 10 mM PTZ was treated three times for 10 min at 30 min intervals between the first and second training session. Quantitative real‐time PCR showed that expression level of hsp70 mRNA increased two to eight fold over that of control in the brain at 0–24 h after termination of PTZ treatment. No change in expression of neuroglobin and fabp‐7 mRNA was shown in PTZ‐treated zebrafish. Our studies suggest that PTZ impairs learning ability in avoidance response and also modifies expression of genes related to the neuroprotection.

Messenger RNA (mRNA) vaccines have exhibited promising potential for infectious disease prevention. Although various delivery methods have been explored, the use of electroporation (EP) for the delivery of naked mRNA has received relatively less attention. In this study, we used mouse models to investigate whether naked mRNA vaccine delivery via intramuscular EP (IM-EP) elicits a protective immune response against lethal viral infection. To achieve this, we injected C57BL/6 mice with naked mRNA encoding the SARS-CoV-2 mRNA vaccine via IM-EP and evaluated the resulting immune responses. IM-EP-mediated delivery of the mRNA vaccine induced robust humoral and cellular immune responses, characterized by elevated SARS-CoV-2 receptor-binding domain (RBD)-specific IgG antibodies, enhanced IFN-γ production by CD8⁺ T cells, and upregulated cytokine expression in the muscle and lymph nodes. Using the K18-hACE2 mouse model, we revealed that IM-EP-mediated delivery of the naked mRNA vaccine effectively protected mice from lethal SARS-CoV-2 infection. Overall, our findings suggest that the delivery of naked mRNA via IM-EP can be an effective strategy for preventing infectious diseases.