Development of a neutralization monoclonal antibody with a broad neutralizing effect against SARS-CoV-2 variants.

BACKGROUND: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has challenged the effectiveness of current therapeutic regimens. Here, we aimed to develop a potent SARS-CoV-2 antibody with broad neutralizing effect by screening a scFv library with the spike protein receptor-binding domain (RBD) via phage display. METHODS: SKAI-DS84 was identified through phage display, and we performed pseudovirus neutralization assays, authentic virus neutralization assays, and in vivo neutralization efficacy evaluations. Furthermore, surface plasmon resonance (SPR) analysis was conducted to assess the physical characteristics of the antibody, including binding kinetics and measure its affinity for variant RBDs. RESULTS: The selected clones were converted to human IgG, and among them, SKAI-DS84 was selected for further analyses based on its binding affinity with the variant RBDs. Using pseudoviruses, we confirmed that SKAI-DS84 was strongly neutralizing against wild-type, B.1.617.2, B.1.1.529, and subvariants of SARS-CoV-2. We also tested the neutralizing effect of SKAI-DS84 on authentic viruses, in vivo and observed a reduction in viral replication and improved lung pathology. We performed binding and epitope mapping experiments to understand the mechanisms underlying neutralization and identified quaternary epitopes formed by the interaction between RBDs as the target of SKAI-DS84. CONCLUSIONS: We identified, produced, and tested the neutralizing effect of SKAI-DS84 antibody. Our results highlight that SKAI-DS84 could be a potential neutralizing antibody against SARS-CoV-2 and its variants.

Suspension culture of Vero cells for the production of adenovirus type 5.

PURPOSE: Most cell culture processes for viral vaccine production are mainly based on adherent cell culture systems using serum, which are associated with expensive and labor-intensive processes to produce large amounts of viral vaccine strains. In this study, we investigated whether Vero cells could be grown in serum-free and shaking suspension conditions. Furthermore, we assessed the ability of the Vero cell suspension culture system to produce adenovirus type 5 (Ad5), compared to that of the adhesive Vero cell culture system. MATERIALS AND METHODS: We tested the feasibility of commercial serum-free media for Vero cell culture. For the adaptation of Vero cells in suspension culture, adhesive Vero cells were added in the early phase of shaking suspension culture, and 50 days after shaking suspension culture, suspension-adapted Vero cells were subcultured continuously. To assess the virus production ability of Vero cells in suspension, the cells were infected with Ad5-green fluorescent protein and evaluated based on their fluorescence intensity. RESULTS: The Vero cells grown in OptiPRO serum-free medium showed no changes in morphology and growth rate, but MRC-5 and FRhk-4 cells showed morphological changes and decreased growth rate, respectively. The Vero cells were well adapted to the suspension culture system. The Vero cells in suspension showed a better Ad5 production ability than the adherent Vero cells. CONCLUSION: Vero cells can be grown in OptiPRO serum-free medium. Further, our suspension culture-adapted Vero cells may be suitable to produce viral vaccine strains due to their high ability to produce viruses such as Ad5.

Effect of apoptosis-associated speck-like protein containing a caspase recruitment domain on vaccine efficacy: Overcoming the effects of its deficiency with aluminum hydroxide adjuvant.

Host factors such as nutritional status and immune cell state are important for vaccine efficacy. Inflammasome activation may be important for triggering vaccine-induced humoral and cell-mediated immune responses. Formulations with alum as a typical adjuvant to overcome the effects of host factors have recently been shown to induce inflammasome activation, which augments vaccine efficacy. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is one of the main components of inflammasomes, but it is not clear whether ASC affects the vaccine-induced immune response. Herein, we used two types of vaccines: inactivated influenza vaccine not formulated with alum, and HPV vaccine formulated with alum. We gave the vaccines to ASC knockout (ASC-/- ) mice to investigate the role of ASC in vaccine efficacy. Influenza vaccine-immunized ASC-/- mice did not show antibody titers in week 2 after the first vaccination. After boosting, the antibody titer in ASC-/- mice was about half that in wild type (WT) mice. Furthermore, a cytotoxic T-lymphocyte response against influenza vaccine was not induced in ASC-/- mice. Therefore, vaccinated ASC-/- mice did not show effective protection against viral challenge. ASC-/- mice immunized with alum-formulated HPV vaccine showed similar antibody titers and T-cell proliferation compared with immunized WT mice. However, the HPV vaccine without alum induced up to threefold lower titers of HPV-specific antibody titers in ASC-/- mice compared with those in WT mice. These findings suggest that alum in vaccine can overcome the ASC-deficient condition.

Sublingual immunization with Japanese encephalitis virus vaccine effectively induces immunity through both cellular and humoral immune responses in mice.

The Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis. Although there are four classes of vaccines against JEV, all of them are administered by s.c or i.m injection. Here, the effectiveness of sublingual (s.l.) administration of a JEV live-attenuated vaccine or recombinant modified vaccinia virus Ankara (MVA) vaccine, including JEV prM/E, was investigated. The mice were immunized three times i.m. or s.c. One week after the final immunization by both s.l. and i.m. routes, the titers of IgG1 induced by the recombinant MVA vaccine were higher than those induced by the live-attenuated vaccine, whereas the titers of IgG2a induced by the live-attenuated vaccine were higher than those induced by the recombinant MVA vaccine. However, both vaccines induced neutralizing antibodies when given by either s.l. or i.m. routes, indicating that both vaccines induce appropriate Th1 and Th2 cell responses through the s.l. and i.m. routes. Moreover, both vaccines protected against induction of proinflammatory cytokines and focal spleen white pulp hyperplasia after viral challenge. Virus-specific IFN-γ+ CD4+ and CD8+ T cells appeared to increase in mice immunized via both s.l. and i.m. routes. Interestingly, virus-specific IL-17+ CD4+ T cells increased significantly only in the mice immunized via the s.l. route; however, the increased IL-17 did not affect pathogenicity after viral challenge. These results suggest that s.l. immunization may be as useful as i.m. injection for induction of protective immune responses against JEV by both live-attenuated and recombinant MVA vaccines.

Apios americana Medik Extract Alleviates Lung Inflammation in Influenza Virus H1N1- and Endotoxin-Induced Acute Lung Injury.

Apios americana Medik (hereinafter Apios) has been reported to treat diseases, including cancer, hypertension, obesity, and diabetes. The therapeutic effect of Apios is likely to be associated with its anti-inflammatory activity. This study was conducted to evaluate the protective effects of Apios in animal models of acute lung injury induced by lipopolysaccharide (LPS) or pandemic H1N1 2009 influenza A virus (H1N1). Mice were exposed to LPS or H1N1 for 2-4 days to induce acute lung injury. The treatment groups were administered Apios extracts via oral injection for 8 weeks before LPS treatment or H1N1 infection. To investigate the effects of Apios, we assessed the mice for in vivo effects of Apios on immune cell infiltration and the level of pro-inflammatory cytokines in the bronchoalveolar lavage (BAL) fluid, and histopathological changes in the lung. After induction of acute lung injury, the numbers of neutrophils and total cells were lower in the Apios-treated groups than in the non-Apios-treated LPS and H1N1 groups. The Apios groups tended to have lower levels of tumor necrosis factor-a and interleukin-6 in BAL fluid. In addition, the histopathological changes in the lungs were markedly reduced in the Apios-treated groups. These data suggest that Apios treatment reduces LPS- and H1N1-induced lung inflammation. These protective effects of Apios suggest that it may have therapeutic potential in acute lung injury.

Catalytic wet oxidation of ammonia: why is N2 formed preferentially against NO3 -?

The role of catalyst and the reason for the preferential formation of N(2) in the catalytic oxidation reaction of ammonia in water over a Ru (3wt.%)/TiO(2) catalyst were elucidated. It was verified that the catalyst in the reaction had no direct relevance to the selective formation of N(2), but was responsible only for the oxidation of aqueous ammonia, NH(3)(aq), finally giving a molecule of nitrous acid. The preferential production of N(2) was experimentally demonstrated due to the homogeneous aqueous phase reaction of the nitrous acid-dissociated NO(2)(-) with NH(4)(+) ions. Even under the highly oxidizing condition, NO(2)(-) was much more likely to react with NH(4)(+) to form N(2) than being oxidized over the catalyst to NO(3)(-) as long as NH(4)(+) was available in solution.

Mechanism and kinetics of the catalytic oxidation of aqueous ammonia to molecular nitrogen.

Aqueous phase catalytic oxidation of ammonia has been studied over Ru/TiO2 catalyst in a batch reactor by changing the solution pH, concentration of catalyst in the solution, temperature, and reaction time. The oxidation reaction of ammonia over Ru/TiO2 catalyst has been found to take place exclusively for the aqueous NH3 with a preferred mode in strong alkaline pH region. An oxidation reaction pathway has been proposed as follows: Oxidation of ammonia is initiated by the reaction of aqueous ammonia with catalytically activated oxygen. After undergoing further successive oxidation reactions with activated oxygen, ammonia is finally oxidized to a molecule of nitrous acid. Nitrous acid dissociates into a nitrite ion and a proton. The solution pH is decreased with the protons from the dissociation of HNO2 so that the solution concentration of NH4+ is increased. Molecular nitrogen as a final product is produced from the homogeneous aqueous phase reaction between nitrous ion and ammonium ion. Further reaction of nitrous ion with the activated oxygen leads to the formation of nitrate ion. The reaction pathway proposed has been validated with the changes of solution pH along with the ammonia conversions, and the formation of N2 from the solution containing NO2- and NH4+ ions in equimolar amounts of nitrogen has been confirmed in a separate experiment. The kinetics of aqueous ammonia oxidation reaction has been well represented as a first-order reaction with respect to the concentration of aqueous ammonia, and an apparent rate constant has been obtained as a function of catalyst concentration in solution, oxygen pressure, and reaction temperature.