Process intensification of EB66® cell cultivations leads to high-yield yellow fever and Zika virus production.

A live-attenuated, human vaccine against mosquito-borne yellow fever virus has been available since the 1930s. The vaccine provides long-lasting immunity and consistent mass vaccination campaigns counter viral spread. However, traditional egg-based vaccine manufacturing requires about 12 months and vaccine supplies are chronically close to shortages. In particular, for urban outbreaks, vaccine demand can be covered rarely by global stockpiling. Thus, there is an urgent need for an improved vaccine production platform, ideally transferable to other flaviviruses including Zika virus. Here, we present a proof-of-concept study regarding cell culture-based yellow fever virus 17D (YFV) and wild-type Zika virus (ZIKV) production using duck embryo-derived EB66® cells. Based on comprehensive studies in shake flasks, 1-L bioreactor systems were operated with scalable hollow fiber-based tangential flow filtration (TFF) and alternating tangential flow filtration (ATF) perfusion systems for process intensification. EB66® cells grew in chemically defined medium to cell concentrations of 1.6 × 108 cells/mL. Infection studies with EB66®-adapted virus led to maximum YFV titers of 7.3 × 108 PFU/mL, which corresponds to about 10 million vaccine doses for the bioreactor harvest. For ZIKV, titers of 1.0 × 1010 PFU/mL were achieved. Processes were automated successfully using a capacitance probe to control perfusion rates based on on-line measured cell concentrations. The use of cryo-bags for direct inoculation of production bioreactors facilitates pre-culture preparation contributing to improved process robustness. In conclusion, this platform is a powerful option for next generation cell culture-based flavivirus vaccine manufacturing.

Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins.

Here, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC-MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtoolMS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.

A coupled thermo-mechanical damage model for fired clay bricks based on the unified strength theory.

On the rise request for long-lasting materials, clay materials are in between the well-nigh minerals exploited by production and ecological fields in the making of fired bricks. Clay incessantly expounded to high temperature reacts differently at ambient temperature which critically touches its longevity. In present study, a coupled thermo-mechanical damage model of clay is established. In this model, the Unified Strength Theory (UST) criterion is used as the failure criterion based on the Weibull distribution and the continuous damage theory. The proposed model is validated by uniaxial compression experiment of high-temperature clay. The variation of the two distribution factors (m and W0) in the combined TM damage relationship with temperature is analysed. The results show that the damage evolvement speed of the clay shows a curving form getting closed to one as the temperature rises, indicating that the temperature can delay the development of cumulative damage. The damage fundamental modelling discussed is in accord with the testings curves at the various phases of yielding and pre-apex force. This study leads to an enhanced understanding of high temperature clay mechanics and affords the fundament to heighten clay bricks resourcefulness lastingness.

Data on the estimation of thermomechanical damage for fired clay bricks.

Fired bricks are on high demand in building constructions because of their cheapness, appearance, robustness, isolation achievement and sustainability. To make fired bricks, Constructions and eco-friendly sector used clay materials. However, the major challenge in their utilization is their thermal and mechanical behavior after exposure. Problems occur mainly when permanently subjected to increased temperature which severely influence its durability, and in this case an overall failure mode calculation is essential. In this work a simple approach based on the Unified Strength Theory (UST) criterion was used to estimate the thermomechanical damage. Results of thermomechanical damage values are shown.

The EB66® cell line as a valuable cell substrate for MVA-based vaccines production.

The selection of a cell substrate is a critical step for the development and manufacturing of a viral vaccine candidate. Several parameters such as cell susceptibility and permissiveness to the viral pathogens but also performance in terms of viral antigens quality and production yields are important considerations when identifying the ideal match between a viral vaccine and cell substrate. The modified vaccinia virus Ankara (MVA) is a replication-deficient viral vector that holds great promise as a vaccine platform, however only limited cell substrates have been tested or are available for industrialization. Here we evaluate the duck embryo-derived EB66® cell line as potential cell substrate for MVA production. To this end, we used two recombinant MVA constructs and demonstrated that EB66® cells are propagating the tested MVA viruses very efficiently, while preserving viral attenuation and transgene expression for up to 20 serial passages. Furthermore we developed upstream and downstream processes that enable industrialization of the virus production. In conclusion, we showed that EB66® cells can be used as potent cell substrate for MVA-based vaccines and represent therefore an attractive alternative for vaccine production.

Characterization of a potent human interleukin-11 agonist.

Human interleukin-11 (hIL-11) is a multi-potential cytokine that is involved in numerous biological activities, such as haematopoiesis, osteoclastogenesis, neurogenesis and female fertility, and also displays anti-inflammatory properties. IL-11 is used clinically to treat chemotherapy-induced thrombocytopenia. Because of its broad spectrum of action, improved IL-11 agonists, as well as IL-11 antagonists, could be of interest for numerous clinical applications. IL-11 signalling is dependent on the formation of a tripartite ligand-receptor complex consisting of IL-11, the IL-11R (IL-11 receptor) alpha subunit (responsible for the specificity of the interaction) and gp130 (glycoprotein 130) receptor beta subunit (responsible for signal transduction). The interaction between IL-11 and IL-11Ralpha subunit occurs at its recently assigned site I. We have designed an IL-11 mutein whose hydrophobicity at site I has been increased. The mutein has been characterized in terms of structure, affinity, specificity and bioactivity. Electrophoretic analysis, gel filtration, IR spectroscopy and CD indicate that this new protein is more compact than wild-type IL-11. It binds to IL-11Ralpha with a three-fold-enhanced affinity, and retains the ability to recruit gp130 through site II. However, analysis of its biological activity revealed a complex pattern: although this mutein is 60-400-fold more active than wild-type IL-11 on the proliferation of 7TD1 murine hybridoma cell, it is less active than IL-11 on the proliferation of B9 cells, another murine hybridoma cell line. The results are interpreted on the basis of an IL-11 conformational change induced by the mutations, and the preferential use by the mutein of another unknown transducing receptor chain.

Cell substrates for the production of viral vaccines.

Vaccines have been used for centuries to protect people and animals against infectious diseases. For vaccine production, it has become evident that cell culture technology can be considered as a key milestone and has been the result of decades of progress. The development and implementation of cell substrates have permitted massive and safe production of viral vaccines. The demand in new vaccines against emerging viral diseases, the increasing vaccine production volumes, and the stringent safety rules for manufacturing have made cell substrates mandatory viral vaccine producer factories. In this review, we focus on cell substrates for the production of vaccines against human viral diseases. Depending on the nature of the vaccine, choice of the cell substrate is critical. Each manufacturer intending to develop a new vaccine candidate should assess several cell substrates during the early development phase in order to select the most convenient for the application. First, as vaccine safety is quite naturally a central concern of Regulatory Agencies, the cell substrate has to answer the regulatory rules stringency. In addition, the cell substrate has to be competitive in terms of viral-specific production yields and manufacturing costs. No cell substrate, even the so-called "designer" cell lines, is able to fulfil all the requested criteria for all viral vaccines. Therefore, the availability of a variety of cell substrates for vaccine production is essential because it improves the chance to successfully respond to the current and future needs of vaccines linked to new emerging or re-emerging infectious diseases (e.g. pandemic flu, Ebola, and Chikungunya outbreaks).

Expression of a bioactive recombinant human interleukin-11 in chicken HD11 cell line.

To direct the synthesis and secretion of recombinant human interleukin-11 (rhIL-11) in chicken HD11 cells, a plasmid targeting the c-lysozyme gene has been constructed which contains the mature cytokine cDNA in frame with the lysozyme leader sequence. The upregulation of rhIL-11 mediated by LPS proves the knock-in of hIL-11 cDNA in the lysozyme gene. The bioactivity of the expressed protein is demonstrated and quantified with the hIL-11 dependent 7TD1 and B9 cell lines. The electrophoretic mobility, receptor binding properties and growth promoting effect of the chicken-derived cytokine are identical to those of a rhIL-11 expressed in Escherichia coli. These results describe the secretion of a biologically active rhIL-11 expressed by an avian cellular machinery.