Vero cell-adapted SARS-CoV-2 strain shows increased viral growth through furin-mediated efficient spike cleavage.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes several host proteases to cleave the spike (S) protein to enter host cells. SARS-CoV-2 S protein is cleaved into S1 and S2 subunits by furin, which is closely involved in the pathogenicity of SARS-CoV-2. However, the effects of the modulated protease cleavage activity due to S protein mutations on viral replication and pathogenesis remain unclear. Herein, we serially passaged two SARS-CoV-2 strains in Vero cells and characterized the cell-adapted SARS-CoV-2 strains in vitro and in vivo. The adapted strains showed high viral growth, effective S1/S2 cleavage of the S protein, and low pathogenicity compared with the wild-type strain. Furthermore, the viral growth and S1/S2 cleavage were enhanced by the combination of the Δ68-76 and H655Y mutations using recombinant SARS-CoV-2 strains generated by the circular polymerase extension reaction. The recombinant SARS-CoV-2 strain, which contained the mutation of the adapted strain, showed increased susceptibility to the furin inhibitor, suggesting that the adapted SARS-CoV-2 strain utilized furin more effectively than the wild-type strain. Pathogenicity was attenuated by infection with effectively cleaved recombinant SARS-CoV-2 strains, suggesting that the excessive cleavage of the S proteins decreases virulence. Finally, the high-growth-adapted SARS-CoV-2 strain could be used as the seed for a low-cost inactivated vaccine; immunization with this vaccine can effectively protect the host from SARS-CoV-2 variants. Our findings provide novel insights into the growth and pathogenicity of SARS-CoV-2 in the evolution of cell-cell transmission. IMPORTANCE: The efficacy of the S protein cleavage generally differs among the SARS-CoV-2 variants, resulting in distinct viral characteristics. The relationship between a mutation and the entry of SARS-CoV-2 into host cells remains unclear. In this study, we analyzed the sequence of high-growth Vero cell-adapted SARS-CoV-2 and factors determining the enhancement of the growth of the adapted virus and confirmed the characteristics of the adapted strain by analyzing the recombinant SARS-CoV-2 strain. We successfully identified mutations Δ68-76 and H655Y, which enhance viral growth and the S protein cleavage by furin. Using recombinant viruses enabled us to conduct a virus challenge experiment in vivo. The pathogenicity of SARS-CoV-2 introduced with the mutations Δ68-76, H655Y, P812L, and Q853L was attenuated in hamsters, indicating the possibility of the attenuation of excessive cleaved SARS-CoV-2. These findings provide novel insights into the infectivity and pathogenesis of SARS-CoV-2 strains, thereby significantly contributing to the field of virology.

Intranasal immunization with an RBD-hemagglutinin fusion protein harnesses preexisting immunity to enhance antigen-specific responses.

Intranasal vaccines are anticipated to be powerful tools for combating many infectious diseases, including SARS-CoV-2, because they induce not only systemic immunity but also mucosal immunity at the site of initial infection. However, they are generally inefficient in inducing an antigen-specific immune response without adjuvants. Here, we developed an adjuvant-free intranasal vaccine platform that utilizes the preexisting immunity induced by previous infection or vaccination to enhance vaccine effectiveness. We made RBD-HA, a fusion of the receptor-binding domain (RBD) of spike derived from SARS-CoV-2 as a vaccine target with HA derived from influenza A virus (IAV) as a carrier protein. Intranasal immunization of previously IAV-infected mice with RBD-HA without an adjuvant elicited robust production of RBD-specific systemic IgG and mucosal IgA by utilizing both HA-specific preexisting IgG and CD4+ T cells. Consequently, the mice were efficiently protected from SARS-CoV-2 infection. Additionally, we demonstrated the high versatility of this intranasal vaccine platform by assessing various vaccine antigens and preexisting immunity associated with a variety of infectious diseases. The results of this study suggest the promising potential of this intranasal vaccine platform to address problems associated with intranasal vaccines.

Protocol to isolate temperature-sensitive SARS-CoV-2 mutants and identify associated mutations.

An inability to proliferate at high temperatures typically gives viruses an attenuated phenotype. Here, we present a protocol to obtain and isolate temperature-sensitive (TS) SARS-CoV-2 strains via 5-fluorouracile-induced mutagenesis. We describe steps for the induction of mutations in the wild-type virus and selection of TS clones. We then show how to identify the mutations associated with the TS phenotype, following forward and reverse genetics strategies. For complete details on the use and execution of this protocol, please refer to Yoshida et al. (2022).1.

Identification and Analysis of Monoclonal Antibodies with Neutralizing Activity against Diverse SARS-CoV-2 Variants.

We identified neutralizing monoclonal antibodies against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants (including Omicron variants BA.5 and BA.2.75) from individuals who received two doses of mRNA vaccination after they had been infected with the D614G virus. We named them MO1, MO2, and MO3. Among them, MO1 showed particularly high neutralizing activity against authentic variants: D614G, Delta, BA.1, BA.1.1, BA.2, BA.2.75, and BA.5. Furthermore, MO1 suppressed BA.5 infection in hamsters. A structural analysis revealed that MO1 binds to the conserved epitope of seven variants, including Omicron variants BA.5 and BA.2.75, in the receptor-binding domain of the spike protein. MO1 targets an epitope conserved among Omicron variants BA.1, BA.2, and BA.5 in a unique binding mode. Our findings confirm that D614G-derived vaccination can induce neutralizing antibodies that recognize the epitopes conserved among the SARS-CoV-2 variants. IMPORTANCE Omicron variants of SARS-CoV-2 acquired escape ability from host immunity and authorized antibody therapeutics and thereby have been spreading worldwide. We reported that patients infected with an early SARS-CoV-2 variant, D614G, and who received subsequent two-dose mRNA vaccination have high neutralizing antibody titer against Omicron lineages. It was speculated that the patients have neutralizing antibodies broadly effective against SARS-CoV-2 variants by targeting common epitopes. Here, we explored human monoclonal antibodies from B cells of the patients. One of the monoclonal antibodies, named MO1, showed high potency against broad SARS-CoV-2 variants including BA.2.75 and BA.5 variants. The results prove that monoclonal antibodies that have common neutralizing epitopes among several Omicrons were produced in patients infected with D614G and who received mRNA vaccination.

A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection.

Proteases play critical roles in various biological processes, including apoptosis and viral infection. Several protease biosensors have been developed; however, obtaining a reliable signal from a very low level of endogenous protease activity remains a challenge. In this study, we developed a highly sensitive protease biosensor, named FlipNanoLuc, based on the Oplophorus gracilirostris NanoLuc luciferase. The flipped ß-strand was restored by protease activation and cleavage, resulting in the reconstitution of luciferase and enzymatic activity. By making several modifications, such as introducing NanoBiT technology and CL1-PEST1 degradation tag, the FlipNanoLuc-based protease biosensor system achieved more than 500-fold luminescence increase in the corresponding protease-overexpressing cells. We demonstrated that the FlipNanoLuc-based caspase sensor can be utilized for the detection of staurosporine-induced apoptosis with sixfold increase in luminescence. Furthermore, we also demonstrated that the FlipNanoLuc-based coronavirus 3CL-protease sensor can be used to detect human coronavirus OC43 with tenfold increase in luminescence and severe acute respiratory syndrome-coronavirus-2 infections with 20-fold increase in luminescence by introducing the stem-loop 1 sequence to prevent the virus inducing global translational shutdown.

Versatile live-attenuated SARS-CoV-2 vaccine platform applicable to variants induces protective immunity.

Live-attenuated vaccines are generally highly effective. Here, we aimed to develop one against SARS-CoV-2, based on the identification of three types of temperature-sensitive (TS) strains with mutations in nonstructural proteins (nsp), impaired proliferation at 37°C-39°C, and the capacity to induce protective immunity in Syrian hamsters. To develop a live-attenuated vaccine, we generated a virus that combined all these TS-associated mutations (rTS-all), which showed a robust TS phenotype in vitro and high attenuation in vivo. The vaccine induced an effective cross-reactive immune response and protected hamsters against homologous or heterologous viral challenges. Importantly, rTS-all rarely reverted to the wild-type phenotype. By combining these mutations with an Omicron spike protein to construct a recombinant virus, protection against the Omicron strain was obtained. We show that immediate and effective live-attenuated vaccine candidates against SARS-CoV-2 variants may be developed using rTS-all as a backbone to incorporate the spike protein of the variants.

Could live attenuated vaccines better control COVID-19?

In an effort to control the COVID-19 pandemic, large-scale vaccination is being implemented in various countries using anti-SARS-CoV-2 vaccines based on mRNAs, adenovirus vectors, and inactivated viruses. However, there are concerns regarding adverse effects, such as the induction of fever attributed to mRNA vaccines and pre-existing immunity against adenovirus vectored vaccines or their possible involvement in the development of thrombosis. The induction of antibodies against the adenovirus vector itself constitutes another hindrance, rendering boosting vaccinations ineffective. Additionally, it has been questioned whether inactivated vaccines that predominantly induce humoral immunity are effective against newly arising variants, as some isolated strains were found to be resistant to the serum from COVID-19-recovered patients. Although the number of vaccinated people is steadily increasing on a global scale, it is still necessary to develop vaccines to address the difficulties and concerns mentioned above. Among the various vaccine modalities, live attenuated vaccines have been considered the most effective, since they closely replicate a natural infection without the burden of the disease. In our attempt to provide an additional option to the repertoire of COVID-19 vaccines, we succeeded in isolating temperature-sensitive strains with unique phenotypes that could serve as seeds for a live attenuated vaccine. In this review article, we summarize the characteristics of the currently approved SARS-CoV-2 vaccines and discuss their advantages and disadvantages. In particular, we focus on the novel temperature-sensitive variants of SARS-CoV-2 that we have recently isolated, and their potential application as live-attenuated vaccines. Based on a thorough evaluation of the different vaccine modalities, we argue that it is important to optimize usage not only based on efficacy, but also on the phases of the pandemic. Our findings can be used to inform vaccination practices and improve global recovery from the COVID-19 pandemic.

Fluorescence and physical properties of thulium and erbium co-doped dental zirconia.

The fluorescence and physical properties of thulium and erbium co-doped dental zirconia were investigated. The high-translucency yttria-stabilized dental zirconia specimens co-doped with Tm2O3 powder 0.8 wt% and Er2O3 powder at proportions from 0.1 to 0.8 wt% were used. The specimens co-doped with Tm2O3 powder 0.8 wt% and Er2O3 from 0.3 to 0.5 wt% exhibited the fluorescence similar to that of natural tooth. All the specimens had a tetragonal peak and no major change in the Vickers hardness, fracture toughness and bending strength on addition of Tm2O3 and Er2O3. These results suggest that the method of co-doping trace amounts of Tm2O3 and Er2O3 into high-translucency dental zirconia powder can effectively improve the esthetics of zirconia monolithic fixed dental prothesis.

In vitro investigation of fracture load and aging resistance of high-speed sintered monolithic tooth-borne zirconia crowns.

OBJECTIVES: The purpose of this study was to evaluate the fit, fracture load and aging resistance of the monolithic zirconia tooth-borne crowns with conventional and high-speed sintering. METHODS: The Y-TZP block was machined and sintered with conventional and high-speed sintering furnace. The marginal and internal gap between the crown and abutment was measured using a microscope and a fit checking material. A total of 28 crowns were further divided into an undegraded and a degraded group. An accelerated aging test was carried out on the degraded group. The crown was cemented and a fracture resistance was tested. X-ray diffraction was used to evaluate the crystalline structure. The data were analyzed with Student's t-test, and a one-way ANOVA and Tukey's multiple comparison test. RESULTS: There was no significant difference in mean marginal gap between the two groups. The mean internal gap was significantly greater in the speed sintering than in the conventional sintering (P <0.001). The mean fracture load of the conventional sintering crowns was not significantly different from that of speed sintering crowns after aging. The occurrence of monoclinic crystals of degraded crown was significantly higher than that of undegraded crown both in the conventional (P <0.001) and speed-sintering group (P <0.001). CONCLUSIONS: It was concluded that the monolithic zirconia crowns produced by high-speed sintering showed no significant difference in the marginal gap and the fracture load after aging compared to conventional sintering. Therefore, the high-speed sintering seems a valid method of producing tooth-borne monolithic zirconia crowns.

Fluorescence of thulium-doped translucent zirconia.

The fluorescence and physical properties of thulium-doped zirconia were investigated. A standard grade of zirconia (TZ-3Y-E) and two translucent dental zirconia materials (Zpex and Zpex Smile) were examined. The specimens were prepared by addition of 0-1.5 wt% Tm2O3 and then sintering. When exposed to UV light, the Tm2O3-doped zirconia exhibited blue fluorescence with a peak wavelength of 460 nm. The fluorescence intensity of Zpex and Zpex Smile was higher than that of TZ-3Y-E, with Zpex being more intense than Zpex Smile. Zpex exhibited maximum fluorescence intensity when doped with 0.8 wt% Tm2O3. XRD analysis revealed that TZ-3Y-E and Zpex contained primarily tetragonal zirconia, while Zpex Smile contained largely cubic phase zirconia. There were no changes observed in the microstructure or physical properties of the zirconia specimens when doped with Tm2O3.

Fabrication of a radiopaque fit-testing material to evaluate the three-dimensional accuracy of dental prostheses.

OBJECTIVES: The objective of this study was to fabricate a radiopaque prosthetic fit-testing material, and to develop methodology to evaluate the fitting accuracy of prostheses three-dimensionally (3D) using a combination of the silicone replica technique and micro-computed tomography (µCT). METHODS: Eight types of prototype specimens of fit-testing materials were prepared by adding contrast agents (zirconia, alumina, and barium-glass) to a commercially available fit-testing material. These specimens were evaluated on their mechanical properties, X-ray absorption coefficients, reproducibility of cement space, and suitability for 3D analysis by µCT. Then, silicone replicas made from prototype specimens were assessed for accurate 3D morphology. Subsequently, color-mapping analyses of the silicone replicas were performed according to replica thickness, and the results were compared with stereomicroscopic images. RESULTS: The mechanical properties, X-ray absorption coefficients, and reproducibility of the cement space demonstrated that prototypes containing 20wt% zirconia (Zr-20) or barium glass (diameter 2µm; Ba2-20) were useful as fit-testing materials. However, the morphology of the Ba2-20 silicone replica was unable to be accurately described using µCT because of its low X-ray absorption threshold. Zr-20, however, could be clearly observed on µCT imaging. Furthermore, color-mapping analysis of the µCT images demonstrated that Zr-20 was the most suitable for 3D observation of prosthetic fit. SIGNIFICANCE: This method could allow any professional to evaluate the fit of any type of dental prosthesis, such as inlays, crowns, and fixed and removable dentures. This study demonstrated that the technique presented in the current study is able to accurately describe the abutment-crown prosthetic discrepancy based on silicone replicas.

Action mechanism of 6, 6'-dihydroxythiobinupharidine from Nuphar japonicum, which showed anti-MRSA and anti-VRE activities.

BACKGROUND: Multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), cause serious infections at clinical sites, for which the development of new drugs is necessary. We screened candidates for new antibiotics and investigated its action mechanism. METHODS: An antimicrobial compound was isolated from an extract of Nuphar japonicum. Its chemical structure was determined by NMR, MS, and optical rotation. We measured its minimum inhibitory concentration (MIC) using the microdilution method. The effects of the compound on DNA gyrase and DNA topoisomerase IV were investigated with DNA supercoiling, decatenation, and cleavage assay. RESULTS: We isolated and identified 6,6'-dihydroxythiobinupharidine as the antimicrobial compound. The MIC of this compound was 1-4 µg/mL against various MRSA and VRE strains. We also demonstrated that this compound inhibited DNA topoisomerase IV (IC50 was 10-15 µM), but not DNA gyrase in S. aureus, both of which are known to be the targets of quinolone antibiotics and necessary for DNA replication. However, this compound only exhibited slight cross-resistance to norfloxacin-resistant S. aureus, which indicated that DTBN might inhibit other targets besides topoisomerase IV. These results suggest that 6,6'-dihydroxythiobinupharidine may be a potent candidate or seed for novel antibacterial agents. CONCLUSIONS: DTBN from N. japonicum showed anti-MRSA and anti-VRE activities. DTBN might be involved in the inhibition of DNA topoisomerase IV. GENERAL SIGNIFICANCE: DTBN might be useful as a seed compound. The information on the inhibition mechanism of DTBN will be useful for the modification of DTBN towards developing novel anti-MRSA and anti-VRE drug.