Yancen Guo

Background The emergence and rapid spread of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), poses a significant threat to human health and public safety. While next-generation sequencing (NGS) is capable of detecting and tracking new COVID-19 variants for disease diagnosis and prevention, its high cost and time-consuming nature limit its widespread use. In this study, our aim was to develop a highly adaptable and accurate RT-PCR method for identifying the Delta or BA.1 variants in inactivated COVID-19 vaccine. We devised three two-plex RT-PCR methods targeting specific mutation sites: S: Δ156–157, S: N211-, L212I, and S: Δ142–144, Y145D. The RT-PCR method targeting the S: Δ156–157 mutation site was able to distinguish the Delta variant from other COVID-19 virus strains, while the RT-PCR methods targeting the S: N211-, L212I or S: Δ142–144, Y145D mutation sites were able to distinguish the BA.1 variant from other COVID-19 virus strains. We separately validated these three two-plex RT-PCR methods, and the results demonstrated good linearity, repeatability, reproducibility, and specificity for each method. Moreover, all three methods can be applied in the production of SARS-CoV-2 variant inactivated vaccines, enabling the identification of Delta or BA.1 variants in virus cultures as well as in inactivated vaccine stocks. This study presents a systematic approach to identify COVID-19 variants using multiple RT-PCR methods. We successfully developed three two-plex RT-PCR methods that can identify Delta and BA.1 variants based on specific mutation sites, and we completed the validation of these three methods.

Influenza is a potentially fatal acute respiratory viral disease caused by the influenza virus. Influenza viruses vary in antigenicity and spread rapidly, resulting in seasonal epidemics. Vaccination is the most effective strategy for lowering the incidence and fatality rates of influenza-related disorders, and it is also an important method for reducing seasonal influenza infections. Mammalian Madin–Darby canine kidney (MDCK) cell lines are recommended for influenza virus growth, and such cell lines have been utilized in several commercial influenza vaccine productions. The limit dilution approach was used to screen ATCC-MDCK cell line subcellular strains that are especially sensitive to H1N1, H3N2, BV, and BY influenza viruses to increase virus production, and research on influenza virus culture media was performed to support influenza virus vaccine development. We also used RNA sequencing to identify differentially expressed genes and a GSEA analysis to determine the biological mechanisms underlying the various levels of susceptibility of cells to influenza viruses. MDCK cell subline 2B6 can be cultured to increase titer and the production of the H1N1, H3N2, BV, and BY influenza viruses. MDCK-2B6 has a significantly enriched and activated in ECM receptor interaction, JAK-STAT signaling, and cytokine receptor interaction signaling pathways, which may result in increased cellular susceptibility and cell proliferation activity to influenza viruses, promote viral adsorption and replication, and elevate viral production, ultimately. The study revealed that MDCK-2B6 can increase the influenza virus titer and yield in vaccine production by increasing cell sensitivity and enhancing proliferative activity.

Due to a large number of mutations in the spike protein and immune escape, the Omicron variant (B.1.1.529) has become a predominant variant of concern (VOC) strain. To prevent the disease, we developed a candidate inactivated vaccine (Omicron COVID-19 Vaccine (Vero Cell), Inactivated). To evaluate the safety of the vaccine, we tested the repeat-dose toxicity in Sprague-Dawley (SD) rats. The doses were administered randomly to three groups: physiological saline solution (control), aluminum adjuvant in PBS solution adjuvant (adjuvant group), and low-dose and high-dose omicron vaccines (vaccine group) for 6 weeks. The SD rats were allowed to recover for 4 weeks after withdrawal. We evaluated the physiological condition of the rats, including their ophthalmological condition, body weight, food intake, body temperature, blood biochemistry, urine, neutralizing antibody, inflammation at the injection site, and organs weight. In summary, no dose-dependent adverse toxicological changes were observed, and a recovery trend was obvious, which proved the preclinical safety of the candidate omicron vaccine and provided evidence for clinical trials in humans.

In response to the fast-waning immune response and the great threat of the Omicron variant of concern (VOC) to the public, we report the pilot-scale production of an inactivated Omicron vaccine candidate that induces high levels of neutralizing antibody titers to protect against the Omicron virus. Here, we demonstrate that the inactivated Omicron vaccine is safe and effective in recalling immune responses to the HB02, Omicron, and Delta viruses after one or two doses of BBIBP-CorV. In addition, the efficient productivity and good genetic stability of the manufactured inactivated vaccine is proved. These results support the further evaluation of the Omicron vaccine in a clinical trial.

It has been reported that the novel coronavirus (COVID-19) has caused more than 286 million cases and 5.4 million deaths to date. Several strategies have been implemented globally, such as social distancing and the development of the vaccines. Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have appeared, such as Alpha, Beta, Gamma, Delta, and Omicron. With the rapid spread of the novel coronavirus and the rapidly changing mutants, the development of a broad-spectrum multivalent vaccine is considered to be the most effective way to defend against the constantly mutating virus. Here, we evaluated the immunogenicity of the multivalent COVID-19 inactivated vaccine. Mice were immunized by multivalent COVID-19 inactivated vaccine, and the neutralizing antibodies in serum were analyzed. The results show that HB02 + Delta + Omicron trivalent vaccine could provide broad spectrum protection against HB02, Beta, Delta, and Omicron virus. Additionally, the different multivalent COVID-19 inactivated vaccines could enhance cellular immunity. Together, our findings suggest that the multivalent COVID-19 inactivated vaccine can provide broad spectrum protection against HB02 and other virus variants in humoral and cellular immunity, providing new ideas for the development of a broad-spectrum COVID-19 vaccine.