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.

Understanding quantitative polymerase chain reaction bioanalysis issues before validation planning: Japan Bioanalysis Forum discussion group.

Investigating the biodistribution of cell and gene therapy products may play an important role in evaluating their safety and pharmacology. As quantitative polymerase chain reaction (qPCR) is often used for these analyses, it is essential to improve the reliability of bioanalysis performed using qPCR. In this report, the authors discuss the use of qPCR in nonclinical studies, as it can be used to detect target DNA/RNA and it is quantitative and applicable for long-term analysis. The authors also discuss points to consider during bioanalysis using qPCR and present appropriate validation items and their criteria. The authors anticipate the discussion provided herein to contribute to the development of validation and sample analysis for pharmaceuticals analyzed using qPCR.

Evaluation of hepatocyte growth factor plasmid therapeutic effect by 99mTc-hexakis-2-methyoxy-isobutylisonitrile blood flow scintigraphy in a rat model of hind limb ischemia.

OBJECTIVE: This study was conducted in an attempt to use blood flow scintigraphy with 99mTc-hexakis-2-methyoxy-isobutylisonitrile (99mTc-MIBI) for the evaluation of the angiogenic effect of hepatocyte growth factor (HGF) plasmid in a rat model of hind limb ischemia. MATERIALS AND METHODS: The femoral artery of the left hind limb of each rat was ligated to create a model of hind limb ischemia. Three weeks later, HGF plasmid (1.5 mg/1.1 ml/body) or saline (1.1 ml/body) was administered intramuscularly into three sites of the ischemic hind limb. Two and 4 weeks after the treatment, blood flow through the hind limb was measured by 99mTc-MIBI scintigraphy. In addition, the number of capillary endothelial cells obtained by immunostaining for CD31 was counted. RESULTS: After 99mTc-MIBI scintigraphy in the HGF plasmid-treated group, the blood flow ratio increased significantly from the pretreatment ratio 63.8 to 73.4%, 2 weeks after treatment (P<0.05) and to 97.8%, 4 weeks after treatment (P<0.05). The number of CD31-positive endothelial cells was significantly higher in the HGF plasmid-treated group than in the control group. CONCLUSIONS: The experimental study using a rat model of hind limb ischemia showed usefulness of 99mTc-MIBI scintigraphy to evaluate the angiogenic effect of HGF plasmid treatment.