Production, purification, and characterization of recombinant rabies virus glycoprotein expressed in PichiaPink™ yeast.

The commonly used host for industrial production of recombinant proteins Pichia pastoris, has been used in this work to produce the rabies virus glycoprotein (RABV-G). To allow a constitutive expression and the secretion of the expressed recombinant RABV-G, the PichiaPink™ commercialized expression vectors were modified to contain the constitutive GAP promoter and the α secretion signal sequences. Recombinant PichiaPink™ strains co-expressing the RABV-G and the protein chaperone PDI, have been then generated and screened for the best producer clone. The influence of seven carbon sources on the expression of the RABV-G, has been studied under different culture conditions in shake flask culture. An incubation temperature of 30°C under an agitation rate of 250 rpm in a filling volume of 10:1 flask/culture volume ratio were the optimal conditions for the RABV-G production in shake flask for all screened carbon sources. A bioreactor Fed batch culture has been then carried using glycerol and glucose as they were good carbon sources for cell growth and RABV-G production in shake flask scale. Cells were grown on glycerol during the batch phase then fed with glycerol or glucose defined solutions, a final RABV-G concentration of 2.7 µg/l was obtained with a specific product yield (YP/X) of 0.032 and 0.06 µg/g(DCW) respectively. The use of semi-defined feeding solution enhanced the production and the YP/X to 12.9 µg/l and 0.135 µg/g(DCW) respectively. However, the high cell density favored by these carbon sources resulted in oxygen limitation which influenced the glycosylation pattern of the secreted RABV-G. Alternatively, the use of sucrose as substrate for RABV-G production in large scale culture, resulted in less biomass production and a YP/X of 0.310 µg/g(DCW) was obtained. A cation exchange chromatography was then used for RABV-G purification as one step method. The purified protein was correctly folded and glycosylated and able to adopt trimeric conformation. The knowledges gained through this work offer a valuable insight into the bioprocess design of RABV-G production in Pichia pastoris to obtain a correctly folded protein which can be used during an immunization proposal for subunit Rabies vaccine development.

Development of an efficient veterinary rabies vaccine production process in the avian suspension cell line AGE1.CR.pIX.

BACKGROUND: Mass vaccination of dogs as important rabies reservoir is proposed to most effectively reduce and eliminate rabies also in humans. However, a minimum coverage of 70% needs to be achieved for control of the disease in zoonotic regions. In numerous developing countries, dog vaccination rate is still dangerously low because of economic constraints and due to a high turnover in dog populations. Improved vaccine production processes may help to alleviate cost and supply limitations. In this work, we studied and optimized the replication and vaccine potency of PV rabies virus strain in the muscovy-duck derived AGE1.CR and AGE1.CR.pIX suspension cell lines. RESULTS: The BHK-21-adapted PV rabies virus strain replicated efficiently in the avian cell lines without requirement for prior passaging. CR.pIX was previously shown to augment heat shock responses and supported slightly higher infectious titers compared to the parental CR cell line. Both cell lines allowed replication of rabies virus also in absence of recombinant IGF, the only complex component of the chemically defined medium that was developed for the two cell lines. After scale-up from optimization experiments in shake flask to production in 7-l bioreactors peak virus titers of 2.4 × 108 FFU/ml were obtained. The potency of inactivated rabies virus harvest according to the NIH test was 3.5 IU/ml. Perfusion with the chemically defined medium during the virus replication phase improved the potency of the vaccine twofold, and increased the number of doses 9.6 fold. CONCLUSION: This study demonstrates that a rabies vaccine for animal vaccination can be produced efficiently in the AGE1.CR.pIX suspension cell line in a scalable process in chemically defined medium.

Production and characterization of a fusion form of hepatitis E virus tORF2 capsid protein in Escherichia coli.

Hepatitis E virus (HEV) is a nonenveloped virus causing an emerging zoonotic disease posing a severe threat to the public health in the world, especially to pregnant women. In this study, a truncated form (aa 368-606) of the open reading frame 2 of the capsid protein (tORF2-HEV), a major structural protein of HEV, was expressed in Escherichia coli. This work characterizes for the first time, the fused Glutathione-S-Transferase-tagged tORF2 (GST-tORF2) and tORF2-HEV forms in E. coli. The fusion protein was purified by affinity chromatography with a purity higher than 90% and to yield about 27% after thrombin digestion. The purified GST-tORF2 protein was then characterized by western blot, using anti-GST antibodies, and CD spectroscopy. The GST-tORF2 and tORF2-HEV proteins were shown to be efficient to develop an ELISA test to detect anti-HEV IgG in mice sera immunized with a recombinant full length ORF2 protein. Sera showed a significant increase of the absorbance signal at 450 nm, in plate wells coated with a quantity of 0.5, 1 and 2 µg of proteins. ELISA plates coated with the purified GST-tORF2 and tORF2-HEV showed similar response when compared to the HEV ELISA where total insect cell lysate, infected with the recombinant baculovirus expressing full ORF2, was used as positive control.

Post-traumatic pulmonary embolism: incidence, physiopathology, risk factors of early occurrence, and impact outcome. A narrative review.

BACKGROUND: Venous thromboembolism (VTE) is a well-established complication of trauma. So far, the factors that are related to early post-traumatic pulmonary embolism (PE) occurrence have been given little attention. AIMS: We have conducted this literature review in order to analyze the incidence and the physiopathology of post-traumatic PE among intensive care unit (ICU) trauma patients, analyze the incidence of early post-traumatic PE, and elucidate risk factors associated with post-traumatic PE. Moreover, we aim to study the impact/outcome of post-traumatic PE in the ICU. METHODS: We used the PubMed and EMBASE databases and entered the following key words in MeSH research: Deep vein thrombosis (DVT), Post-traumatic Pulmonary embolism, Early pulmonary-embolism, risk factors, and Prognosis. RESULTS: The incidence of PE among trauma patients varies considerably, ranging from 0.35% to 24%. The incidence of early post-traumatic PE varies widely from 10 to 42%. After a traumatic injury, many factors have been found to be responsible for the formation of DVT and PE. In addition to the risk factors of hypercoagulability described by Virchow in his original triad, inflammation acting via endothelial damage may be considered as a fourth factor. The literature review showed that lower limb fractures and age are the most frequent factors associated with PE (particularly in early PE). The heterogeneity among studies limits reliable conclusions regarding the true risk factors for the timing of the occurrence of post-traumatic PE. Fatality from pulmonary embolism (PE) is close to 50% in some series. Moreover, high mortality rates, a high rate of nosocomial infections, and a prolonged stay in an ICU and/or in a hospital were found to be associated with the development of PE. CONCLUSION: Post-traumatic PE is frequent in ICUs. Inflammation acting via endothelial damage may be considered as a fourth factor in addition to the Virchow's triad of risk factors for venous thrombosis. Fractures of the lower extremities, obesity, and age happen to be the most frequent factors associated with PE (in particular early PE). PE development was associated with high rates of mortality, nosocomial infections, and a prolonged stay in an ICU and/or in a hospital. Therefore, prevention is warranted.

Establishing a Robust Manufacturing Platform for Recombinant Veterinary Vaccines: An Adenovirus-Vector Vaccine to Control Newcastle Disease Virus Infections of Poultry in Sub-Saharan Africa.

Developing vaccine technology platforms to respond to pandemic threats or zoonotic diseases is a worldwide high priority. The risk of infectious diseases transmitted from wildlife and domestic animals to humans makes veterinary vaccination and animal health monitoring highly relevant for the deployment of public health global policies in the context of "one world, one health" principles. Sub-Saharan Africa is frequently impacted by outbreaks of poultry diseases such as avian influenza and Newcastle Disease (ND). Here, an adenovirus-vectored vaccine technology platform is proposed for rapid adaptation to ND or other avian viral threats in the region. Ethiopian isolates of the Newcastle Disease virus (NDV) were subjected to sequence and phylogenetic analyses, enabling the construction of antigenically matched vaccine candidates expressing the fusion (F) and hemagglutinin-neuraminidase (HN) proteins. A cost-effective vaccine production process was developed using HEK293 cells in suspension and serum-free medium. Productive infection in bioreactors (1-3L) at 2 × 106 cells/mL resulted in consistent infectious adenoviral vector titers of approximately 5-6 × 108 TCID50/mL (approximately 1011VP/mL) in the harvest lysates. Groups of chickens were twice immunized with 1 × 1010 TCID50 of the vectors, and full protection against a lethal NDV challenge was provided by the vector expressing the F antigen. These results consolidate the basis for a streamlined and scalable-vectored vaccine manufacturing process for deployment in low- and medium-income countries.

Acute Pancreatitis Induced by Diabetic Ketoacidosis with Major Hypertriglyceridemia: Report of Four Cases.

Acute pancreatitis (AP) is a real clinical challenge. Acute pancreatitis remains a common cause of emergency department consultations and a major cause for hospitalization. Gallstones and drinking a lot of alcohol are the most frequent causes of AP. Moreover, AP can be induced by diabetic ketoacidosis (DKA) complicated by hypertriglyceridemia. We report 4 cases of DKA with hypertriglyceridemia complicated by AP in previously undiagnosed diabetes patients. All of our patients presented to the emergency ward with abdominal pain. Their physical exam showed epigastric tenderness. An abdominal CT scan was performed for each patient, showing an AP grade E. Laboratory samples showed high serum glucose levels. They had metabolic acidosis with elevated anion gap. They had high lipasemia and amylasemia. Their lipid panel was disturbed with a high level of cholesterol (from 12.8 mmol/l to 33 mmol/l) and triglyceridemia (from 53 to 133 mmol/l). Our patients were admitted into our ICU where they received fluid resuscitation and intravenous insulin, and their triglycerides rates decreased gradually. Two patients recovered to a good health state, and the two others developed septic shock, requiring the use of large-spectrum antibiotics, and acute kidney injury (AKI) with refractory metabolic acidosis, requiring hemodialysis. Despite the intensive treatment, they developed an unrecoverable multiorgan failure. Through our case series, we aim to highlight the importance of making an early diagnosis, which can be difficult in some situations due to overlapping signs; however, it is crucial for a good recovery. A good understanding of the pathway of hypoinsulinemic states causing hypertriglyceridemia then AP is important because it is the key to best management.

Impacts of ramadan on intensive care unit admission patterns and outcomes.

BACKGROUND: Ramadan fasting is a religious obligation for healthy adult Muslims. Even though those unable to fast are exempt, many individuals refuse this authorization and insist to fast. This may lead to life threatening conditions and an increase in intensive care unit (ICU) demand. AIM: To investigate the impacts of lifestyle changes during Ramadan on ICU admission patterns and outcomes. METHODS: It was a retrospective study carried out in the medical ICU of Farhat HACHED teaching hospital (Sousse, Tunisia). Patients who were admitted to the ICU during Ramadan (G2), Chaaban (G1), and Shawal (G3) over a period of 10 years were included. Demographic, clinical features and outcomes were compared. RESULTS: During the review period, 748 patients were included (G1=257; G2=230 and G3=261). Compared to Chaaban, during Ramadan and Shawal, the percentages of admitted patients with, chronic kidney disease (CKD) (2.3, 3.5 and 7.3%, respectively) and for hypovolemic shock (1.6, 6.1 and 5.0%, respectively) were significantly higher. Furthermore, compared to Chaaban, during Ramadan and Shawal, patients were more likely to have inverted urinary sodium to potassium ratio (28.3, 48.7, 36.8% respectively). There was no significant difference in length-of-stay nor in mortality between the three months' periods. CONCLUSION: While there were no differences in any studied outcomes in patients admitted to ICU before, during or after Ramadan, there was a significant increase in patients presenting with past history of CKD, hypovolemic shock and inverted urinary sodium to potassium ratio.

Purification of rabies virus produced in Vero cells grown in serum free medium.

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. Vaccination against rabies is still the sole efficient way to fight against the disease. Cell culture vaccines are recommended by World Health Organization (WHO) for pre and post exposure prophylaxis; among them Vero cell rabies vaccines which are used worldwide. In this work we studied the purification of inactivated rabies virus produced in Vero cells grown in animal component free conditions, using different methods. Cells were grown in VP-SFM medium in stirred bioreactor, then infected at an MOI of 0.05 with the LP2061 rabies virus strain. Collected harvests were purified by zonal centrifugation, and by chromatography supports, namely the Capto Core 700 and the monolithic CIM-QA column. Generated data were compared in terms of residual DNA level, host cell proteins (HCP) level and the overall recovery yield. Rabies virus purification using the monolithic column resulted in the highest antigen recovery yield, equal to 94%. Capto Core 700 showed a lower yield, about 84%; whereas the purification yield by zonal centrifugation was equal to 60%. In terms of host cell residual DNA removal, zonal centrifugation was the most efficient method; the removal yield was equal to 88.5%; elimination of host cell DNA was slightly lower when using the monolithic CIM-QA (equal to 73%). Whereas Capto Core 700 showed the lowest level (49.2%). Host cell protein removal varied between 92.6% for the monolithic column and 78.6% for the zonal centrifugation. Capto Core 700 eliminated 86.5% of HCP.

Adaptation of Vero cells to suspension growth for rabies virus production in different serum free media.

Vero cells are nowadays widely used in the production of human vaccines. 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 the costs of vaccine manufacturing 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 based on subsequent passages. The best one that relies on cell adaption to IPT-AFM an in-house developed animal component free medium was then chosen for further studies. Besides, as aggregates have been observed, the improvement of IPT-AFM composition and mechanical dissociation were also investigated. In addition to IPT-AFM, three chemically defined media (CD293, Hycell CHO and CD-U5) and two serum free media (293SFMII and SFM4CHO) were tested to set up a serum free culture of the suspension-adapted Vero cells (VeroS) in shake flasks. Cell density levels higher than 2 × 106 cells/mL were obtained in the assessed conditions. The results were comparable to those obtained in spinner culture of adherent Vero cells grown on Cytodex 1 microcarriers. Cell infection with LP-2061 rabies virus strain at an MOI (Multiplicity of Infection) of 0.1 and a cell density of 8 ±â€¯0.5 × 105 cells/mL resulted in a virus titer higher than 107 FFU/mL in all media tested. Nevertheless, the highest titer equal to 5.2 ±â€¯0.5 × 107 FFU/mL, was achieved in IPT-AFM containing a reduced amount of Ca++ and Mg++. Our results demonstrate the suitability of the obtained VeroS cells to produce rabies virus at a high titer, and pave the way to develop VeroS cells bioreactor process for rabies vaccine production.

Investigating the effect of carbon source on rabies virus glycoprotein production in Pichia pastoris by a transcriptomic approach.

Several factors affect protein expression in Pichia pastoris, one among them is the carbon source. In this work, we studied the effect of this factor on the expression level of rabies virus glycoprotein (RABV-G) in two recombinant clones harboring seven copies of the gene of interest. The expression was driven either by the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or the inducible alcohol oxidase1 (AOX1) promoter. Clones were compared in terms of cell physiology and carbon source metabolism. The transcription levels of 16 key genes involved in the central metabolic pathway, the methanol catabolism, and the oxidative stress were investigated in both clones. Cell size, as a parameter reflecting cell physiological changes, was also monitored. Our results showed that when glucose was used as the sole carbon source, large cells were obtained. Transcript levels of the genes of the central metabolic pathway were also upregulated, whereas antioxidative gene transcript levels were low. By contrast, the use of methanol as a carbon source generated small cells and a shift in carbon metabolism toward the dissimilatory pathway by the upregulation of formaldehyde dehydrogenase gene and the downregulation of those of the central metabolic. These observations are in favor of the use of glucose to enhance the expression of RABV-G in P. pastoris.

Expression of rabies virus glycoprotein in the methylotrophic yeast Pichia pastoris.

Rabies is a fatal disease that can be prevented by vaccination. Different approaches were investigated to develop novel human rabies vaccines with improved features compared to the current available vaccines, among them is the use of heterologous gene expression technology. Here, we describe the expression of the surface rabies virus glycoprotein (RABV-G), which is the major antigen responsible for the induction of protective immunity, in Pichia pastoris. Six transformants were selected according to their gene copy number as determined by real time qPCR. Upon induction by methanol, low level of RABV-G was secreted into the culture medium, around 60 ng/mL. To understand the effect of foreign gene dosage on cellular physiology of P. pastoris, transcriptional analysis of key genes involved in unfolded protein response (UPR) and endoplasmic reticulum associated degradation (ERAD) pathway was performed. Results showed that these pathways were highly activated; misfolded RABV-G was degraded in the cytosol via the ERAD mechanism. To study the functionality of the secreted RABV-G, in vitro competitive neutralizing assay was conducted. Data showed the secreted recombinant RABV-G had enabled a reduction of the neutralizing activity of human immune rabies serum, indicating that the secreted recombinant protein had reached its correct conformational form.

pH and Not Cell Morphology Modulate pLIP2 Induction in the Dimorphic Yeast Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica has become an emerging cell factory for recombinant proteins production. Expression vectors involving LIP2 promoter (pLIP2) have been developed and used successfully. However, the relationship between dimorphic transition (i.e., cell morphology) and pLIP2 regulation is still unclear and must be assessed to improve process robustness. This requests to discriminate the effect of cell morphology from that of effectors, such as pH, that trigger the dimorphic transition. This was performed using gene reporter system based on ß-galactosidase activity and DsRed fluorescence, single-cell analysis by flow cytometry, and quantification of gene expression. Our results clearly pointed out that cell morphology has not effect on the regulation of pLIP2. By contrast, pH modification yielded to phenotypic heterogeneity, potentially leading to a lack of robustness of the cell population. Taken altogether, our results demonstrated that, under appropriate environmental conditions (e.g., pH being an important factor), Y. lipolytica could be considered as a robust and reliable host for recombinant protein production.

Molecular optimization of rabies virus glycoprotein expression in Pichia pastoris.

In this work, different approaches were investigated to enhance the expression rabies virus glycoprotein (RABV-G) in the yeast Pichia pastoris; this membrane protein is responsible for the synthesis of rabies neutralizing antibodies. First, the impact of synonymous codon usage bias was examined and an optimized RABV-G gene was synthesized. Nevertheless, data showed that the secretion of the optimized RABV-G gene was not tremendously increased as compared with the non-optimized one. In addition, similar levels of RABV-G were obtained when α-factor mating factor from Saccharomyces cerevisiae or the acid phosphatase PHO1 was used as a secretion signal. Therefore, sequence optimization and secretion signal were not the major bottlenecks for high-level expression of RABV-G in P. pastoris. Unfolded protein response (UPR) was induced in clones containing high copy number of RABV-G expression cassette indicating that folding was the limiting step for RABV-G secretion. To circumvent this limitation, co-overexpression of five factors involved in oxidative protein folding was investigated. Among these factors only PDI1, ERO1 and GPX1 proved their benefit to enhance the expression. The highest expression level of RABV-G reached 1230 ng ml(-1). Competitive neutralizing assay confirmed that the recombinant protein was produced in the correct conformational form in this host.

Large-scale adenovirus and poxvirus-vectored vaccine manufacturing to enable clinical trials.

Efforts to make vaccines against infectious diseases and immunotherapies for cancer have evolved to utilize a variety of heterologous expression systems such as viral vectors. These vectors are often attenuated or engineered to safely deliver genes encoding antigens of different pathogens. Adenovirus and poxvirus vectors are among the viral vectors that are most frequently used to develop prophylactic vaccines against infectious diseases as well as therapeutic cancer vaccines. This mini-review describes the trends and processes in large-scale production of adenovirus and poxvirus vectors to meet the needs of clinical applications. We briefly describe the general principles for the production and purification of adenovirus and poxvirus viral vectors. Currently, adenovirus and poxvirus vector manufacturing methods rely on well-established cell culture technologies. Several improvements have been evaluated to increase the yield and to reduce the overall manufacturing cost, such as cultivation at high cell densities and continuous downstream processing. Additionally, advancements in vector characterization will greatly facilitate the development of novel vectored vaccine candidates.

Development of a vectored vaccine against hepatitis E virus.

Hepatitis E virus is a non-enveloped ssRNA virus [1] that causes human acute hepatitis through primarily fecal and oral transmission [2]. Currently, no commercial hepatitis E (HEV) vaccine is available. In the absence of an appropriate cell culture system for HEV propagation, HEV pseudocapsids (ORF2 protein) have been produced either in Escherichia coli or in insect cells and they have been shown to protect monkeys against virus challenge and to be effective in the prevention of natural HEV infection of humans. In this work, we propose to develop a novel candidate vaccine against hepatitis E infection using adeno-associated virus (AAV) as a vector expressing the gene of the truncated capsid protein of HEV (aa 112-aa 660). rAAV will be produced in Sf9 cells using the baculovirus expression vector system. For this purpose, construction of recombinant baculoviruses was performed and viral stocks of BacRep, BacCap for serotypes 2, 5 and 6 were prepared in Sf9 cells. The recombinant baculovirus coding for the truncated capsid protein of HEV (BacITRHEVORF2) was also constructed, the virus titer was equal to 5.41×10(9) PFU/mL, at the third passage. Transduction of HEK 293 EBNA cells with rAAV was carried out; the production of HEVORF2 was confirmed by Western blot. Optimization of rAAV production in Sf9 cells is currently ongoing.

An animal component free medium that promotes the growth of various animal cell lines for the production of viral vaccines.

IPT-AFM is a proprietary animal component free medium that was developed for rabies virus (strain LP 2061) production in Vero cells. In the present work, we demonstrated the versatility of this medium and its ability to sustain the growth of other cell lines and different virus strains. Here, three models were presented: Vero cells/rabies virus (strain LP 2061), MRC-5 cells/measles virus (strain AIK-C) and BHK-21 cells/rabies virus (strain PV-BHK21). The cell lines were first adapted to grow in IPT-AFM, by progressive reduction of the amount of serum in the culture medium. After their adaptation, BHK-21 cells grew in suspension by forming clumps, whereas MRC-5 cells remained adherent. Then, kinetics of cell growth were studied in agitated cultures for both cell lines. In addition, kinetics of virus replication were investigated.

Detection of cell surface hydrophobicity, biofilm and fimbirae genes in salmonella isolated from tunisian clinical and poultry meat.

BACKGROUND: The aim of this study was to evaluate the ability of 15 serotypes of Salmonella to form biofilm on polystyrene, polyvinyl chloride (PVC) and glass surfaces. . METHODS: Initially slime production was assessed on CRA agar and hydrophobicity of 20 Salmonella strains isolated from poultry and human and two Salmonella enterica serovar Typhimurium references strains was achieved by microbial adhesion to n-hexadecane. In addition, biofilm formation on polystyrene, PVC and glass surfaces was also investigated by using MTT and XTT colorimetric assay. Further, distribution of Salmonella enterotoxin (stn), Salmonella Enteritidis fimbrial (sef) and plasmid encoded fimbrial (pef) genes among tested strains was achieved by PCR. RESULTS: Salmonella strains developed red and white colonies on CRA and they are considered as hydrophilic with affinity values to n-hexadecane ranged between 0.29% and 29.55%. Quantitative biofilm assays showed that bacteria are able to form biofilm on polystyrene with different degrees and 54.54% of strains produce a strong biofilm on glass. In addition, all the strains form only a moderate (54.54%) and weak (40.91%) biofilm on PVC. PCR detection showed that only S. Enteritidis harbour Sef gene, whereas Pef and stn genes were detected in S. Kentucky, S. Amsterdam, S. Hadar, S. Enteritidis and S. Typhimurium. CONCLUSION: Salmonella serotypes are able to form biofilm on hydrophobic and hydrophilic industrial surfaces. Biofilm formation of Salmonella on these surfaces has an increased potential to compromise food safety and potentiate public health risk.

Development of an in situ detachment protocol of Vero cells grown on Cytodex1 microcarriers under animal component-free conditions in stirred bioreactor.

Subcultivation of Vero cells grown in a proprietary animal component-free medium named IPT-AFM, on microcarriers, was studied. TrypLE Select, a non-animal-derived protease, was used as an alternative to trypsin for cell passaging. We first studied the effect of increasing concentrations of TrypLE Select toward cell growth and then studied the inactivation of the protease using either soybean trypsin inhibitor (STI) or the soy hydrolysate Hypep 1510, in six-well plates. Data showed that cell growth was impaired by residual level of TrypLE Select; STI was identified as an efficient agent to neutralize this effect. To restore cell growth and inactivate TrypLE Select, STI should be added to the medium at least at 0.2 g L(-1). Cells were also grown in spinner flask on 2 g L(-1) Cytodex1 in IPT-AFM. In these conditions, the cell detachment yield was equal to 78 ± 8 %. Furthermore, cells exhibited a typical growth profile when using the dislodged cells to seed a new culture. A cell detachment yield of 70 ± 19 % was also achieved when the cells were grown in a 2-L stirred bioreactor in IPT-AFM, on 3 g L(-1) Cytodex1. This protocol can be of great interest to scale-up the process of Vero cells cultivation in IPT-AFM on Cytodex1 from one stirred bioreactor culture to another.

Proteome analysis of virus-host cell interaction: rabies virus replication in Vero cells in two different media.

The use of Vero cells for rabies vaccine production was recommended from the WHO in 2005. A controlled production process is necessary to reduce the risk of contaminants in the product. One step towards this is to turn away from animal-derived components (e.g. serum, trypsin, bovine serum albumin) and face a production process in animal component-free medium. In this study, a proteomic approach was applied, using 2-D differential gel electrophoresis and mass spectrometry to compare rabies virus propagation in Vero cells under different cultivation conditions in microcarrier culture. Protein alterations were investigated for uninfected and infected Vero cells over a time span from 1 to 8 days post-infection in two different types of media (serum-free versus serum-containing media). For mock-infected cells, proteins involved in stress response, redox status, protease activity or glycolysis, and protein components in the endoplasmic reticulum were found to be differentially expressed comparing both cultivation media at all sampling points. For virus-infected cells, additionally changes in protein expression involved in general cell regulation and in calcium homeostasis were identified under both cultivation conditions. The fact that neither of these additional proteins was identified for cells during mock infection, but similar protein expression changes were found for both systems during virus propagation, indicates for a specific response of the Vero cell proteome on rabies virus infection.