Suspension Vero cell culture technology for high titer production of viral vaccines

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The continuous Vero cell line has been commercially used, after propagation on microcarriers, for the production of rabies, polio, enterovirus 71, hantaan, more recent COVID19 and other vaccines. Vero cell culture technologies were also explored for productions of many more viral vaccines over the last two decades. The growth of Vero cells is anchorage-dependent, and cells need to be dissociated enzymatically or mechanically for the process of subcultivation. This process is labor intensive and complicated in process scale-up. Adaptation of Vero cells to grow in suspension will significantly simplify scale-up and manufacturing processes. Development of advanced suspension Vero culture technology to improve product titer will further reduce the cost of goods. We previously reported a successful adaptation of adherent Vero cells originated from ATCC CCL-81 to grow in suspension in serum-free and animal component-free media developed in-house. The suspension adapted cells were found to retain their genetic stability and to be non-tumorigenic. Present work continues the development and optimization of cell culture process and feeding strategy to improve the growth of suspension Vero cell and the production of vesicular stomatitis virus (VSV) and herpes simplex virus-1 (HSV-1). Data from this study showed the suspension adapted Vero cells retained similar VSV productivity to that obtained in adherent culture; volumetric productivity of VSV increased with the increasing cell density at infection in batch culture. However, the maximum cell density in batch culture was about 2.5x106 cells/mL, and was not improved significantly despite tremendous effort dedicated to improve culture conditions such as supplementing various nutrients in batch culture. As a result, perfusion culture was employed as an approach to increase cell density in the culture, which in turn increased the VSV productivity up to one log, at 1.1x1010 TCID50/mL when the culture infected at 7x106 cells/mL. High titer production of HSV-1 in the Vero culture is more challenging. The virus productivity is not only limited by the maximum cell density in batch culture, but also reduced by inhibitory metabolites secreted in the culture even at low cell density such as 1x106 cells/mL. Media replacement before virus infection is essential to achieve a high HSV-1 yield. As such, perfusion culture was a preferred mode for high titer production of HSV-1, which improved the HSV-1 titer also by up to one log to 1.8 x109 TCID50/ in a culture infected at 5x106 cells/mL when comparing to a control shake flask culture infected at 1x106 cells/mL. Experimental data also demonstrated that perfusion Vero culture was robust and reproducible. This study demonstrates that batch or perfusion suspension Vero culture is a much simplified process than current adherent culture technology for manufacturing of viral vaccines, and offers great potentials in reducing the cost of goods. The suspension Vero culture developed in our institute has generated tremendous interests from industry and academia, and are being tested by many different organizations

Development of suspension adapted Vero cell culture process technology for production of viral vaccines

Abstract Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost. Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house. The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40–44 h. Much higher cell density (8 × 10 6 cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process. Both adherent and suspension Vero cells from various stages were tested for their authenticity using short tandem repeat analysis. Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability. Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic. The viral productivity of suspension Vero cells was evaluated by using vesicular stomatitis virus (VSV) as a model. The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture. In addition, the suspension culture could be infected at higher cell densities, thus improving the volumetric virus productivity. More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 × 10 6 cells/mL

Development of suspensions adapted Vero cell culture process for production of viruses

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The continuous Vero cell line has been commercially used, after propagation on microcarriers, for the production of rabies, polio, enterovirus 71 and hantaan virus vaccines. Vero cell culture technologies are also explored for productions of many more viral vaccines over the last two decades. The growth of Vero cells is anchorage-dependent, and cells need to be dissociated enzymatically or mechanically for the process of subcultivation. This process is labor intensive and complicated in process scale-up. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly. Here we report on the adaptation of adherent Vero cells to grow in suspension using a serum-free and animal component-free medium developed in-house. The maximum cell density and cell doubling time of the suspension adapted Vero cells in batch culture grown in the in-house developed medium were similar to or better than what was observed for the adherent Vero cells grown in commercial media. The growth of suspension adapted Vero culture was successfully scaled up to 3 L bioreactor. The Vero cells from various stages (both adherent and adapted) were tested for their authenticity using a Short Tandem Repeat (STR) analysis. The testing result indicates that all Vero cell samples have 100% concordance with the Vero DNA control sample, indicating the suspension adapted cells maintained their genetic stability. Productions of vesicular stomatitis virus (VSV) and influenza virus in adherent culture and suspension adapted culture were compared, showing the suspension adapted Vero cell retained similar viral productivity. The volumetric productivity of VSV in the suspension culture was even higher, and was further increased by almost 200 times when culture was infected at higher cell density and with medium replacement before the virus infection. In contrast, the VSV production decreased when the adherent culture was infected at higher cell density. Additional process development revealed that the maximum cell density in batch culture was doubled, reaching 6x106 cells/mL, when the culture medium was replaced during the process of batch culture, which indicates potential for further increases in product titer

Development and characterization of a murine hepatoma model expressing hepatitis Cvirus (HCV) non-structural antigens for evaluating HCV vaccines

Hepatitis C (HCV) is a highly prevalent blood-borne virus with infection of 2-3% of world population and high rate of chronicity (\u3e70%) leading to chronic hepatitis, which often progress to cirrhosis and hepatocellular carcinoma. HCV- specific immune responses consisting of CD4 and CD8 T cells and virus neutralizing antibodies have been shown to eliminate HCV infections in humans and chimpanzees. Therefore, vaccines that can induce potent and durable anti-HCV T and B cell responses may have the potential to clear chronic HCV infections. A number of HCV vaccines have been tested clinically with limited success. One of the major limitations in developing effective HCV therapies is the lack of effective and reliable animal models due to the narrow host range of the HCV virus. The study described herein reports the generation of a murine hepatoma cell line expressing HCV non-structural proteins and its use in a metastatic tumor setting to test anti-tumor efficacy of bacterial and viral vector vaccines expressing the HCV non-structural genes. HCV-recombinant hepatoma cells formed large solid-mass tumors when implanted into syngeneic mice, allowing the testing of HCV vaccines for immunogenicity and anti-tumor efficacy. Using this model, we tested the therapeutic potential of recombinant anti-HCV-specific vaccines based on two fundamentally different attenuated pathogen vaccine systems - attenuated Salmonella and recombinant adenoviral vector based vaccine. Attenuated Salmonella secreting HCV antigens limited growth of the HCV-recombinant tumors when used in a therapeutic vaccination setting. The inhibition of tumor growth by Salmonella vector-based vaccines was significantly reduced in mice co-injected with an anti-CD8 antibody, suggesting a role by CD8+ cells in the vaccine efficacy. The model was also used to compare replication deficient and replication-competent but non-infectious adenoviral vectors expressing non-structural HCV antigens. Results showed overall greater survival and reduced weight loss with the replication-competent vector compared to the non-replicating vector. Our results demonstrate the novel recombinant murine hepatoma model expressing HCV non-structural antigens as a useful model for evaluating therapeutic vaccines against HCV. Vaccines that are capable of inducing potent anti-HCV immune responses that are capable of controlling aggressive and metastatic tumor growth in this model would likely have the potential to control chronic viral infections such as HCV. This novel approach is particularly interesting for the development of therapeutic vaccines

Evaluation of recombinant adenovirus vectors and adjuvanted protein as a heterologous prime-boost strategy using HER2 as a model antigen.

Induction of strong antigen-specific cell-mediated and humoral responses are critical to developing a successful therapeutic vaccine. Herein, using HER2 as a model antigen, we aim to evaluate a therapeutic vaccine protocol that elicits anti-tumor antibody and cytotoxic T cells to HER2/neu antigen. Replication-competent (ΔPS AdV) and non-replicating recombinant adenoviral vectors (AdV) expressing a rat HER2/neu (ErbB2) oncogene, were generated and compared for four different doses and over four time points for their ability to induce antigen-specific T and B cell responses in mice. Although ΔPS AdV:Her2 vector was shown to induce more durable antigen-specific CD8⁺ T cell responses, overall, the AdV:Her2 vector induced broader T and B cell responses. Hence the AdV:Her2 vector was used to evaluate a heterologous prime-boost vaccination regimen using rat HER2 protein encapsulated in archaeosomes composed of a semi-synthetic glycolipid (sulfated S-lactosylarchaeol, SLA; and lactosylarchaeol, LA) (SLA/LA:HER2enc) or admixed with archaeosomes composed of SLA alone (SLA:HER2adm). We first tested AdV:Her2 using a prime-boost approach with SLA/LA:HER2enc, and thereafter evaluated a sub-optimal AdV:Her2 dose in a heterologous prime-boost approach with SLA:HER2adm. A single administration of AdV:Her2 alone induced strong cell-mediated immune responses, whereas SLA/LA:HER2enc alone induced strong antigen-specific IgG titers. In mice primed with a suboptimal dose of AdV:Her2, strong CD8⁺ T-cell responses were observed after a single dose which were not further augmented by protein boost. AdV:Her2 induced CD4⁺ specific T-cell responses were augmented by SLA:HER2adm. Homologous vaccination using SLA:HER2adm induced strong antigen-specific antibody responses. However, the overall magnitude of the responses was similar with three doses of SLA:HER2adm or Ad:HER2 prime followed by two doses of SLA:HER2adm. We demonstrate that AdV:Her2 is capable of inducing strong antigen-specific CD8⁺ T cell responses, even at a low dose, and that these responses can be broadened to include antigen-specific antibody responses by boosting with SLA adjuvanted proteins without compromising CD8 T cell responses elicited by AdV priming

Wild Bird Influenza Survey, Canada, 2005

Of 4,268 wild ducks sampled in Canada in 2005, real-time reverse transcriptase–PCR detected influenza A matrix protein (M1) gene sequence in 37% and H5 gene sequence in 5%. Mallards accounted for 61% of samples, 73% of M1-positive ducks, and 90% of H5-positive ducks. Ducks hatched in 2005 accounted for 80% of the sample

A Method to Generate and Rescue Recombinant Adenovirus Devoid of Replication-Competent Particles in Animal-Origin-Free Culture Medium

Adenoviruses are promising vectors for vaccine production and gene therapy. Despite all the efforts in removing animal-derived components such as fetal bovine serum (FBS) during the production of adenovirus vector (AdV), FBS is still frequently employed in the early stages of production. Conventionally, first-generation AdVs (E1 deleted) are generated in different variants of adherent HEK293 cells, and plaque purification (if needed) is performed in adherent cell lines in the presence of FBS. In this study, we generated an AdV stock in SF-BMAdR (A549 cells adapted to suspension culture in serum-free medium). We also developed a limiting dilution method using the same cell line to replace the plaque purification assay. By combining these two technologies, we were able to completely remove the need for FBS from the process of generating and producing AdVs. In addition, we demonstrated that the purified AdV stock is free of any replication-competent adenovirus (RCA). Furthermore, we demonstrated that our limiting dilution method could effectively rescue an AdV from a stock that is highly contaminated with RCA

DNA delivery to cells in culture: generation of adenoviral libraries for high-throughput functional screening

In functional genomics, the use of expression libraries of DNA variants in combination with potent screening techniques is a powerful tool for gene discovery. They allow study of gene and protein function, generation of peptide variants with novel properties, as well as identification of functional short DNA and RNA motifs. In proteomics, generation of large expression libraries of protein variants with random substitutions ('directed evolution') and further screening for novel or improved functions has been commonly used for isolation of proteins with novel characteristics, for improving enzymes, for rapid isolation of antibodies, and for functional protein studies. Most commonly, peptide libraries are expressed and screened in prokaryotic systems. Such systems have the advantage of rapid and simple generation of clones expressing single variants, allow high diversity (up to 10(11)), and can be combined with phage- or cell-surface display technique (2). The main disadvantage of bacterial systems is the absence of posttranslational modifications and native folding of many mammalian proteins, leading to limited applications, particularly when enzyme-substrate-, protein-protein, or protein-RNA interactions are to be studiedEnglish14970582NRC publication: Ye

Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors

To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious viral particles are assembled, there is no risk of viral shedding. The complementary cell lines for this developed AdV proved to be suboptimal for the production of viral particles and require the presence of fetal bovine serum (FBS) to grow. In the current study, we produced both stable pools and clones using adherent and suspension cells expressing the PS gene. The best adherent cell pool can be used in the early stages for the generation of protease-deleted adenovirus, plaque purification, and titration. Using this, we produced over 3400 infectious viral particles per cell. Additionally, the best suspension subclone that was cultured in the absence of FBS yielded over 4000 infectious viral particles per cell. Harvesting time, culture media, and concentration of the inducer for the best suspension subclone were further characterized. With these two types of stable cells (pool and subclone), we successfully improved the titer of protease-deleted adenovirus in adherent and suspension cultures and eliminated the need for FBS during the scale-up production. Eight lots of SC-AdV were produced in the best suspension subclone at a scale of 2 to 8.2 L. The viral and infectious particle titers were influenced by the virus backbone and expressed transgene