Development of highly adaptable RT-PCR methods for identifying Delta and BA.1 variants in inactivated COVID-19 vaccines.

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.

Nonclinical safety and immunogenicity assessment of a combined DTacP vaccine in animal models.

The (diphtheria, tetanus, and pertussis [acellular, component] [DTacP]) vaccine is a combined vaccine designed to prevent three potentially fatal diseases including pertussis, tetanus, and diphtheria in both children and adults. We utilized advanced technology to develop a novel DTacP vaccine that was previously unavailable in China. The nonclinical studies were performed to evaluate the immunogenicity, potential toxicity, and local tolerance of the vaccine in animal models. In the immunogenicity study, three batches of the vaccine were intraperitoneally administered to National Institutes of Health (NIH) mice, resulting in 100% seropositivity for all three batches. Additionally, antibody levels notably increased as the immunization dosage increased. In acute toxicity study, no mortality was observed among the animals during the 14-day observation period, and no abnormalities in clinical signs were reported. Active systemic anaphylaxis assessment in guinea pigs showed no evidence of serious allergic reactions in the vaccine groups. In the repeat-dose toxicity study, where five intramuscular doses were administered every 2 weeks, gross autopsy and histopathological examination revealed no vaccine-related systemic pathological changes in rats, with dose site irritant reactions mostly recovered at the end of recovery period. In conclusion, the vaccine demonstrated good local and systemic tolerance, supporting its clinical development.

The Screening and Mechanism of Influenza-Virus Sensitive MDCK Cell Lines for Influenza Vaccine Production.

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.

Alum/CpG adjuvant promotes immunogenicity of inactivated SARS-CoV-2 Omicron vaccine through enhanced humoral and cellular immunity.

The SARS-CoV-2 Omicron variant, which was classified as a variant of concern (VOC) by the World Health Organization on 26 November 2021, has attracted worldwide attention for its high transmissibility and immune evasion ability. The existing COVID-19 vaccine has been shown to be less effective in preventing Omicron variant infection and symptomatic infection, which brings new challenges to vaccine development and application. Here, we evaluated the immunogenicity and safety of an Omicron variant COVID-19 inactivated vaccine containing aluminum and CpG adjuvants in a variety of animal models. The results showed that the vaccine candidate could induce high levels of neutralizing antibodies against the Omicron variant virus and binding antibodies, and significantly promoted cellular immune response. Meanwhile, the vaccine candidate was safe. Therefore, it provided more foundation for the development of aluminum and CpG as a combination adjuvant in human vaccines.

Integrated Multi-Omic Characterization of the Detachment Process of Adherent Vero Cells with Animal-Based and Animal-Origin-Free Enzymes.

Cell detachment techniques using animal-derived enzymes are necessary for the production of biopharmaceuticals that are made with the help of adherent cell cultures, although the majority of protein therapeutics (>USD 100 billion of income per year) are made under suspension cultures that do not require animal-derived proteins for manufacture. In this study, we establish the optimal Vero cell detachment process, and analyze physiological changes during cell detachment at the cellular and molecular levels. Using flow cytometry, we find that animal-based enzymes are more likely to induce apoptosis than animal-origin-free enzymes. We analyze the levels of RNAs, proteins, and metabolites in cells treated with two detachment strategies, and identify 1237 differentially expressed genes, 2883 differential proteins, and 210 differential metabolites. Transcriptomic analysis shows that animal-origin-free enzymes have a less significant effect on gene expression levels. Combined with proteomic analysis, animal-based enzymes affect the oxidative phosphorylation process and reduce the mRNA and protein levels of Cytochrome C Oxidase Assembly Protein 17 (COX17), which is a Cytochrome C Oxidase Copper Chaperone involved in the mitochondrial respiratory chain. Metabolomics analysis indicates that the levels of spermine and spermidine, which are involved in the glutathione metabolism pathway and apoptosis inhibition, are significantly reduced. Therefore, COX17, spermine, and spermidine may be biomarkers for evaluating the cell subculture process. In conclusion, we have deeply characterized the cell subculture process through multi-omics, which may provide important guidance for research and process evaluation to optimize cell detachment processes.

Safety and immunogenicity of a combined DTacP-sIPV-Hib vaccine in animal models.

The DTacP-sIPV-Hib combination vaccine can replace the single-component acellular pertussis, diphtheria, tetanus, polio, and Haemophilus influenzae type B vaccines. In this study, we evaluated the safety and immunogenicity of a newly developed DTacP-sIPV-Hib combination vaccine in animal models. We used 40 mice and 46 cynomolgus monkeys to evaluate acute and long-term toxicity. Thirty-six guinea pigs were used for sensitization assessment. For immunogenicity assessment, 50 NIH mice and 50 rats were equally randomized to receive 3 doses of 3 different batches of the tested vaccine at an interval of 21 d, or physiological saline solution (0.5 mL). Orbital blood was collected at an interval of 21 d post inoculation to detect related antibody titers or neutralizing antibody titers against poliovirus. Gross autopsy and histopathological examination revealed no abnormal toxicity or irritation in mice and cynomolgus monkeys. Sensitization assessment in guinea pigs indicated the lack of evident allergic symptoms in the high- and low-dose vaccine groups within 30 min after repeated stimulation. The DTacP-sIPV-Hib combination vaccine induced significant immune responses in mice, rats, and cynomolgus monkeys, with 100% seroconversion rates after 3 doses. The DTacP-sIPV-Hib combination vaccine is safe and immunogenic in animal models. Three doses of the vaccine elicited satisfactory antibody responses in mice, rats, and cynomolgus monkeys.

Immunogenicity Evaluating of the Multivalent COVID-19 Inactivated Vaccine against the SARS-CoV-2 Variants.

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.

Vaccination with Omicron Inactivated Vaccine in Pre-vaccinated Mice Protects against SARS-CoV-2 Prototype and Omicron Variants.

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.