Towards a virus production platform based on VeroS cells grown on chemically defined media

Vero cells are nowadays widely used in the production of vaccines. However, they are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce the costs of vaccine manufacturing to develop vaccines affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective. We had previously adapted Vero cells to suspension growth for rabies virus (strain LP2061) production in different serum free media (Rourou et al. 2019). In this work, we describe the use of a chemically defined medium (CDM) provided by Xell AG to assess the growth of suspension adapted Vero cells, VeroS, and the production of both rabies and Bluetongue Serotype 1 (BTV-1) viruses. The viruses were first adapted to VeroS grown on CDM by successive passages. Then, kinetics of cell growth and virus production were studied in shake flask cultures. Please click Download on the upper right corner to see the full abstract

Adaptation of Vero cells to suspension culture and rabies virus production on different SERUM free media

Vero cells are nowadays widely used in the production of high quality vaccines for humans and animals. They are considered as one of the most productive and flexible continuous cell lines available for vaccine manufacturing. However, these cells are anchorage dependent, which greatly complicates upstream processing and process scale-up. Moreover, there is a recognized need to reduce costs of vaccine manufacturing in order to develop vaccines that are affordable worldwide. The use of cell lines adapted to suspension growth contributes to reach this objective. The current work describes the adaptation of Vero cells to suspension culture in different serum free media according to multiple protocols. Then, the suspension adapted Vero cells (VeroS) were infected with rabies virus (strain LP-2061). In addition to IPT-AFM (an in-house animal component free medium described in Rourou et al. 2009a; 2009b and 2014), five commercial formulations were analyzed for the establishment of serum free culture of VeroS. The cultures were performed in erlenmeyer flasks at 37ºC, 5% CO2. The Cell doubling (CD) for VeroS adapted to IPT-AFM was 2,1+0.7; the average specific growth rate (μ) reached 0,016+ 0,003 h-1 and the maximum cell density (Xmax) amounted to 2,16+0,9x106 cells/mL. Through adaptation, the cells behave differently in the different media. A comparative study was performed and IPT-AFM showed promising results. Kinetics of rabies virus replication in VeroS cells grown in IPT_AFM showed that rabies virus was able to replicate in VeroS cells and to achieve a virus titer of 6x107 FFU/mL at day 3 post infection. These data show that the VeroS cell line can be considered as a suitable cell line for large scale rabies virus production. Please click Additional Files below to see the full abstract

Four-dimensional differential equations for the leading divergences of dimensionally-regulated loop integrals

We invent an automated method for computing the divergent part of Feynman integrals in dimensional regularization. Our method exploits simplifications from four-dimensional integration-by-parts identities. Leveraging algorithms from the literature, we show how to find simple differential equations for the divergent part of Feynman integrals. We illustrate the method by an application to heavy quark effective theory at three loops.Comment: second version with minor change

The Use of Ferritin as a Carrier of Peptides and Its Application for Hepcidin

Hepcidin a 25-amino-acid and highly disulfide bonded hormone, is the central regulator of iron homeostasis. In this chapter we propose ferritin as a peptide carrier to promote the association of the hybrid hepcidin/ferritin nanoparticle with a particular cell or tissue for therapeutic or diagnostic use. Indeed, human ferritin H-chain fused directly (on its 5’end) with camel mature hepcidin was cloned into the pASK-43 plus vector and expressed using BL21 (DE3) pLys E. coli strain. The transformed E.coli produced efficiently hepcidin-ferritin construct (hepcH), consisting of 213 amino acids with a molecular weight of 24 KDa. The recovered product is a ferritin exposing hepcidin on outer surface. The hepcH monomer was characterized by immunoblotting using a monoclonal antibody specific for human ferritin and a polyclonal antibody specific for hepcidin-25. The results were also confirmed by MALDI-TOF mass spectrometry. The recombinant native human ferritin and the commercial human hepcidin-25 were used as controls in this experiment. The assembly of hepcH, as an heteropolymer molecule, was performed in presence of denatured human ferritin-H and -L chains. After cysteine oxidation of the recombinant nanoparticles, cellular binding assays were performed on mammalian cells such as mouse monocyte–macrophage cell line J774, HepG2 and COS7

An adenovirus-based vaccine manufacturing technology platform for mucosal or parenteral immunization against poultry diseases in sub-Saharan Africa

Developing novel vaccine technology platforms to respond to emergency situations such as pandemic threats or zoonotic diseases is a worldwide high priority. Public health at a global scale is frequently influenced by the risk of transmission of infectious diseases from wildlife and domestic animals. Thus, veterinary vaccination and animal health monitoring are highly relevant for the deployment of a preventative global policy in the context of “one world, one health”. In regions such as Sub-Saharan Africa, farmers’ activities are frequently affected by the impact of diseases in poultry such as avian influenza and Newcastle disease (ND). ND is one of the most critical, with several outbreaks per year. Currently, protection is provided by vaccination with live vaccines produced only in embryonated eggs, with limitations related to egg supply and the possibility of virus shedding by vaccinated poultry, leading to disease in non-vaccinated birds. The purpose of this work was to develop an adenovirus (Ad) vectored vaccine platform technology suitable for the rapid adaptation to ND or other avian viral threats. The project involved the phylogenetic analysis of local isolates of Newcastle disease virus (NDV) and the construction of adenoviral vectors expressing the F and HN antigens from NDV genotype VI, either as individual antigens or in bicistronic vectors. Remarkably, adenoviral rescue and generation of primary stocks was streamlined by developing a novel procedure for single step amplification in suspension cultures. Please click Download on the upper right corner to see the full abstract

Optimization of rAd5 vectored Newcastle vaccine production in HEK293 at high cell densities

Newcastle disease (ND) is a highly contagious and often severe global spread syndrome that affects birds including domestic poultry. It is caused by a virus belonging to the paramyxoviridae family. Indeed, an outbreak of ND can be quite severe, and is considered as a constant threat to the industry and food security worldwide. The disease can be controlled through the administration of effective vaccines. Immunizations with inactivated or live vaccines, although protective, have some eminent disadvantages. The aims of this work is the development of a vectored vaccine using a non-replicative human adenovirus vector, expressing the F antigen from Newcastle Disease Virus (rAd-F-ND) in bioreactor. The recombinant vaccine is produced using the HEK293 cell line. HEK293 cultures were carried out in suspension, first in shake flasks and then in stirred bioreactor at 37°C, 5% CO2 and 150 rpm in chemically defined media. The virus titers were determined by qPCR. To improve rAd-F-ND virus productions in HEK-293 cells, we studied the effects of the following parameters in shake flask cultures: culture media (Hycell Trans FX-H and Xell-GM), cell density, multiplicity of infection (MOI) and feed (Xell-FS , Xell-GM and cell Boost 5). Please click Download on the upper right corner to see the full abstract

Production of Inactivated Influenza H5N1 Vaccines from MDCK Cells in Serum-Free Medium

BACKGROUND: Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system. PRINCIPAL FINDING: The current study has evaluated the performance of cell adaptation switched from serum-containing (SC) medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3 × 10(6) cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA) units/50 µL and 7.1 ± 0.3 × 10(8) pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera. CONCLUSIONS: The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production

Evaluation du test immunoenzymatique CEDIA pour la recherche de benzodiazépines dans les liquides biologiques

CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF