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

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

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

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

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

Recombinant adenoviruses expressing the E2 protein of bovine viral diarrhea virus induce humoral and cellular immune responses

UI - 99365986NRC publication: Ye

Recombinant adenoviruses expressing the E2 protein of bovine viral diarrhea virus induce humoral and cellular immune responses

UI - 99365986NRC publication: Ye

The emergence of porcine circovirus 2b genotype (PCV-2b) in swine in Canada

Since late 2004, the swine industry in the province of Quebec has experienced a significant increase in death rate related to postweaning multisystemic wasting syndrome (PMWS). To explain this phenomenon, 2 hypotheses were formulated: 1) the presence of a 2nd pathogen could be exacerbating the porcine circovirus 2 (PCV-2) infection, or 2) a new and more virulent PCV-2 strain could be infecting swine. In 2005, 13 PMWS cases were submitted to the Quebec provincial diagnostic laboratory and PCV-2 was the only virus that could be found consistently by PCR in all 13 samples. The PCR detection results obtained for other viruses revealed the following: 61.5% were positive for porcine reproductive and respiratory syndrome virus, 30.8% for swine influenza virus, 15.4% for porcine parvovirus, 69.2% for swine torque teno virus (swTTV), 38.5% for swine hepatitis E virus (swHEV) and 84.6% for Mycoplasma hyorhinis; transmissible gastroenteritis virus and porcine respiratory coronavirus (TGEV/PRCV) was not detected. Sequences of the entire genome revealed that these PCV-2 strains belonged to a genotype (named PCV-2b) that has never been reported in Canada. Further sequence analyses on 83 other Canadian PCV-2 positive cases submitted to the provincial diagnostic laboratory during years 2005 and 2006 showed that 79.5% of the viral sequences obtained clustered in the PCV-2b genotype. The appearance of the PCV-2b genotype in Canada may explain the death rate increase related to PMWS, but this relationship has to be confirmed

Induction of humoral and cellular immune responses against the nucleocapsid of bovine viral diarrhea virus by an adenovirus vector with an inducible promoter

UI - 99373439NRC publication: Ye