Establishment of multicenter COVID-19 therapeutics preclinical test system in Republic of Korea.

Throughout the recent COVID-19 pandemic, South Korea led national efforts to develop vaccines and therapeutics for SARS-CoV-2. The project proceeded as follows: 1) evaluation system setup (including Animal Biosafety Level 3 (ABSL3) facility alliance, standardized nonclinical evaluation protocol, and laboratory information management system), 2) application (including committee review and selection), and 3) evaluation (including expert judgment and reporting). After receiving 101 applications, the selection committee reviewed pharmacokinetics, toxicity, and efficacy data and selected 32 final candidates. In the nonclinical efficacy test, we used golden Syrian hamsters and human angiotensin-converting enzyme 2 transgenic mice under a cytokeratin 18 promoter to evaluate mortality, clinical signs, body weight, viral titer, neutralizing antibody presence, and histopathology. These data indicated eight new drugs and one repositioned drug having significant efficacy for COVID-19. Three vaccine and four antiviral drugs exerted significant protective activities against SARS-CoV-2 pathogenesis. Additionally, two anti-inflammatory drugs showed therapeutic effects on lung lesions and weight loss through their mechanism of action but did not affect viral replication. Along with systematic verification of COVID-19 animal models through large-scale studies, our findings suggest that ABSL3 multicenter alliance and nonclinical evaluation protocol standardization can promote reliable efficacy testing against COVID-19, thus expediting medical product development.

Rag2 Deficiency Enhances Susceptibility to Systemic Mouse Adenovirus Type 1 Infection.

INTRODUCTION: Recombination-activating gene (Rag) 1 and Rag2, which are essential in V(D)J recombination, play a crucial role in B- and T-cell maturation. METHOD: We investigated the effects of Rag2 deficiency in clustered regularly interspaced short palindromic repeats/Cas9-mediated FVB-Rag2 knockout (KO) and wild-type (WT) mice infected with mouse adenovirus type 1 (MAV-1) via the intranasal route. RESULTS: MAV-1 infection caused more severe histopathological changes in FVB-Rag2 KO mice than in WT mice. FVB-Rag2 KO mice exhibited moderate to severe inflammation on day 4 and severe inflammation on day 8 post infection. In contrast, WT mice showed mild inflammation on day 4 and mild to severe inflammation on day 8 post infection, including interstitial pneumonia and inflammatory cell infiltration in the lungs and liver. Viral loads in the spleen and kidneys were significantly higher in FVB-Rag2 KO mice than in WT mice on day 8 post infection. Levels of cytokines and chemokines, including macrophage inflammatory protein-1α, induced protein 10, interferon (IFN)-α, IFN-γ, and tumor necrosis factor alpha, were upregulated in the spleens of FVB-Rag2 KO mice compared with those of WT mice. The upregulation of several cytokines occurred concurrently with the histopathological changes. MAV-1 infection induced more severe systemic infection in FVB-Rag2 KO mice than in WT mice. CONCLUSION: In mice, Rag2 deficiency induces inflammatory cell recruitment via the upregulation of cytokine and chemokine levels. The MAV-1 infection model can be utilized to assess the efficacy and safety of therapeutic agents for human adenoviral diseases.

Differences between immunodeficient mice generated by classical gene targeting and CRISPR/Cas9-mediated gene knockout.

Immunodeficient mice are widely used for pre-clinical studies to understand various human diseases. Here, we report the generation of four immunodeficient mouse models using CRISPR/Cas9 system without inserting any foreign gene sequences such as NeoR cassettes and their characterization. By eliminating any possible effects of adding a NeoR cassette, our mouse models may allow us to better elucidate the in vivo functions of each gene. Our FVB-Rag2-/-, B6-Rag2-/-, and BALB/c-Prkdc-/- mice showed phenotypes similar to those of the earlier immunodeficient mouse models, including a lack of mature B cells and T cells and an increase in the number of CD45+DX-5+ natural killer cells. However, B6-Il2rg-/- mice had a unique phenotype, with a lack of mature B cells, increased number of T cells, and decreased number of natural killer cells. Additionally, serum immunoglobulin levels in all four immunodeficient mouse models were significantly reduced when compared to those in wild-type mice with the exception of IgM in B6-Il2rg-/- mice. These results indicate that our immunodeficient mouse models are a robust tool for in vivo studies of the immune system and will provide new insights into the variation in phenotypic outcomes resulting from different gene-targeting methodologies.

Protective Role of the Toll-Like Receptor 5 Agonist KMRC011 against Murine Colitis Induced by Citrobacter rodentium and Dextran Sulfate Sodium.

This study aimed to identify the therapeutic ability of a novel toll-like receptor (TLR) 5 agonist, KMRC011, on ulcerative colitis induced by Citrobacter rodentium and dextran sulfate sodium in a C57BL/6N mouse model. Ulcerative colitis was induced in the mice by the oral administration of 1% dextran sulfate sodium in sterile drinking water for seven days ad libitum, followed by C. rodentium infection on the seventh day by intra-gastric administration (DSS-CT group). KMRC011 was administered intramuscularly at both 24 h and 15 min before (Treatment 1 group), and at both 15 min and 24 h after (Treatment 2 group) the C. rodentium infection. The length of the large intestine and histopathological counts were significantly greater and mucosal thickness was significantly thinner in the Treatment 1 group compared to the DSS-CT and Treatment 2 groups. Il-6 and Il-10 mRNA expression levels were upregulated, while Ifn-γ and Tnf-α mRNA expression levels were significantly downregulated in the Treatment 1 group, compared to the DSS-CT group. NF-κB p65 expression level was elevated due to ulcerative colitis in the DSS-CT group, but was significantly downregulated in the Treatment 1 group. Overall, KMRC011 showed protective effects against murine colitis by inhibiting NF-κB signaling.

Short-term carcinogenicity study of N-methyl-N-nitrosourea in FVB-Trp53 heterozygous mice.

Carcinogenicity tests predict the tumorigenic potential of various substances in the human body by studying tumor induction in experimental animals. There is a need for studies that explore the use of FVB/N-Trp53em2Hwl/Korl (FVB-Trp53+/-) mice, created by TALEN-mediated gene targeting in Korea, in carcinogenicity tests. This study was performed to determine whether FVB-Trp53+/- mice are a suitable model for short-term carcinogenicity studies. To compare the carcinogenicity at different concentrations, 25, 50, and 75 mg/kg of N-methyl-N-nitrosourea (MNU), a known carcinogen, were administered intraperitoneally to FVB-Trp53+/- and wild-type male mice. After 26 weeks, the survival rate was significantly reduced in FVB-Trp53+/- mice compared to the wild-type mice in the 50 and 75 mg/kg groups. The incidence of thymic malignant lymphoma (TML) in the 50 and 75 mg/kg groups was 54.2 and 59.1% in FVB-Trp53+/- male mice, respectively. TML metastasized to the lungs, spleen, lymph nodes, liver, kidney, and heart in FVB-Trp53+/- male mice. Furthermore, the incidence of primary lung tumors, such as adenomas and adenocarcinomas, was 65.4, 62.5, and 45.4% in the FVB-Trp53+/- mice of the 25, 50, and 75 mg/kg groups, respectively. The main tumor types in FVB-Trp53+/- mice were TML and primary lung tumors, regardless of the dose of MNU administered. These results suggest that systemic tumors may result from malfunctions in the p53 gene and pathway, which is an important factor in the pathogenesis of human cancers. Therefore, FVB-Trp53 heterozygous mice are suitable for short-term carcinogenicity tests using positive carcinogens, and that the best result using MNU, a positive carcinogen, might have a single dose of 50 mg/kg.

COVID-19 spike polypeptide vaccine reduces the pathogenesis and viral infection in a mouse model of SARS-CoV-2.

The SARS-CoV-2 coronavirus, which causes a respiratory disease called COVID-19, has been declared a pandemic by the World Health Organization (WHO) and is still ongoing. Vaccination is the most important strategy to end the pandemic. Several vaccines have been approved, as evidenced by the ongoing global pandemic, but the pandemic is far from over and no fully effective vaccine is yet available. One of the most critical steps in vaccine development is the selection of appropriate antigens and their proper introduction into the immune system. Therefore, in this study, we developed and evaluated two proposed vaccines composed of single and multiple SARS-CoV-2 polypeptides derived from the spike protein, namely, vaccine A and vaccine B, respectively. The polypeptides were validated by the sera of COVID-19-vaccinated individuals and/or naturally infected COVID-19 patients to shortlist the starting pool of antigens followed by in vivo vaccination to hACE2 transgenic mice. The spike multiple polypeptide vaccine (vaccine B) was more potent to reduce the pathogenesis of organs, resulting in higher protection against the SARS-CoV-2 infection.

Clinical evaluation of toll-like receptor-5 agonist for radiation-induced oral mucositis in beagle dogs.

This study aimed to evaluate the clinical safety and validate the radiomitigative effect of KMRC011, against radiation-induced oral mucositis in beagle dogs. Clinical safety was evaluated by assessing tolerability, complete blood tests, and plasma biochemistry after drug administration. The radiomitigative effect of KMRC011 was evaluated macropathologically and histopathologically after inducing oral mucositis iatrogenically using 20 Gy irradiation. The plasma concentration of interleukin-6 was measured via enzyme-linked immunosorbent assay, as a biomarker of KMRC011 bioreactivity. Decreased tolerability, increased neutrophil count, hepatic enzyme concentration, C-reactive protein concentration, and interleukin-6 concentration after the administration was observed and ceased within 24 h without additional treatment. Although all animals included in the present study developed severe mucositis in the late course of the study, animals administered KMRC011 showed less erythema, ulcer, inflammatory infiltration. These results suggest that KMRC011 may be used as an adjuvant for radiotherapy without severe adverse effects, especially during short-term radiotherapy, such as hypofractionated radiotherapy or stereotactic radiotherapy.

Development of transgenic models susceptible and resistant to SARS-CoV-2 infection in FVB background mice.

Coronavirus disease (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is currently spreading globally. To overcome the COVID-19 pandemic, preclinical evaluations of vaccines and therapeutics using K18-hACE2 and CAG-hACE2 transgenic mice are ongoing. However, a comparative study on SARS-CoV-2 infection between K18-hACE2 and CAG-hACE2 mice has not been published. In this study, we compared the susceptibility and resistance to SARS-CoV-2 infection between two strains of transgenic mice, which were generated in FVB background mice. K18-hACE2 mice exhibited severe weight loss with definitive lethality, but CAG-hACE2 mice survived; and differences were observed in the lung, spleen, cerebrum, cerebellum, and small intestine. A higher viral titer was detected in the lungs, cerebrums, and cerebellums of K18-hACE2 mice than in the lungs of CAG-hACE2 mice. Severe pneumonia was observed in histopathological findings in K18-hACE2, and mild pneumonia was observed in CAG-hACE2. Atrophy of the splenic white pulp and reduction of spleen weight was observed, and hyperplasia of goblet cells with villi atrophy of the small intestine was observed in K18-hACE2 mice compared to CAG-hACE2 mice. These results indicate that K18-hACE2 mice are relatively susceptible to SARS-CoV-2 and that CAG-hACE2 mice are resistant to SARS-CoV-2. Based on these lineage-specific sensitivities, we suggest that K18-hACE2 mouse is suitable for highly susceptible model of SARS-CoV-2, and CAG-hACE2 mouse is suitable for mild susceptible model of SARS-CoV-2 infection.

Laboratory information management system for COVID-19 non-clinical efficacy trial data.

BACKGROUND: As the number of large-scale studies involving multiple organizations producing data has steadily increased, an integrated system for a common interoperable format is needed. In response to the coronavirus disease 2019 (COVID-19) pandemic, a number of global efforts are underway to develop vaccines and therapeutics. We are therefore observing an explosion in the proliferation of COVID-19 data, and interoperability is highly requested in multiple institutions participating simultaneously in COVID-19 pandemic research. RESULTS: In this study, a laboratory information management system (LIMS) approach has been adopted to systemically manage various COVID-19 non-clinical trial data, including mortality, clinical signs, body weight, body temperature, organ weights, viral titer (viral replication and viral RNA), and multiorgan histopathology, from multiple institutions based on a web interface. The main aim of the implemented system is to integrate, standardize, and organize data collected from laboratories in multiple institutes for COVID-19 non-clinical efficacy testings. Six animal biosafety level 3 institutions proved the feasibility of our system. Substantial benefits were shown by maximizing collaborative high-quality non-clinical research. CONCLUSIONS: This LIMS platform can be used for future outbreaks, leading to accelerated medical product development through the systematic management of extensive data from non-clinical animal studies.

C1qa deficiency in mice increases susceptibility to mouse hepatitis virus A59 infection.

BACKGROUND: Mouse hepatitis virus (MHV) A59 is a highly infectious pathogen and starts in the respiratory tract and progresses to systemic infection in laboratory mice. The complement system is an important part of the host immune response to viral infection. It is not clear the role of the classical complement pathway in MHV infection. OBJECTIVES: The purpose of this study was to determine the importance of the classical pathway in coronavirus pathogenesis by comparing C1qa KO mice and wild-type mice. METHODS: We generated a C1qa KO mouse using CRISPR/Cas9 technology and compared the susceptibility to MHV A59 infection between C1qa KO and wild-type mice. Histopathological and immunohistochemical changes, viral loads, and chemokine expressions in both mice were measured. RESULTS: MHV A59-infected C1qa KO mice showed severe histopathological changes, such as hepatocellular necrosis and interstitial pneumonia, compared to MHV A59-infected wild-type mice. Virus copy numbers in the olfactory bulb, liver, and lungs of C1qa KO mice were significantly higher than those of wild-type mice. The increase in viral copy numbers in C1qa KO mice was consistent with the histopathologic changes in organs. These results indicate that C1qa deficiency enhances susceptibility to MHV A59 systemic infection in mice. In addition, this enhanced susceptibility effect is associated with dramatic elevations in spleen IFN-γ, MIP-1 α, and MCP-1 in C1qa KO mice. CONCLUSIONS: These data suggest that C1qa deficiency enhances susceptibility to MHV A59 systemic infection, and activation of the classical complement pathway may be important for protecting the host against MHV A59 infection.