Preparation of equine anti-SARS-CoV-2 immunoglobulin special IgG and special F (ab')2

After immunizing healthy horses with SARS-CoV-2 virus-like particles (VLPs) as immunogens, immunized horse serum was collected. The total IgG in the serum was separated by affinity chromatography, and then digested with pepsin to obtain immunoglobulin F(ab')2, the IgG and F(ab')2 using an immunochro-matographic column that binds to the RBD protein to obtain a highly specific horse Anti-SARS-CoV-2 IgG and F(ab')2. It's concentration of IgG and F(ab')2 is 2.36 mg/mL and 1.05 mg/mL, whi le the recovery rates were 11% and 4.89%, and the purities of prepared IgG and F(ab')2 were 91% and 96%. Semi-inhibited concentrations of pseudovirus (IC50) were 1.406 g/mL and 0.862 g/mL. These results show that a high purity, specificity, activity of specific IgG and F(ab')2 against SARS-CoV-2 was prepared successfully, which laid a foundation for preparing safe and efficient anti-SARS-CoV-2 therapeutic antibody drugs.

Integrative Analysis of DNA Methylation and Gene Expression Data Identifies Potential Biomarkers and Functional Epigenetic Modules for SARS-CoV-2.

To integrate gene expression and DNA methylation data and find the potential role of DNA methylation in the invasion and replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We first conducted differential expression and methylation analysis between the coronavirus disease of 2019 (COVID-19) and healthy controls. FEM was employed to identify functional epigenetic modules, from which a diagnostic model for COVID-19 was built. SKA1 and WSB1 modules were identified, with SKA1 module enriched in COVID-19 replication and transcription, and WSB1 module related to ubiquitin-protein activity. The differentially expressed or differentially methylated genes in these two modules could be used to distinguish COVID-19 from healthy controls, with AUC reaching 1 and 0.98 for SKA1 and WSB1 modules, respectively. Two epigenetically activated genes (CENPM and KNL1) from the SKA1 module were upregulated in HPV- or HBV-positive tumor samples and were found to be significantly associated with the survival of tumor patients. In conclusion, the identified FEM modules and potential signatures play an essential role in the replication and transcription of coronavirus.

PEGylation Prolongs the Half-Life of Equine Anti-SARS-CoV-2 Specific F(ab')2.

Therapeutic antibodies-F(ab')2 obtained from hyperimmune equine plasma could treat emerging infectious diseases rapidly because of their high neutralization activity and high output. However, the small-sized F(ab')2 is rapidly eliminated by blood circulation. This study explored PEGylation strategies to maximize the half-life of equine anti-SARS-CoV-2 specific F(ab')2. Equine anti-SARS-CoV-2 specific F(ab')2 were combined with 10 KDa MAL-PEG-MAL in optimum conditions. Specifically, there were two strategies: Fab-PEG and Fab-PEG-Fab, F(ab')2 bind to a PEG or two PEG, respectively. A single ion exchange chromatography step accomplished the purification of the products. Finally, the affinity and neutralizing activity was evaluated by ELISA and pseudovirus neutralization assay, and ELISA detected the pharmacokinetic parameters. The results displayed that equine anti-SARS-CoV-2 specific F(ab')2 has high specificity. Furthermore, PEGylation F(ab')2-Fab-PEG-Fab had a longer half-life than specific F(ab')2. The serum half-life of Fab-PEG-Fab, Fab-PEG, and specific F(ab')2 were 71.41 h, 26.73 h, and 38.32 h, respectively. The half-life of Fab-PEG-Fab was approximately two times as long as the specific F(ab')2. Thus far, PEGylated F(ab')2 has been prepared with high safety, high specificity, and a longer half-life, which could be used as a potential treatment for COVID-19.

Nucleic acid visualization assay for Middle East Respiratory Syndrome Coronavirus (MERS-CoV) by targeting the UpE and N gene.

Since its first emergence in 2012, cases of infection with Middle East respiratory syndrome coronavirus (MERS-CoV) have continued to occur. At the end of January 2020, 2519 laboratory confirmed cases with a case-fatality rate of 34.3% have been reported. Approximately 84% of human cases have been reported in the tropical region of Saudi Arabia. The emergence of MERS-CoV has highlighted need for a rapid and accurate assay to triage patients with a suspected infection in a timely manner because of the lack of an approved vaccine or an effective treatment for MERS-CoV to prevent and control potential outbreaks. In this study, we present two rapid and visual nucleic acid assays that target the MERS-CoV UpE and N genes as a panel that combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF). This test panel was designed to improve the diagnostic accuracy through dual-target screening after referencing laboratory testing guidance for MERS-CoV. The limit of detection was 1.2×101 copies/µl viral RNA for the UpE assay and 1.2 copies/µl viral RNA for the N assay, with almost consistent with the sensitivity of the RT-qPCR assays. The two assays exhibited no cross-reactivity with multiple CoVs, including the bat severe acute respiratory syndrome related coronavirus (SARSr-CoV), the bat coronavirus HKU4, and the human coronaviruses 229E, OC43, HKU1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, the panel does not require sophisticated equipment and provides rapid detection within 30 min. This panel displays good sensitivity and specificity and may be useful to rapidly detect MERS-CoV early during an outbreak and for disease surveillance.