A rapid response vaccine manufacturing platform as a countermeasure to epidemic threats

The preparedness and rapid response to emerging infectious diseases outbreaks remains challenging due to limited market incentives. The development and manufacturing of vaccines are today facing bottlenecks in development time as well as in cost-effectiveness in order to be able to react rapidly and deliver low-cost, high-quality vaccines. 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

Comparaison of rabies virus purification using different methods

Rabies is a viral zoonosis caused by negative-stranded RNA viruses of the Lyssavirus genus. It can affect all mammals including humans. Dogs are the main source of human rabies deaths, contributing up to 99% of all rabies transmissions to humans. Infection causes tens of thousands of deaths every year, mainly in Asia and Africa. Most of these victims are children under the age of 15. Vaccination against rabies is still the sole way to fight against the disease. The aim of this work is to compare the purity of rabies vaccine purified by zonal centrifugation and chromatographic methods, in terms of residual DNA level, host cell protein (HCP) level and the overall recovery yield. For this purpose, Vero cells were grown under animal component free conditions, on Cytodex 1 microcarriers in VP-SFM medium. Vero cell growth and virus production were previously optimized; studies were conducted in a 7-L bioreactor. Virus replication phase was conducted using perfusion culture mode, viral harvests obtained through the culture were clarified, inactivated by BPL (Beta-propiolactone) and then pooled. The pooled harvests were purified by zonal centrifugation on a sucrose density gradient. The fractions of interest (11 in total) were pooled and checked for their antigenic activity according to the NIH potency test. They showed an activity of 61 UI/ml. The yield obtained was around 60%. To improve the overall yield, we have tested during a previous work several chromatography matrices (Sephacryl S200, Sephacryl S300, Sepharose 4FF,...). However the yield obtained was not high, around 40% in the best case. To improve this performance, we tested the Capto Core 700 (GE Healthcare Life Sciences) which a matrice that had a dual-functionality: size separation and binding chromatography, and was specially designed for the purification of large biological products such as viruses. The use of this matrice to purify rabies virus resulted in a yield of 84.5%, which was 2-fold and 1.5 fold higher than that obtained using chromatographic columns or zonal centrifugation, respectively. We also tested Monolytic chromatographic (CIMmultus™ QA-8 Advanced Composite Column) from BIA Separation. Such kind of media represents a new generation of chromatographic matrices with efficient mass transfer and better hydrodynamic properties. This allows fast and efficient separation of large molecules such as DNA and viruses. In our case, we were able to increase the purification yield to values close to 94%. It was the highest yield obtained compared to other methods used. Currently the content of the purified fractions collected using the different methods is analyzed to estimate the efficiency of DNA and HCP removal

High level production and purification of human interferon α2b in high cell density culture of Pichia pastoris.

Human interferon α2b gene was cloned in the methylotrophic yeast Pichia pastoris under the control of the AOX1 methanol inducible promoter. To optimise the volumetric productivity, we performed different fed-batch studies in a 5-L bioreactor. We demonstrated that hIFNα2b was highly sensitive to proteases activity during high cell density culture. The target protein was totally degraded 20 h after the start of methanol feeding. Replacement of culture medium with fresh medium after glycerol fed-batch culture mode as well as medium enrichment with casamino acids at 0.1% and EDTA at 10 mM, had significantly improved hIFNα2b expression and prevented its proteolysis. Moreover, to further improve hIFNα2b production, three different methanol fed-batch strategies had been assayed in high cell density culture. The optimal strategy resulted in a production level of 600 mg/l while residual methanol level was maintained below 2 g/l. Clarification of culture supernatant through a 0.1 μm hollow fiber cartridge showed that almost 95% of the target protein was retained within the retentate. Triton X-100 or NaCl addition to the culture harvest before microfiltration had improved the recovery yield of this step. rhIFNα2b was further purified by cation exchange on Sepharose SP resin followed by gel permeation on Sephacryl S-100. The overall yield of the process was equal to 30% (180 mg/l). The biological activity of the purified protein based on the antiviral activity test was 1.5 × 108 IU/mg. The optimised process has a great potential for large scale production of fully functional hIFNα2b.pre-print3123 K

Design of an efficient medium for heterologous protein production in Yarrowia lipolytica: case of human interferon alpha 2b

<p>Abstract</p> <p>Background</p> <p>The non conventional yeast <it>Yarrowia lipolytica </it>has aroused a strong industrial interest for heterologous protein production. However most of the studies describing recombinant protein production by this yeast rely on the use of complex media, such media are not convenient for large scale production particularly for products intended for pharmaceutical applications. In addition medium composition can also affect the production yield. Hence it is necessary to design an efficient medium for therapeutic protein expression by this host.</p> <p>Results</p> <p>Five different media, including four minimal media and a complex medium, were assessed in shake flasks for the production of human interferon alpha 2b (hIFN α2b) by <it>Y. lipolytica </it>under the control of POX2 promoter inducible with oleic acid. The chemically defined medium SM4 formulated by Invitrogen for <it>Pichia pastoris </it>growth was the most suitable. Using statistical experimental design this medium was further optimized. The selected minimal medium consisting in SM4 supplemented with 10 mg/l FeCl₃, 1 g/l glutamate, 5 ml/l PTM1 (<it>Pichia </it>Trace Metals) solution and a vitamin solution composed of myo-inositol, thiamin and biotin was called GNY medium. Compared to shake flask, bioreactor culture in GNY medium resulted in 416-fold increase of hIFN α2b production and 2-fold increase of the biological activity.</p> <p>Furthermore, SM4 enrichment with 5 ml/l PTM1 solution contributed to protect hIFN α2b against the degradation by the 28 kDa protease identified by zymography gel in culture supernatant. The screening of the inhibitory effect of the trace elements present in PTM1 solution on the activity of this protease was achieved using a Box-Behnken design. Statistical data analysis showed that FeCl₃and MnSO₄had the most inhibitory effect.</p> <p>Conclusion</p> <p>We have designed an efficient medium for large scale production of heterologous proteins by <it>Y. lipolytica</it>. The optimized medium GNY is suitable for the production of hIFN α2b with the advantage that no complex nitrogen sources with non-defined composition were required.</p

Virulence gene expression, proteins secreted and morphological alterations of Vibrio parahaemolyticus and Vibrio alginolyticus in response to long-term starvation in seawater

International audienceIn this study, we incubated Vibrio parahaemolyticus and Vibrio alginolyticus (marine food-borne pathogens bacteria) in seawater for 8 months to study their morphologic, proteomic and genetic responses to starvation. The atomic force micrographs of stressed strains showed a reduction of the cells size and an evolution to two coccoid-shape forms whose length is less than 0.4 mu m and between 0.5 and 1 mu m. Extracellular protein patterns and gelatinase profiles of stressed bacteria were also altered. Indeed, these modifications were manifested by the appearance and/or disappearance of bands as well as in the level of expression of certain proteins. In addition, we also searched for the presence of eight Vibrio cholerae virulence genes: toxR, toxS, toxRS, ctxA, zot, ace, toxT, and Virulence Pathogenicity Island (VPI) in the genome of investigated strains. The expression level of VPI gene studied by reverse transcriptase polymerase chain reaction was decreased, whereas the mRNA quantities of toxR, toxS, and ace in starved Vibrio remained stable

Manganese Induces Oxidative Stress, Redox State Unbalance and Disrupts Membrane Bound ATPases on Murine Neuroblastoma Cells In Vitro: Protective Role of Silymarin

Manganese (Mn) is an essential trace element required for ubiquitous enzymatic reactions. Chronic overexposure to this metal may promote potent neurotoxic effects. The mechanism of Mn toxicity is not well established, but several studies indicate that oxidative stress play major roles in the Mn-induced neurodegenerative processes. Silymarin (SIL) has antioxidant properties and stabilizes intracellular antioxidant defense systems. The aim of this study was to evaluate the toxic effects of MnCl2 on the mouse neuroblastoma cell lines (Neuro-2A), to characterize the toxic mechanism associated with Mn exposure and to investigate whether SIL could efficiently protect against neurotoxicity induced by Mn. A significant increase in LDH release activity was observed in Neuro-2A cells associated with a significant decrease in cellular viability upon 24 h exposure to MnCl2 at concentrations of 200 and 800 μM (P < 0.05) when compared with control unexposed cells. In addition, exposure cells to MnCl2 (200 and 800 μM), increases oxidant biomarkers and alters enzymatic and non enzymatic antioxidant systems. SIL treatment significantly reduced the levels of LDH, nitric oxide, reactive oxygen species and the oxidants/antioxidants balance in Neuro-2A cells as compared to Mn-exposed cells. These results suggested that silymarin is a powerful antioxidant through a mechanism related to its antioxidant activity, able to interfere with radical-mediated cell death. SIL may be useful in diseases known to be aggravated by reactive oxygen species and in the development of novel treatments for neurodegenerative disorders such as Alzheimer or Parkinson diseases

Development of a vaccine production platform for poultry diseases in Africa: Newcastle Disease Virus non-replicative adenovirus-vectored vaccine

Poultry are a vital village livestock playing an important economic and nutritional role in the livelihoods of poor rural households in developing countries, including the Sub-Saharan Africa. Poultry production in Africa is threatened by infectious diseases such as Newcastle Disease (ND), which is highly contagious and endemic, with recurrent outbreaks that provoke heavy losses every year. ND is caused by the Newcastle disease virus (NDV), a negative-sense single-stranded RNA virus from the genus Avulavirus, family Paramyxoviridae. In particular, ND is one of the major problems in village chickens in most parts of Ethiopia where commercial poultry is routinely vaccinated with inactivated or live vaccines. Available ND vaccines are produced in specific pathogen free chicken embryonated eggs, whose supply is expensive and imported from Europe. The development and execution of the present project, funded by the Canadian International Development Research Center and presently in its initial phase, aims towards the implementation at the National Veterinary Institute (NVI), Ethiopia, of a technological platform for the production of veterinary vaccines based on the development of recombinant non-replicating adenoviral vectors, using the human adenovirus serotype 5 (Ad5). The ND adenovirus vaccine proposed, expressing protective antigens from ND virus (NDV), will provide an efficient and cost-effective system to address the limitations associated with the current vaccines such as efficacy and virus shedding in flocks of vaccinated birds. The key success factor of the project relies on the development of a robust and cost-effective production platform using serum-free suspension HEK293 adapted cells expressing maximized rAd5 product yields. This will be achieved by augmenting the production cell mass and the cellular productivity beyond cell densities of 6 million cells per mL. Critical parameters and operating conditions impacting the yield and quality of the Ad vaccine will be identified and elevated in a rational process operating strategy that will lead to high-cell density productive infection in bioreactors. Process development and scale-up will be followed by a downstream processing, evaluation of immunogenicity, formulation and stability assays, and protective capacity assessments after viral challenge in the target animals. Recombinant adenoviruses have been generated carrying the NDV coding sequences for the fusion (F) or the hemagglutinin-neuraminidase (HN) proteins, and also for co-expression of both genes in a bicistronic construction. Phylogenetic analyses were primarily conducted to ensure a high degree of sequence identity of the genes cloned with the genoype of locally circulating strains. Recombinant protein expression was also designed and analyzed under different regulatory sequences aiming for selection of the most immunogenic variant. Following the initial phases of project execution, the subsequent steps will define the final parameters for high-cell density infection and rAd5 production for the animal studies. Here we discuss in detail the completed and upcoming project steps as well as the different strategies implemented to achieve the set objectives supporting the main goal of sustainable technology transfer and capacity building of the NVI in Ethiopia

Development of a cultivation process for the enhancement of human interferon alpha 2b production in the oleaginous yeast, Yarrowia lipolytica

<p>Abstract</p> <p>Background</p> <p>As an oleaginous yeast, <it>Yarrowia lipolytica </it>is able to assimilate hydrophobic substrates. This led to the isolation of several promoters of key enzymes of this catabolic pathway. Less is known about the behavior of <it>Y. lipolytica </it>in large bioreactors using these substrates. There is therefore a lack of established know-how concerning high cell density culture protocols of this yeast. Consequently, the establishment of suitable induction conditions is required, to maximize recombinant protein production under the control of these promoters.</p> <p>Results</p> <p>Human interferon α2b (huIFN α2b) production in <it>Yarrowia lipolytica </it>was used as a model for the enhancement of recombinant protein production under the control of the oleic acid (OA)-inducible promoter POX2. Cell viability and heterologous protein production were enhanced by exponential glucose feeding, to generate biomass before OA induction. The optimal biomass level before induction was determined (73 g L^-1), and glucose was added with oleic acid during the induction phase. Several oleic acid feeding strategies were assessed. Continuous feeding with OA at a ratio of 0.02 g OA per g dry cell weight increased huIFNα2b production by a factor of 1.88 (425 mg L^-1) and decreased the induction time (by a factor of 2.6, 21 h). huIFN α2b degradation by an aspartic protease secreted by <it>Y. lipolytica </it>was prevented by adding pepstatin (10 μM), leading to produce a 19-fold more active huIFN α2b (26.2 × 10⁷IU mg^-1).</p> <p>Conclusion</p> <p><it>Y. lipolytica</it>, a generally regarded as safe (GRAS) microorganism is one of the most promising non conventional yeasts for the production of biologically active therapeutic proteins under the control of hydrophobic substrate-inducible promoter.</p