Yuxiu Zhao

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.

The Haemophilus influenzae type B (Hib) conjugate vaccine is the most effective way to prevent Hib infection in infants and young children, and it is designed to induce the production of antibodies against polyribosylribitol phosphate (PRP) to protect babies from infection. However, the mechanism of immunity induced by the Hib vaccine is not fully understood. Recently, with the development of the combination diphtheria and tetanus toxoids and acellular pertussis vaccines (DTaP), increasing numbers of manufacturers have begun to develop DTaP-based combination vaccines, like the combination vaccine diphtheria and tetanus toxoids and acellular pertussis and Hib conjugate vaccine (DTaP-Hib), which contains adjuvants. However, the Hib vaccine does not contain adjuvants. It was theorized that the Hib antigen has poor compatibility with aluminum adjuvants for unclear reasons. Therefore, understanding the mechanism of the Hib-vaccine-induced immune response and the influence of adjuvants on the Hib vaccine is of great significance. In this paper, we immunized BalBc mice with either the Hib vaccine or the Hib vaccine that adsorbs aluminum adjuvants (Hib-Al). Here, we analyzed the anti-PRP antibody level and immune response of different cells using cell and cytokine levels. We found that the Hib vaccine could induce a humoral and cellular immune response, and the Hib-Al vaccine could induce greater quantities of IFN-γ, IL-4, and IL-6 and more antigen-specific antibodies through B cells, Th1, Th2, and ILC3s in the spleen. Together, our findings demonstrate the serologic responses and immune response in terms of cell and cytokine levels induced by the Hib vaccine, and they also imply that the addition of aluminum hydroxide adjuvant could enhance the function of the Hib vaccine, which preliminarily reveals the mechanism of immune response induced by the Hib-related vaccine.

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.

Coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread to more than 230 countries and territories worldwide since its outbreak in late 2019. In less than three years, infection by SARS-CoV-2 has resulted in over 600 million cases of COVID-19 and over 6.4 million deaths. Vaccines have been developed with unimaginable speed, and 11 have already been approved by the World Health Organization and given Emergency Use Listing. The administration of several first-generation SARS-CoV-2 vaccines has successfully decelerated the spread of COVID-19 but not stopped it completely. In the ongoing fight against viruses, genetic mutations frequently occur in the viral genome, resulting in a decrease in vaccine-induced antibody neutralization and widespread breakthrough infection. Facing the evolution and uncertainty of SARS-CoV-2 in the future, and the possibility of the spillover of other coronaviruses to humans, the need for vaccines with a broad spectrum of antiviral variants against multiple coronaviruses is recognized. It is imperative to develop a universal coronavirus or pan-coronavirus vaccine or drug to combat the ongoing COVID-19 pandemic as well as to prevent the next coronavirus pandemic. In this review, in addition to summarizing the protective effect of approved vaccines, we systematically summarize current work on the development of vaccines aimed at suppressing multiple SARS-CoV-2 variants of concern as well as multiple coronaviruses.

The production efficiency of a cell substrate directly affects the yield of target products such as viruses, while its density is mainly regulated by the type of culture medium and culture conditions. In this study, Vero cells were used as model cells for systematic medium screening, and a high-efficiency medium for biological drug production was identified. Through the results of cell proliferation by a cell counting kit (CCK)-8 assay, 5-Ethynyl-2′-deoxyuridine(EdU) assay, real-time quantitative PCR (RT-qPCR) and Western blotting, we found that adding an appropriate amount of vitamin B to the conventional basic medium can significantly improve and maintain the high-density growth of Vero cells. In addition, the molecular mechanism of the high-density culture of Vero cells promoted by B vitamins is explained for the first time by using the systems multi-omics analysis methods. Here, we determined that B vitamins regulate cell proliferation through the synthesis and metabolism of unsaturated fatty acids, affecting the productivity of cell substrate in industrial production. This study provides an important tool for the screening of key components of cell-based high-efficiency medium.

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019, the virus has been mutating continuously, resulting in the continuous emergence of variants and creating challenges for epidemic prevention and control. Here, we immunized mice with different vaccine candidates, revealing the immune, protein, and metabolomic changes that take place in vaccines composed of different variants. We found that the prototype strain and Delta- and Omicron-variant inactivated vaccine candidates could all induce a high level of neutralizing antibodies and cellular immunity responses in mice. Next, we found that the metabolic and protein profiles were changed, showing a positive association with immune responses, and the level of the change was distinct in different inactivated vaccines, indicating that amino acid variations could affect metabolomics and proteomics. Our findings reveal differences between vaccines at the metabolomic and proteomic levels. These insights provide a novel direction for the immune evaluation of vaccines and could be used to guide novel strategies for vaccine design.

Since the beginning of the COVID-19 pandemic, numerous variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged, including five variants of concern (VOC) strains listed by the WHO: Alpha, Beta, Gamma, Delta and Omicron. Extensive studies have shown that most of these VOC strains, especially the currently dominant variant Omicron, can escape the host immune response induced by existing COVID-19 vaccines to different extents, which poses considerable risk to the health of human beings around the world. In the present study, we developed a vaccine based on inactivated SARS-CoV-2 and an adjuvant consisting of aluminum hydroxide (alum) and CpG. The immunogenicity and safety of the vaccine were investigated in rats. The candidate vaccine elicited high titers of SARS-CoV-2-spike-specific IgG antibody and neutralizing antibody in immunized rats, which not only neutralize the original SARS-CoV-2, but also showed great cross-neutralization activity against the Beta, Delta 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.

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.