Immune response induced in rodents by anti-CoVid19 plasmid DNA vaccine via pyro-drive jet injector inoculation.

There is an urgent need to stop the coronavirus disease 2019 (COVID-19) pandemic through the development of efficient and safe vaccination methods. Over the short term, plasmid DNA vaccines can be developed as they are molecularly stable, thus facilitating easy transport and storage. pVAX1-SARS-CoV2-co was designed for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) S protein. The antibodies produced led to immunoreactions against the S protein, an anti-receptor-binding-domain, and a neutralizing action of the pVAX1-SARS-CoV2-co, as previously confirmed. To promote the efficacy of the pVAX1-SARS-CoV2-co vaccine a pyro-drive jet injector (PJI) was used. An intradermally adjusted PJI demonstrated that the pVAX1-SARS-CoV2-co vaccine injection caused a high production of anti-S protein antibodies, triggered immunoreactions, and neutralized the actions against SARS-CoV-2. A high-dose pVAX1-SARS-CoV2-co intradermal injection using PJI did not cause any serious disorders in the rat model. A viral challenge confirmed that intradermally immunized mice were potently protected from COVID-19. A pVAX1-SARS-CoV2-co intradermal injection using PJI is a safe and promising vaccination method for overcoming the COVID-19 pandemic.

Preclinical study of a DNA vaccine targeting SARS-CoV-2.

To fight against the worldwide COVID-19 pandemic, the development of an effective and safe vaccine against SARS-CoV-2 is required. As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFNγ expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies by a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, and neutralization assays using pseudo-virus, and live SARS-CoV-2. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits. Finally, DNA vaccine protected hamsters from SARS-CoV-2 infection. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic.

AJP001, a novel helper T-cell epitope, induces a humoral immune response with activation of innate immunity when included in a peptide vaccine.

Vaccine design requires well-tailored formulations including a T-cell epitope and adjuvants. We identified a novel cationic peptide, AJP001, which possesses a strong affinity for murine MHC class II alleles (H2-IEd and H2-IAd) and low affinity for H2-IAb. We designed an AJP001 and epitope peptide-conjugated vaccine, AJP001-angiotensin (Ang) II, which was intracutaneously administered to mice three times at 2-week intervals. Indeed, the AJP001-Ang II vaccine induced antibody production against Ang II in BALB/cA mice but not in C57BL/6 mice. To estimate the T-cell-dependent immunogenicity of the AJP001 conjugate vaccine in human cells, naïve human peripheral blood mononuclear cells (PBMCs) were exposed to AJP001-Ang II, and T-cell proliferation was evaluated by analyzing cell division using flow cytometric measurement of carboxyfluorescein succinimidyl ester (CFSE) dye dilution. To activate the immune response, the innate immune system must be activated by adjuvant treatment. Interestingly, treatment with AJP001 induced IL-1ß and IL-18 secretion via NLRP3 inflammasome activation and induced TNF-α and IL-6 production through an NF-κB-dependent pathway in human and mouse macrophages. These results suggest that AJP001 behaves as a T-cell epitope in mice and humans and is a useful tool for the formulation of peptide vaccines without the addition of adjuvants.

Pyridoxamine scavenges protein carbonyls and inhibits protein aggregation in oxidative stress-induced human HepG2 hepatocytes.

Introduction of carbonyl groups into amino acid residues is a hallmark for oxidative damage to proteins by reactive oxygen species (ROS). Protein carbonylation can have deleterious effects on cell function and viability, since it is generally unrepairable by cells and can lead to protein dysfunction and to the production of potentially harmful protein aggregates. Meanwhile, pyridoxamine (PM) is known to scavenge various toxic carbonyl species derived from either glucose or lipid degradation through nucleophilic addition. PM is also demonstrated to catalyze non-enzymatic transamination reactions between amino and α-keto acids. Here, we found that PM scavenges protein carbonyls in oxidized BSA with concomitant generation of pyridoxal and recovers oxidized lysozyme activity. Moreover, we demonstrated that the treatment of H2O2-exposed HepG2 hepatocytes with PM significantly reduced levels of cellular carbonylated proteins and aggregated proteins, and also improved cell survival rate. Our results suggest that PM may have potential efficacy in ameliorating ROS-mediated cellular dysfunction.

[Examination of three-detector high-speed-rotation SPECT acquisition under breathhold in body: clinical application of 99mTc-MAA].

In traditional pulmonary perfusion single photon emission computed tomography (SPECT), respiratory lung motion and cyclically varying changes in lung volume during image acquisition inherently degrade the image sharpness of ill-defined perfusion defects. However, because of the lack of an adequate fast imaging technique, perfusion SPECT has never been acquired under breathhold conditions, whereas breathhold images are commonly used for pulmonary magnetic resonance (MR) and computed tomographic (CT) images. Although a high-speed imaging technique combined with a multidetector SPECT system may enable SPECT images to be obtained during a short period of breathholding, image quality would be degraded owing to decreased radioactivity counts and increased statistical noise. To resolve this problem, we developed an innovative SPECT imaging technique using a triple-head SPECT system and the high-speed-detector rotation-multiplied projection (HSRMP) technique, where a single SPECT image was reconstructed from multiple respiratory dimensional breathhold projection data obtained at the same angle. HSRMP provided noiseless high-quality perfusion SPECT images by compensating for decreased radioactivity counts caused by high-speed imaging, and significantly improved image quality and perfusion defect clarity compared with traditional non-breathhold SPECT images.

[Measurement of effective dose of the assisting person in diagnostic X-ray examination].

We measured the effective dose received by the person assisting the patient at diagnostic X-ray examination. Measurement was done when a patient's chest, abdomen, lumbar vertebrae, hip joint, skull, cervical vertebrae, or knee joint was examined by radiography. A body phantom including human bones exposed to radiation was used in the role of the patient. Some exposure conditions for these measurements were the same as those used routinely in computed radiography. Effective dose was measured directly with an ionization survey meter. As a result, the effective dose of the person assisting with axial projection of the hip joint was 124 microSv, which was higher than that for other regions and projections. If the assisting person helped a patient without using any protective device, the effective dose would be low enough to ignore. However, because medical staff are frequently exposed to radiation, optimal protection is crucial to prevent unnecessary radiation.