CDC20 is a potential target gene to inhibit the tumorigenesis of MDCK cells.

MDCK is currently the main cell line used for influenza vaccine production in culture. Previous studies have reported that MDCK cells possess tumorigenic ability in nude mice. Although complete cell lysis can be ensured during vaccine production, host cell DNA released after cell lysis may still pose a risk for tumorigenesis. Greater caution is needed in the production of human vaccines; therefore, the use of gene editing to establish cells incapable of forming tumors may significantly improve the safety of influenza vaccines. Knowledge regarding the genes and molecular mechanisms that affect the tumorigenic ability of MDCK cells is crucial; however, our understanding remains superficial. Through monoclonal cell screening, we previously obtained a cell line, CL23, that possesses significantly reduced cell proliferation, migration, and invasion abilities, and tumor-bearing experiments in nude mice showed the absence of tumorigenic cells. With a view to exploring tumorigenesis-related genes in MDCK cells, DIA proteomics was used to compare the differences in protein expression between wild-type (M60) and non-tumorigenic (CL23) cells. Differentially expressed proteins were verified at the mRNA level by RT-qPCR, and a number of genes involved in cell tumorigenesis were preliminarily screened. Immunoblotting further confirmed that related protein expression was significantly reduced in non-tumorigenic cells. Inhibition of CDC20 expression by RNAi significantly reduced the proliferation and migration of MDCK cells and increased the proliferation of the influenza virus; therefore, CDC20 was preliminarily determined to be an effective target gene for the inhibition of cell tumorigenicity. These results contribute to a more comprehensive understanding of the mechanism underlying cell tumorigenesis and provide a basis for the establishment of target gene screening in genetically engineered non-tumorigenic MDCK cell lines.

Viral Vector-Based Gene Therapy.

Gene therapy is a technique involving the modification of an individual's genes for treating a particular disease. The key to effective gene therapy is an efficient carrier delivery system. Viral vectors that have been artificially modified to lose their pathogenicity are used widely as a delivery system, with the key advantages of their natural high transduction efficiency and stable expression. With decades of development, viral vector-based gene therapies have achieved promising clinical outcomes. Currently, the three key vector strategies are based on adeno-associated viruses, adenoviruses, and lentiviruses. However, certain challenges, such as immunotoxicity and "off-target", continue to exist. In the present review, the above three viral vectors are discussed along with their respective therapeutic applications. In addition, the major translational challenges encountered in viral vector-based gene therapies are summarized, and the possible strategies to address these challenges are also discussed.

AddaVax Formulated with PolyI:C as a Potential Adjuvant of MDCK-based Influenza Vaccine Enhances Local, Cellular, and Antibody Protective Immune Response in Mice.

Poor immune responses to inactivated influenza vaccine can be improved by effective and safe adjuvants to increase antibody titers and cellular protective response. In our study, AddaVax and PolyI:C combined adjuvant (AP adjuvant) were used for influenza vaccine development. After immunizing BALB/c mice and Wistar rats intramuscularly, Split inactivated H3N2 vaccine adjuvanted with AP elicited higher serum hemagglutination-inhibition antibodies and IgG titers. We demonstrated that AP induced a transient innate immune cytokines production at the injection site, induced H3N2 uptake by DCs, increased recruitment of monocytes and DCs in LNs, and promoted H3N2 vaccine migration; AP facilitated vaccines to induce a vigorous adaptive immune response. Besides, AP showed good safety as shown by lymph nodes (LNs) size, spleens index of BALB/c mice, and weight changes and C-reaction protein level of BALB/c mice and Wistar rats after repeated administration of high-dose vaccine with or without adjuvant. These findings indicate that AP is a potential novel adjuvant and can be used as a safe and effective adjuvant for MDCK-based influenza inactivated vaccine to induce cellular and antibody protective response.

Correction: Nian et al. Coping Strategies for Pertussis Resurgence. Vaccines 2023, 11, 889.

The authors would like to make the following corrections to this published paper [...].

Immunosuppressants exert antiviral effects against influenza A(H1N1)pdm09 virus via inhibition of nucleic acid synthesis, mRNA splicing, and protein stability.

Influenza A virus (IAV) poses a threat to patients receiving immunosuppressive medications since they are more susceptible to infection with severe symptoms, and even death. Understanding the direct effects of immunosuppressants on IAV infection is critical for optimizing immunosuppression in these patients who are infected or at risk of influenza virus infection. We profiled the effects of 10 immunosuppressants, explored the antiviral mechanisms of immunosuppressants, and demonstrated the combined effects of immunosuppressants with the antiviral drug oseltamivir in IAV-infected cell models. We found that mycophenolic acid (MPA) strongly inhibits viral RNA replication via depleting cellular guanosine pool. Treatment with 6-Thioguanine (6-TG) promoted viral protein degradation through a proteasomal pathway. Filgotinib blocked mRNA splicing of matrix protein 2, resulting in decreased viral particle assembly. Furthermore, combined treatment with immunosuppressants and oseltamivir inhibits IAV viral particle production in an additive or synergic manner. Our results suggest that MPA, 6-TG, and filgotinib could be the preferential choices for patients who must take immunosuppressants but are at risk of influenza virus infection.

Enhanced Downstream Processing for a Cell-Based Avian Influenza (H5N1) Vaccine.

H5N1 highly pathogenic avian influenza virus (HPAIV) infections pose a significant threat to human health, with a mortality rate of around 50%. Limited global approval of H5N1 HPAIV vaccines, excluding China, prompted the need to address safety concerns related to MDCK cell tumorigenicity. Our objective was to improve vaccine safety by minimizing residual DNA and host cell protein (HCP). We developed a downstream processing method for the cell-based H5N1 HPAIV vaccine, employing CaptoTM Core 700, a multimodal resin, for polishing. Hydrophobic-interaction chromatography (HIC) with polypropylene glycol as a functional group facilitated the reversible binding of virus particles for capture. Following the two-step chromatographic process, virus recovery reached 68.16%. Additionally, HCP and DNA levels were reduced to 2112.60 ng/mL and 6.4 ng/mL, respectively. Western blot, high-performance liquid chromatography (HPLC), and transmission electron microscopy (TEM) confirmed the presence of the required antigen with a spherical shape and appropriate particle size. Overall, our presented two-step downstream process demonstrates potential as an efficient and cost-effective platform technology for cell-based influenza (H5N1 HPAIV) vaccines.

Human monoclonal antibody F61 nasal spray effectively protected high-risk populations from SARS-CoV-2 variants during the COVID-19 pandemic from late 2022 to early 2023 in China.

Following the national dynamic zero-COVID strategy adjustment, the utilization of broad-spectrum nasal neutralizing antibodies may offer an alternative approach to controlling the outbreak of Omicron variants between late 2022 and early 2023 in China. This study involved an investigator-initiated trial (IIT) to assess the pharmacokinetic, safety and efficacy of the F61 nasal spray. A total of 2,008 participants were randomly assigned to receive F61 nasal spray (24 mg/0.8 mL/dose) or normal saline (0.8 mL/dose) and 1336 completed the follow-up in the IIT. Minimal absorption of F61 antibody into the bloodstream was detected in individuals receiving F61 nasal spray for seven consecutive days. No treatment-emergent adverse reactions of grade 3 severity or higher were reported. In the one-dose cohort, the 7-day cumulative SARS-CoV-2 infection rate was 79.0% in the F61 group and 82.6% in the placebo group, whereas, in the multiple-dose (once daily for 7 consecutive days) cohort, the rates were 6.55% in the F61 group and 23.83% in the placebo group. The laboratory-confirmed efficacy of F61 was 3.78% (-3.74%-10.75%) in the one-dose cohort and 72.19% (57.33%-81.87%) in the multiple-dose cohort. In the real-world study, 60,225 volunteers in four different regions were administered the F61 nasal spray based on the subject's wishes, over 90% efficacy rate was observed against different Omicron variants. The F61 nasal spray, with its favourable safety profile, could be a promising prophylactic monoclonal antibody against SARS-CoV-2 VOCs.

Coping Strategies for Pertussis Resurgence.

Pertussis (whooping cough) is a respiratory disease caused primarily by Bordetella pertussis, a Gram-negative bacteria. Pertussis is a relatively contagious infectious disease in people of all ages, mainly affecting newborns and infants under 2 months of age. Pertussis is undergoing a resurgence despite decades of high rates of vaccination. To better cope with the challenge of pertussis resurgence, we evaluated its possible causes and potential countermeasures in the narrative review. Expanded vaccination coverage, optimized vaccination strategies, and the development of a new pertussis vaccine may contribute to the control of pertussis.

Liposome and QS-21 Combined Adjuvant Induces theHumoral and Cellular Responses of Acellular Pertussis Vaccine in a Mice Model.

The resurgence of pertussis in vaccinated communities may be related to the reduced long-term immunity induced by acellular pertussis vaccines. Therefore, developing improved pertussis vaccine candidates that could induce strong Th1 or Th17 cellular immunity is an urgent need. The use of new adjuvants may well meet this requirement. In this research, we developed a novel adjuvant candidate by combining liposome and QS-21 adjuvant. Adjuvant activity, protective efficacy, the level of neutralizing antibody against PT, and the resident memory T (TRM) cells in lung tissue after vaccination were studied. We then performed B. pertussis respiratory challenge in mice after they received vaccination with traditional aluminum hydroxide and the novel adjuvant combination. Results showed that the liposome + QS-21 adjuvant group had a rapid antibody and higher antibody (PT, FHA, Fim) level, induced anti-PT neutralizing antibody and recruited more IL-17A-secreting CD4+ TRM cells along with IL-17A-secreting CD8+ TRM cells in mice, which provided robust protection against B. pertussis infection. These results provide a key basis for liposome + QS-21 adjuvant as a promising adjuvant candidate for developing an acellular pertussis vaccine that elicits protective immunity against pertussis.

Suspended cell lines for inactivated virus vaccine production.

INTRODUCTION: Inactivated virus vaccines are the most widely used tool to prevent disease. To meet vaccine production demands, increasing attention has been placed on identifying methods to improve vaccine production efficiency. The use of suspended cells can greatly increase vaccine production. Suspension acclimation is a traditional method to convert adherent cells to suspension strains. Furthermore, as genetic engineering technology has developed, increasing attention has focused on the development of suspension cell lines using targeted genetic engineering techniques. AREAS COVERED: This review systematically summarizes and analyzes the development and research progress of various inactivated viral vaccine production suspension cell lines and provides protocols and candidate target genes for the engineered establishment of additional suspension cell lines for vaccine production. EXPERT OPINION: The use of suspended cells can significantly improve the production efficiency of inactivated virus vaccines and other biological products. Presently, cell suspension culture is the key component to improve many vaccine production processes.

Metabolomics profiling reveals differences in proliferation between tumorigenic and non-tumorigenic Madin-Darby canine kidney (MDCK) cells.

Background: Madin-Darby canine kidney (MDCK) cells are a cellular matrix in the production of influenza vaccines. The proliferation rate of MDCK cells is one of the critical factors that determine the vaccine production cycle. It is yet to be determined if there is a correlation between cell proliferation and alterations in metabolic levels. This study aimed to explore the metabolic differences between MDCK cells with varying proliferative capabilities through the use of both untargeted and targeted metabolomics. Methods: To investigate the metabolic discrepancies between adherent cell groups (MDCK-M60 and MDCK-CL23) and suspension cell groups (MDCK-XF04 and MDCK-XF06), untargeted and targeted metabolomics were used. Utilizing RT-qPCR analysis, the mRNA expressions of key metabolites enzymes were identified. Results: An untargeted metabolomics study demonstrated the presence of 81 metabolites between MDCK-M60 and MDCK-CL23 cells, which were mainly affected by six pathways. An analysis of MDCK-XF04 and MDCK-XF06 cells revealed a total of 113 potential metabolites, the majority of which were impacted by ten pathways. Targeted metabolomics revealed a decrease in the levels of choline, tryptophan, and tyrosine in MDCK-CL23 cells, which was in accordance with the results of untargeted metabolomics. Additionally, MDCK-XF06 cells experienced a decrease in 5'-methylthioadenosine and tryptophan, while S-adenosylhomocysteine, kynurenine, 11Z-eicosenoic acid, 3-phosphoglycerate, glucose 6-phosphate, and phosphoenolpyruvic acid concentrations were increased. The mRNA levels of MAT1A, MAT2B, IDO1, and IDO2 in the two cell groups were all increased, suggesting that S-adenosylmethionine and tryptophan may have a significant role in cell metabolism. Conclusions: This research examines the effect of metabolite fluctuations on cell proliferation, thus offering a potential way to improve the rate of MDCK cell growth.

Development of an mRNA vaccine against a panel of heterologous H1N1 seasonal influenza viruses using a consensus hemagglutinin sequence.

Seasonal influenza, causes hundreds of thousands of deaths annually, posing a severe threat to human health. Currently available influenza vaccines are targeted only at specific strains or conserved epitopes; however, these vaccines are not completely efficacious because influenza viruses can undergo mutation during circulation, leading to antigenic mismatch between recommended strains and circulating strains and elusion from the immune system. Therefore, developing an influenza vaccine that is quick, effective, and broadly protective has become crucial, and the integral part of hemagglutinin (HA) remains an ideal target for vaccine development. This study developed a lipid nanoparticle-encapsulated nucleoside-modified mRNA vaccine (mRNA-LNPs) encoding a consensus full-length HA sequence (H1c) and evaluated its protective efficacy and immunogenicity through in vitro and in vivo assays. Following two intramuscular immunizations (2, 10 µg, or 20 µg) at a 3-week interval in BALB/c mice, H1c-mRNA-LNP vaccine induced strong antibodies as shown in the hemagglutination-inhibition test and protective neutralizing antibodies against numerous heterologous H1N1 influenza viruses as shown in the microneutralization assay. Additionally, both Th1- and Th2-biased cellular immune responses were elicited, with the Th1-biased response being stronger. Two doses of the H1c-mRNA-LNP vaccine could neutralize a panel of heterologous H1N1 influenza viruses and could confer protection in mice. Taken together, these findings suggest that the H1c-mRNA-LNP vaccine encoding a consensus full-length HA is a feasible strategy for developing a cross-protective vaccine against a panel of heterologous H1N1 influenza viruses.

TRAF3 deficiency in MDCK cells improved sensitivity to the influenza A virus.

Tumor necrosis factor receptor-associated factor 3 (TRAF3), an adaptor protein, has significant and varying effects on immunity depending on cell types. The role of TRAF3 in Madin-Darby Canine Kidney Epithelial (MDCK) cell resistance to influenza A virus (IVA) remains elusive. In the present study, CRISPR-Cas9 gene editing technology was used to construct the TRAF3 knockout MDCK cells (MDCK-TRAF3-/-). Hemagglutination assay, plaque assay, transcriptome, and quantitative real-time PCR were performed after IVA infection. The results showed that after IVA infection, HA titers and virus titers were promoted, interferon I-related pathways were significantly blocked, and transcription of several antiviral-related genes was significantly decreased in MDCK-TRAF3-/- cells. Thus, our study suggests that TRAF3 gene knockout reduced MDCK cell's resistance to IVA, thereby resulting in a promising way for IVA isolation and vaccine manufacturing.

Safety and immunogenicity of rabies vaccine (PVRV-WIBP) in healthy Chinese aged 10-50 years old: Randomized, blinded, parallel controlled phase III clinical study.

This phase III clinical trial aimed to assess the safety and demonstrate the immunogenicity of a candidate freeze-dried purified Vero cell-based rabies vaccine (PVRV-WIBP) developed for human use. A cohort of 40 participants in stage 1 and 1956 subjects in stage 2 with an age range of 10-50 years were recruited for the phase III clinical trial. For safety analysis in stage 1, 20 participants received either 4-dose or 5-dose regimen of PVRV-WIBP. In stage 2, 1956 subjects were randomly divided into the 5-dose PVRV-WIBP, 5-dose PVRV-LNCD, and 4-dose PVRV-WIBP groups. The serum neutralizing antibody titer against rabies was determined on day 7 or 14 and day 35 or 42. Adverse reactions were recorded for more than 6 months. Most adverse reactions, which were mild and moderate in severity, occurred and resolved within 1 week after each injection in the PVRV-WIBP (4 and 5 doses) and PVRV-LNCD (5 doses) groups. All three groups achieved complete seroconversion 14 days after the initial dose and 14 days after completing the full vaccination schedule, the susceptible subjects in the PVRV-WIBP group (4-dose or 5-dose regimen) displayed higher neutralizing antibody titers against the rabies virus compared to those in the PVRV-LNCD group (5-dose regimen). PVRV-WIBP induced non-inferior immune responses versus PVRV-LNCD as assessed by seroconversion rate. PVRV-WIBP was well tolerated and non-inferior to PVRV-LNCD in healthy individuals aged 10-50 years. The results indicated that PVRV-WIBP (both 4- and 5-dose schedules) could be an alternative to rabies post-exposure prophylaxis.

Development of MDCK-based quadrivalent split seasonal influenza virus vaccine with high safety and immunoprotection: A preclinical study.

Vaccination remains the best prevention strategy against influenza. The MDCK-based influenza vaccine prompted the development of innovative cell culture manufacturing processes. In the present study, we report the effects of multiple administrations of a candidate, seasonal, MDCK-based, quadrivalent split influenza virus vaccine MDCK-QIV in Sprague-Dawley (SD) rats. Moreover, the effects of the vaccine were evaluated in terms of fertility and early embryonic development, embryo-fetal development, and perinatal toxicity in the SD rats and immunogenicity in Wistar rats and BALB/c mice. Regarding the safety profile, MDCK-QIV demonstrated tolerance in local stimulation with repeated dose administration and presented no significant effect on the development, growth, behavior, fertility, and reproductive performance of the adult male rats, maternal rats, and their offspring. MDCK-QIV elicited strong hemagglutination inhibition neutralizing antibody response and protection against the influenza virus in the mouse model. Thus, data supported that MDCK-QIV could be further evaluated in human clinical trial, which is currently underway.

The Epidemiology of Influenza and the Associated Vaccines Development in China: A Review.

Influenza prevention and control has been one of the biggest challenges encountered in the public health domain. The vaccination against influenza plays a pivotal role in the prevention of influenza, particularly for the elderly and small children. According to the epidemiology of influenza in China, the nation is under a heavy burden of this disease. Therefore, as a contribution to the prevention and control of influenza in China through the provision of relevant information, the present report discusses the production and batch issuance of the influenza vaccine, analysis of the vaccination status and vaccination rate of the influenza vaccine, and the development trend of the influenza vaccine in China.

Development of Nasal Vaccines and the Associated Challenges.

Viruses, bacteria, fungi, and several other pathogenic microorganisms usually infect the host via the surface cells of respiratory mucosa. Nasal vaccination could provide a strong mucosal and systemic immunity to combat these infections. The intranasal route of vaccination offers the advantage of easy accessibility over the injection administration. Therefore, nasal immunization is considered a promising strategy for disease prevention, particularly in the case of infectious diseases of the respiratory system. The development of a nasal vaccine, particularly the strategies of adjuvant and antigens design and optimization, enabling rapid induction of protective mucosal and systemic responses against the disease. In recent times, the development of efficacious nasal vaccines with an adequate safety profile has progressed rapidly, with effective handling and overcoming of the challenges encountered during the process. In this context, the present report summarizes the most recent findings regarding the strategies used for developing nasal vaccines as an efficient alternative to conventional vaccines.

Safety and immunogenicity of a quadrivalent influenza vaccine in adults aged 60 years or above: a phase III randomized controlled clinical study.

To control seasonal influenza epidemics in elders, a quadrivalent, inactivated, split-virion influenza vaccine (IIV4) comprising A and B lineages is produced for young individuals and adults aged ≥60 years. In this phase III, randomized, double-blind, active-controlled trial, we compared safety and immunogenicity of IIV4 with a licensed quadrivalent inactivated vaccine (IIV4-HL) produced by Hualan Biological Engineering during the 2019 influenza season. Participants were randomly assigned to receive IIV4 (n = 959) or IIV4-HL (n = 959). Compared to IIV4-HL, geometric mean titers (GMT) of hemagglutination inhibition (HAI) titers and seroconversion rate (SCR) of IIV4 demonstrated better antibody responses in A lineages (H1N1 and H3N2) (P < .01) and equivalent antibody responses in B lineages (B/Yamagata and B/Victoria) (P > .01) in both age groups. After immunization, IIV4 provided a satisfactory SCR and seroprotection rate (SPR) in elders. No discernible variation in immunogenicity was observed between the two age cohorts. In both age groups, IIV4 and IIV4-HL recipients experienced similar levels of solicited and unsolicited adverse events (AEs), and the incidence of AEs was low in both vaccine groups. Most AEs were of mild-to-moderate severity and no grade 3 AEs in IIV4 group, but AEs in adults aged 60-65 were little higher than in adults over 65 years in IIV4 and IIV4-HL groups (IIV4: 14.66% vs. 10.36%; IIV4-HL:14.67% vs. 11.43%). Totally, IIV4 was generally well tolerated and induced high antibody titers against all four influenza strains in elderly, making it a compelling alternative for the elderly aged ≥60 years.Trial registration: Clinical Trials.gov: 2015L00649-2.

Safety and immunogenicity of a quadrivalent, inactivated, split-virion influenza vaccine (IIV4-W) in healthy people aged 3-60 years: a phase III randomized clinical noninferiority trial.

BACKGROUND: A quadrivalent split influenza vaccine IIV4-W against both influenza A and B viruses is urgently needed. METHODS: To evaluate the safety and immunogenicity of IIV4-W in people aged 3-60 years, 2400 participants recruited in a double-blind phase III trial and were randomly assigned to the IIV4-W, TIV1 and TIV2 groups. The immunogenicity indicators were measured at 28 days postvaccination and for 180 days for safety follow-up. RESULTS: Adverse events (AEs) occurred in 162 (20.28%), 116 (14.55%) and 123 (15.41%) participants in the IIV4-W, TIV1 and TIV2 groups, respectively. All these AEs were mild and self-limiting, and no serious AEs related to the vaccines were observed. IIV4-W elicited a non-inferior immune response for matched strains (the lower limit of 95% CI for GMT ratio >0.67, for SCR and SPR difference >-10%) and superior immune response for the additional B strains (the lower limit of 95% CI for GMT ratio >1.5, for SCR difference >10%) versus TIVs. The lower limit of the 95% confidence interval of the GMT increase fold, the seroconversion rate and the seroprotection rate exceeded 2.5, 40% and 70% for the four strains in IIV4-W respectively. CONCLUSIONS: IIV4-W was noninferior to the TIV-matched strains and was superior to the additional B strain. IIV4-W was safe in the participants and elicited high antibody titers.

Preclinical immunogenicity assessment of a cell-based inactivated whole-virion H5N1 influenza vaccine.

In influenza vaccine development, Madin-Darby canine kidney (MDCK) cells provide multiple advantages, including large-scale production and egg independence. Several cell-based influenza vaccines have been approved worldwide. We cultured H5N1 virus in a serum-free MDCK cell suspension. The harvested virus was manufactured into vaccines after inactivation and purification. The vaccine effectiveness was assessed in the Wuhan Institute of Biological Products BSL2 facility. The pre- and postvaccination mouse serum titers were determined using the microneutralization and hemagglutination inhibition tests. The immunological responses induced by vaccine were investigated using immunological cell classification, cytokine expression quantification, and immunoglobulin G (IgG) subtype classification. The protective effect of the vaccine in mice was evaluated using challenge test. Antibodies against H5N1 in rats lasted up to 8 months after the first dose. Compared with those of the placebo group, the serum titer of vaccinated mice increased significantly, Th1 and Th2 cells were activated, and CD8+ T cells were activated in two dose groups. Furthermore, the challenge test showed that vaccination reduced the clinical symptoms and virus titer in the lungs of mice after challenge, indicating a superior immunological response. Notably, early after vaccination, considerably increased interferon-inducible protein-10 (IP-10) levels were found, indicating improved vaccine-induced innate immunity. However, IP-10 is an adverse event marker, which is a cause for concern. Overall, in the case of an outbreak, the whole-virion H5N1 vaccine should provide protection.