9 research outputs found

    Characteristics of rVSV-ZEBOV production kinetics in HEK293 and Vero cells

    Get PDF
    The vesicular stomatitis virus (VSV) can be used as an effective vaccine platform, inducing both cellular and humoral immunity. Because VSV infections of humans are mostly asymptomatic, recombinant VSV (rVSV) can be used as a platform to safely deliver and express foreign antigens. This research study focusses on cell culture production of an rVSV expressing the Ebola virus glycoprotein on its surface (rVSV-ZEBOV). This virus has been demonstrated to be safe to administer to humans. In addition, recent results of a human phase III clinical trial showed that this vaccine can efficiently protect against Ebola virus infection. However, limited data is available in the literature about the growth characteristics of this virus during the production process. In our study, we investigated the influence of multiplicity of infection (MOI), time of infection (TOI), time of harvest (TOH), media components and temperature on the viral titer (TCID50/mL, ddPCR) of rVSV-ZEBOV produced from cell culture. Results are compared between the standard production in the Vero cell line and in a suspension-adapted HEK293-based cell line without serum

    Genomics of Vero cells: Understanding this cell line and its virus-host interactions for improved vaccine production

    Get PDF
    The Vero cell line is the most used continuous cell line for viral vaccine manufacturing with more than 40 years of accumulated experience in the vaccine industry. Additionally, the Vero cell line has shown high affinity for infection by MERS-CoV, SARS-CoV and recently SARS-CoV-2, emerging as an important discovery and screening tool to support the global research and development efforts in this COVID-19 pandemic. Furthermore, Vero cells anchorage-dependent use renders scaling-up challenging and operations very labor intensive which affects cost effectiveness. Thus, efforts to adapt Vero cells to suspension cultures have been invested but hurdles such as the long doubling time and low cell viability remain to be addressed. However, the lack of a reference genome for the Vero cell line has limited our understanding of host-virus interactions underlying such affinity of the Vero cell towards key emerging pathogens, and more importantly our ability to re-design high-yield vaccine production processes using Vero genome editing. In this study, we present an annotated highly contiguous 2.9 Gb assembly of the Vero cell genome. Please click Download on the upper right corner to see the full abstract

    Production of rVSV-ZEBOV in serum-free suspension culture of HEK 293SF cells.

    Get PDF
    Abstract Ebola virus disease is an urgent international priority. Promising results for several vaccine candidates have been reported in non-human primate studies and clinical trials with the most promising being the rVSV-ZEBOV vaccine. In this study, we sought to produce rVSV-ZEBOV in HEK 293SF cells in suspension and serum-free media. The purpose of this study was to establish a process using the HEK 293SF production platform, optimise the production titre, demonstrate scalability and the efficiency of the generated material to elicit an immune reaction in an animal model. Critical process parameters were evaluated to maximize production yield and process robustness and the following operating conditions: 1–2 × 106 cells/mL grown in HyClone HyCell TransFx-H media infected at an MOI of 0.001 with a temperature shift to 34 °C during the production phase and a harvest of the product after 48 h. Using these conditions, scalability in a 3.5 L controlled bioreactor was shown reaching a titre of 1.19 × 108 TCID50/mL at the peak of production, the equivalent of 4165 doses of vaccine per litre. The produced virus was shown to be thermostable in the culture media and, when concentrated, purified and administered to mice, demonstrated the ability to induce a ZEBOV-specific immune response

    Evaluation of novel HIV vaccine candidates using recombinant vesicular stomatitis virus vector produced in serum-free Vero cell cultures.

    Get PDF
    Acquired Immune Deficiency Syndrome (AIDS) in humans is a result of the destruction of the immune system caused by Human Immunodeficiency Virus (HIV) infection. This serious epidemic is still progressing world-wide. Despite advances in treatment, a safe and effective preventive HIV vaccine is desired to combat this disease, and to save millions of lives. However, such a vaccine is not available yet although extensive amounts of resources in research and development have been invested over three decades. In light of the recently approved Ebola virus disease vaccine based on a recombinant vesicular stomatitis virus (rVSV-ZEBOV), we present the results of our work on three novel VSV-vectored HIV vaccine candidates. We describe the design, rescue, production and purification method and evaluate their immunogenicity in mice prior to preclinical studies that will be performed in non-human primates. The production of each of the three candidate vaccines (rVSV-B6-NL4.3Env/SIVtm, rVSV-B6-NL4.3Env/Ebtm and rVSV-B6-A74Env(PN6)/SIVtm) was evaluated in small scale in Vero cells and it was found that production kinetics on Vero cells vary depending on the HIV gp surface protein used. Purified virus preparations complied with the WHO restrictions for the residual DNA and host cell protein contents. Finally, when administered to mice, all three rVSV-HIV vaccine candidates induced an HIV gp140-specific antibody response

    Process Development for Newcastle Disease Virus-Vectored Vaccines in Serum-Free Vero Cell Suspension Cultures

    No full text
    The ongoing COVID-19 pandemic drew global attention to infectious diseases, attracting numerous resources for development of pandemic preparedness plans and vaccine platforms—technologies with robust manufacturing processes that can quickly be pivoted to target emerging diseases. Newcastle Disease Virus (NDV) has been studied as a viral vector for human and veterinary vaccines, but its production relies heavily on embryonated chicken eggs, with very few studies producing NDV in cell culture. Here, NDV is produced in suspension Vero cells, and analytical assays (TCID50 and ddPCR) are developed to quantify infectious and total viral titer. NDV-GFP and NDV-FLS (SARS-CoV-2 full-length spike protein) constructs were adapted to replicate in Vero and HEK293 suspension cultures using serum-free media, while fine-tuning parameters such as MOI, temperature, and trypsin concentration. Shake flask productions with Vero cells resulted in infectious titers of 1.07 × 108 TCID50/mL for NDV-GFP and 1.33 × 108 TCID50/mL for NDV-FLS. Production in 1 L batch bioreactors also resulted in high titers in culture supernatants, reaching 2.37 × 108 TCID50/mL for NDV-GFP and 3.16 × 107 TCID50/mL for NDV-FLS. This shows effective NDV production in cell culture, building the basis for a scalable vectored-vaccine manufacturing process that can be applied to different targets

    Process Development for Newcastle Disease Virus-Vectored Vaccines in Serum-Free Vero Cell Suspension Cultures

    No full text
    The ongoing COVID-19 pandemic drew global attention to infectious diseases, attracting numerous resources for development of pandemic preparedness plans and vaccine platforms—technologies with robust manufacturing processes that can quickly be pivoted to target emerging diseases. Newcastle Disease Virus (NDV) has been studied as a viral vector for human and veterinary vaccines, but its production relies heavily on embryonated chicken eggs, with very few studies producing NDV in cell culture. Here, NDV is produced in suspension Vero cells, and analytical assays (TCID50 and ddPCR) are developed to quantify infectious and total viral titer. NDV-GFP and NDV-FLS (SARS-CoV-2 full-length spike protein) constructs were adapted to replicate in Vero and HEK293 suspension cultures using serum-free media, while fine-tuning parameters such as MOI, temperature, and trypsin concentration. Shake flask productions with Vero cells resulted in infectious titers of 1.07 × 108 TCID50/mL for NDV-GFP and 1.33 × 108 TCID50/mL for NDV-FLS. Production in 1 L batch bioreactors also resulted in high titers in culture supernatants, reaching 2.37 × 108 TCID50/mL for NDV-GFP and 3.16 × 107 TCID50/mL for NDV-FLS. This shows effective NDV production in cell culture, building the basis for a scalable vectored-vaccine manufacturing process that can be applied to different targets
    corecore