A Catalan merchant of the mid-seventeenth century: Narcís Feliu (?-1665)

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Intensified virus production in suspension HEK293SF cells using high inoculum fed-batch or low-perfusion rate cultures with continuous harvest

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Intensification of influenza virus production in fed-batch and perfusion cultures of HEK293SF cells

More than half a million people die every year from complications of seasonal influenza, and vaccination stands as the most effective method to prevent and limit outbreaks of the disease. Constant vaccine development based on emerging strains and worldwide distribution of vaccines is a great challenge for public health and vaccine manufacturers, particularly in a potential pandemic scenario. The limited flexibility of the current egg-based production system combined with recent advances in large-scale cell culture techniques have encouraged the development of cell culture processes for influenza vaccine production. While cell culture offers a valuable alternative, productivities are still low when compared to traditional egg-based systems, requiring extensive efforts in process intensification and suspension cell line development. Please click Download on the upper right corner to see the full abstract

Proteomic characterization of influenza H1N1 Gag virus-like particles and extracellular vesicles produced in HEK-293SF

One of the major concerns associated with the use of influenza virus-like particles (VLPs) produced in cell culture as vaccine candidates is their heterogeneous composition. Enveloped VLPs take up the host cell membrane at the budding site carrying out with them not only the viral antigenic proteins but also host cell proteins. In addition, the intrinsic nature of the cells to produce membrane derived vesicles which have similar size to the VLPs and can also contain the antigenic proteins, makes the VLP purification process challenging. Certainly, the expression system and the viral recombinant proteins employed will determine the nature of the proteins within the VLPs. To further characterize cell culture produced-influenza VLPs and contribute to enable their approval as vaccine candidates, the composition and biogenesis of VLPs need to be better understood. In this study we have characterized, by nanoscale liquid chromatography tandem mass spectrometry (n-LC-MS/MS), influenza H1N1 Gag-VLPs produced in human embryonic kidney cells adapted to serum-free medium (HEK-293SF). The cells stably express HA and NA, and the VLPs production occurs following transient transfection with a plasmid containing the gag gene of HIV-1 fused to GFP. Extracellular vesicles (EVs) produced by the unmodified HEK-293SF were also characterized by n-LC-MS/MS. A total of 73 host cell proteins were identified in the VLPs, whereas 98 were detected in the extracellular vesicles. From that, 32 host cell proteins were unique to VLPs while 41 proteins were found in both. Importantly, nucleolin was the most abundant host cell differential protein identified in VLPs while lactotransferrin and heat shock protein 90 were the most present in EVs. This study provides a detailed proteomic description of the VLPs and EVs produced in HEK-293SF as well as a critical discussion of the function of each protein incorporated in both nanoparticles species. The outcome of this research also sheds light on unique target proteins differentially identified either in VLPs and EVs that could potentially be exploited for the development of novel purification protocols to separate EVs from VLPs

Critical assessment of influenza VLP production in Sf9 and HEK293 expression systems

Background: Each year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus' fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen's eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers. Methods: In this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles. Results: For the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles. Conclusions: This study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine. © Thompson et al.; licensee BioMed Central

Biografías imprescindibles. nº 12: Felipe II. Un rey tolerante frente a la leyenda negra

El monarca que seguramente encarna mejor el máximo esplendor del Imperio español hubo de hacer la guerra, pero ansiaba la paz; respaldó a la Inquisición, pero no la utilizó en beneficio propio; combatió a los enemigos interiores, pero se esforzó por mantener las autonomías de los distintos reinos. El autor presenta el perfil de un rey manchado por la Leyenda Negra, que no fue tan fanático ni tan intransigente como la historia se ha empeñado en mostrar