301. Efficient Delivery of Nuclear and Cytoplasmic Proteins Fused to HIV-1 Gag Polypeptide By Means of Virus-Like Particles

The expression of the retroviral polypeptide Gag can induce the formation of virus-like particles (VLP). Upon expression, the polypeptide is targeted to the cell membrane and incorporated in the VLP during membrane budding. Chimeric proteins consisting of Gag polypeptide fused to different proteins were engineered and the VLP produced were used as vehicles for protein delivery. One major advantage of this approach is the low mutation risk due to the absence of DNA transfer and genomic integration. In this study, the C-Terminus of Gag from HIV-1 was fused to the green fluorescent protein (GFP), a chimeric transactivator (cTA) and a reprogamming factor (KLF4). VLP were produced by transfection using a stable cell line (293SF-pacLV) that expresses VSVg and Gag-pol. Analysis of the supernatants from producing cells by western blot confirmed the presence of Gag-GFP, -cTA and -KLF4. Confocal microscopy showed that the vast majority of the cells (> 90%) treated with VLP-GFP/polybrene complexes was successfully transduced. The cells also displayed a GFP signal almost exclusively localized inside the cytoplasm. Additional VSVg expression during the production facilitated the endosomal escape of VLP-GFP in transduced cells. The insertion of a nuclear localisation signal (NLS) shifted the localization of the GFP to the cell nucleus demonstrating that a nuclear protein could be successfully delivered by VLP. The experiment was thus repeated using two transcription factors. Lentiviral vectors were used to make two stable pools of HEK293 cells each containing a specific GFP reporter cassette. The GFP gene was regulated either by the CR5 promoter (specifically activated by the cTA) or by a minimal promoter fused to KLF4 transcription response elements (TRE). Transduction of the CR5-GFP pool with VLP-cTA/polybrene complexes showed a powerful activation of the reporter (365-fold compared to the negative control) as measured by flow cell cytometry two days post-transduction. Surprisingly, no activation was observed in the TRE-GFP pool three days after transduction by VLP-KLF4/polybrene complexes. Evidence obtained by transfection suggested that the Gag fusion inhibits KLF4 activity. To augment activity, the activation domain of VP16 was fused to KLF4. Transfection of a plasmid encoding Gag-VP16KLF4 strongly activated the reporter by a factor of 126-fold (6-fold higher than wild type KLF4). The ability of VLP produced with Gag-VP16KLF4 to activate transcription is currently under investigation. In summary, VLP based on HIV-1 Gag can deliver nuclear and cytoplasmic proteins directly into cells with a low mutation risk. Therefore, our platform for VLP production could be useful for several applications including cell reprogramming and genome editing oriented toward cell therapies of diseases like Duchenne muscular dystrophy

Effects of hypoxia on benthic macrofauna and bioturbation in the Estuary and Gulf of St. Lawrence, Canada

The bottom water in the 4300 m deep Lower St. Lawrence Estuary (LSLE) is persistently hypoxic in contrast to the normoxic bottom waters in the Gulf of St. Lawrence (GSL). We photographed the seabed at 11 stations in the Estuary and Gulf of St. Lawrence (EGSL) during the summers 2006 and 2007 and analysed the images to identify bioturbation traces (lebensspuren) and benthic macrofauna. The objective was to identify the environmental variables that influence the density and diversity of benthic macrofauna and bioturbation traces, and the differences that exist among regions with high, medium and low oxygen levels in the bottom water. The bottom water oxygen concentration is the variable that best explains the densities of total-traces as well as surface-traces. However, the density of these traces was higher in hypoxic regions than in well-oxygenated regions. The higher density of traces in the hypoxic region of the LSLE is mainly due to the activities of the surface deposit feeder Ophiura sp., which occurs in large numbers in this region. Possible explanations explored are stress behaviour of the organisms in response to hypoxia and different benthic macrofauna community structures between the hypoxic regions of the LSLE and the normoxic regions of the GSL. In the former, surface deposit feeders and low-oxygen tolerant species dominate over suspension feeders and low-oxygen intolerant species

Characteristics of rVSV-ZEBOV production kinetics in HEK293 and Vero cells

The vesicular stomatitis virus (VSV) can be used as an effective vaccine platform, inducing both cellular and humoral immunity. Because VSV infections of humans are mostly asymptomatic, recombinant VSV (rVSV) can be used as a platform to safely deliver and express foreign antigens. This research study focusses on cell culture production of an rVSV expressing the Ebola virus glycoprotein on its surface (rVSV-ZEBOV). This virus has been demonstrated to be safe to administer to humans. In addition, recent results of a human phase III clinical trial showed that this vaccine can efficiently protect against Ebola virus infection. However, limited data is available in the literature about the growth characteristics of this virus during the production process. In our study, we investigated the influence of multiplicity of infection (MOI), time of infection (TOI), time of harvest (TOH), media components and temperature on the viral titer (TCID50/mL, ddPCR) of rVSV-ZEBOV produced from cell culture. Results are compared between the standard production in the Vero cell line and in a suspension-adapted HEK293-based cell line without serum

555. Development of a Post-Exposure Treatment for Ebola Virus Infections Based on AAV Vectors and Zmapp Antibody Cocktail

The recent Ebola outbreak in West Africa has been the deadliest in the history. To prevent future recurrence of such outbreak, better treatments and effective vaccines against Ebola virus are desirable. Among such promising treatments, the Zmapp cocktail containing neutralizing antibodies (13C6, 2G4 and 4G7) has successfully treated some patients. However, the feasibility of using it on large populations especially in developing countries is questionable. To address this potential issue, we propose to employ recombinant vectors derived from adeno-associated virus (rAAV). There are several advantages of using rAAV: because of 1) their safety profile; 2) only one injection (or a few) would be required; 3) the high stability of lyophilized rAAVs at ambient temperature and; 4) the panel of available serotypes. Because of these interesting features, we are currently developing a treatment based on three rAAVs to deliver the genes for the Zmapp cocktail of antibodies. We have already produced at small scale a rAAV expressing the 2G4 antibody. The DNA sequences for the heavy chain and light chains were codon-optimized for better expression in humans and were designed to be expressed from the same gene. A strong promoter (CAG) resistant to silencing in vivo was chosen to drive gene expression of the antibody. The rAAV were produced by transfection using our patented cGMP compatible HEK293 cell line. The production was performed in suspension culture in the absence of serum. Secretion of 2G4 antibody by rAAV transduced cells (HEK293 and CHO cells) was confirmed. The results demonstrated that rAAV-CAG-2G4 was functional and allowed for the correct assembly of the heavy and light chains of 2G4. Purification of 200 mL of rAAV-CAG-2G4 production was performed by ultracentrifugation on an iodixanol density-step gradient. Two other rAAVs coding 13C6 and 4G7 antibodies are in the processed of being constructed and produced in a similar manner. We are also in the process of comparing the efficacy of two serotypes of AAV (9 and DJ) in mice by intranasal delivery. Using the best serotype, the rAAVs will be produced and purified from a starting suspension culture of 20 L. Their efficacy for treating Ebola infections will then be evaluated in a mouse model infected by the virus

Characterization of HA and NA-containing VLPs produced in suspension cultures of HEK 293 cells

Virus like particles (VLPs) can be formulated into promising vaccines to prevent influenza infection. In addition of having a structure and composition that mimic the wild type virus, VLPs are safe since they are devoid of viral genes and consequently are not infectious. One approach to scale up the manufacturing of VLPs is to produce them in a serum-free suspension culture using a stable mammalian cell line. Importantly, with VLPs synthetized by mammalian cells, the post-translational modifications of the surface antigens should be similar to the wild type virus, and therefore should trigger a potent and specific immune response for the pathogen. As a proof of concept, we first established a cell line that was stably expressing hemagglutinin (HA) and neuraminidase (NA) proteins of influenza (subtype H1N1) using our patented cGMP human embryonic kidney (HEK293) cell line (clone 293SF-3F6). Transcription of the genes for these two glycoproteins was regulated by the inducible cumate transcription gene-switch. Next, to establish our capability to produce VLPs, we compared the formation of VLPs using these cells after forced expression of two scaffold proteins: Gag from the human immunodeficiency virus and M1 protein from influenza A (H1N1). In addition, monitoring of the VLPs was facilitated by fusing the Gag protein to the green fluorescent protein (GFP). VLP production was therefore initiated by transient transfection of plasmid encoding Gag or M1 and by addition of cumate to the culture medium. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion. The presence of HA an NA within the VLP fraction was demonstrated by western blot and quantified by dot blot. Interestingly, VLPs were produced more efficiently in the presence of Gag, indicating that Gag is a better scaffolding protein than M1 in this context. Under the electron microscope, the Gag-VLPs appeared as vesicles of 100 to 150 nm of diameter, containing a denser internal proteinous ring, which is a typical morphology for VLPs produced through Gag expression. The production of Gag-VLPs was also validated in a 3-L stirred tank bioreactor in serum-free medium. The immunogenicity of the VLPs is currently under investigation in a murine model for influenza. In conclusion, VLPs containing HA and NA can be manufactured in serum free suspension culture of HEK293 cells through forced expression of Gag. The efficacy of these VLPs for vaccination remains to be demonstrated

Suspension Vero cell culture technology for high titer production of viral vaccines

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The continuous Vero cell line has been commercially used, after propagation on microcarriers, for the production of rabies, polio, enterovirus 71, hantaan, more recent COVID19 and other vaccines. Vero cell culture technologies were also explored for productions of many more viral vaccines over the last two decades. The growth of Vero cells is anchorage-dependent, and cells need to be dissociated enzymatically or mechanically for the process of subcultivation. This process is labor intensive and complicated in process scale-up. Adaptation of Vero cells to grow in suspension will significantly simplify scale-up and manufacturing processes. Development of advanced suspension Vero culture technology to improve product titer will further reduce the cost of goods. We previously reported a successful adaptation of adherent Vero cells originated from ATCC CCL-81 to grow in suspension in serum-free and animal component-free media developed in-house. The suspension adapted cells were found to retain their genetic stability and to be non-tumorigenic. Present work continues the development and optimization of cell culture process and feeding strategy to improve the growth of suspension Vero cell and the production of vesicular stomatitis virus (VSV) and herpes simplex virus-1 (HSV-1). Data from this study showed the suspension adapted Vero cells retained similar VSV productivity to that obtained in adherent culture; volumetric productivity of VSV increased with the increasing cell density at infection in batch culture. However, the maximum cell density in batch culture was about 2.5x106 cells/mL, and was not improved significantly despite tremendous effort dedicated to improve culture conditions such as supplementing various nutrients in batch culture. As a result, perfusion culture was employed as an approach to increase cell density in the culture, which in turn increased the VSV productivity up to one log, at 1.1x1010 TCID50/mL when the culture infected at 7x106 cells/mL. High titer production of HSV-1 in the Vero culture is more challenging. The virus productivity is not only limited by the maximum cell density in batch culture, but also reduced by inhibitory metabolites secreted in the culture even at low cell density such as 1x106 cells/mL. Media replacement before virus infection is essential to achieve a high HSV-1 yield. As such, perfusion culture was a preferred mode for high titer production of HSV-1, which improved the HSV-1 titer also by up to one log to 1.8 x109 TCID50/ in a culture infected at 5x106 cells/mL when comparing to a control shake flask culture infected at 1x106 cells/mL. Experimental data also demonstrated that perfusion Vero culture was robust and reproducible. This study demonstrates that batch or perfusion suspension Vero culture is a much simplified process than current adherent culture technology for manufacturing of viral vaccines, and offers great potentials in reducing the cost of goods. The suspension Vero culture developed in our institute has generated tremendous interests from industry and academia, and are being tested by many different organizations

Fluorescent labeling in semi-solid medium for selection of mammalian cells secreting high-levels of recombinant proteins

<p>Abstract</p> <p>Background</p> <p>Despite the powerful impact in recent years of gene expression markers like the green fluorescent protein (GFP) to link the expression of recombinant protein for selection of high producers, there is a strong incentive to develop rapid and efficient methods for isolating mammalian cell clones secreting high levels of marker-free recombinant proteins. Recently, a method combining cell colony growth in methylcellulose-based medium with detection by a fluorescently labeled secondary antibody or antigen has shown promise for the selection of Chinese Hamster Ovary (CHO) cell lines secreting recombinant antibodies. Here we report an extension of this method referred to as fluorescent labeling in semi-solid medium (FLSSM) to detect recombinant proteins significantly smaller than antibodies, such as IGF-E5, a 25 kDa insulin-like growth factor derivative.</p> <p>Results</p> <p>CHO cell clones, expressing 300 μg/ml IGF-E5 in batch culture, were isolated more easily and quickly compared to the classic limiting dilution method. The intensity of the detected fluorescent signal was found to be proportional to the amount of IGF-E5 secreted, thus allowing the highest producers in the population to be identified and picked. CHO clones producing up to 9.5 μg/ml of Tissue-Plasminogen Activator (tPA, 67 kDa) were also generated using FLSSM. In addition, IGF-E5 high-producers were isolated from 293SF transfectants, showing that cell selection in semi-solid medium is not limited to CHO and lymphoid cells. The best positive clones were collected with a micromanipulator as well as with an automated colony picker, thus demonstrating the method's high throughput potential.</p> <p>Conclusion</p> <p>FLSSM allows rapid visualization of the high secretors from transfected pools prior to picking, thus eliminating the tedious task of screening a high number of cell isolates. Because of its rapidity and its simplicity, FLSSM is a versatile method for the screening of high producers for research and industry.</p

Development of suspension adapted Vero cell culture process technology for production of viral vaccines

Abstract Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost. Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house. The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40–44 h. Much higher cell density (8 × 10 6 cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process. Both adherent and suspension Vero cells from various stages were tested for their authenticity using short tandem repeat analysis. Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability. Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic. The viral productivity of suspension Vero cells was evaluated by using vesicular stomatitis virus (VSV) as a model. The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture. In addition, the suspension culture could be infected at higher cell densities, thus improving the volumetric virus productivity. More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 × 10 6 cells/mL

Scalable lentiviral vector production using stable producer cell lines in perfusion mode

Lentiviral vectors (LVs) are becoming an important tool in gene and cell therapy and are being utilized in several clinical studies for rare and more frequent genetic and acquired diseases, as well as in cancer therapies. However, two major challenges need to be overcome in order to generate enough material to treat patients: First, current production platforms result in low titers (stable producer cell lines from adherent cell lines) or are not amenable to large scale production (LV produced by transfection). Next, LVs are known to have a low temperature stability. To address these two challenges, the National Research Council Canada has developed packaging cell lines and stable producer cell lines for the production of LVs which can grow in suspension in serum-free media and produce LV in the 106 TU/ml range without optimization. Furthermore, productions are performed in perfusion mode in order to operate at high cell densities and address the low LV stability. Please click Additional Files below to see the full abstrac

Development of suspensions adapted Vero cell culture process for production of viruses

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The continuous Vero cell line has been commercially used, after propagation on microcarriers, for the production of rabies, polio, enterovirus 71 and hantaan virus vaccines. Vero cell culture technologies are also explored for productions of many more viral vaccines over the last two decades. The growth of Vero cells is anchorage-dependent, and cells need to be dissociated enzymatically or mechanically for the process of subcultivation. This process is labor intensive and complicated in process scale-up. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly. Here we report on the adaptation of adherent Vero cells to grow in suspension using a serum-free and animal component-free medium developed in-house. The maximum cell density and cell doubling time of the suspension adapted Vero cells in batch culture grown in the in-house developed medium were similar to or better than what was observed for the adherent Vero cells grown in commercial media. The growth of suspension adapted Vero culture was successfully scaled up to 3 L bioreactor. The Vero cells from various stages (both adherent and adapted) were tested for their authenticity using a Short Tandem Repeat (STR) analysis. The testing result indicates that all Vero cell samples have 100% concordance with the Vero DNA control sample, indicating the suspension adapted cells maintained their genetic stability. Productions of vesicular stomatitis virus (VSV) and influenza virus in adherent culture and suspension adapted culture were compared, showing the suspension adapted Vero cell retained similar viral productivity. The volumetric productivity of VSV in the suspension culture was even higher, and was further increased by almost 200 times when culture was infected at higher cell density and with medium replacement before the virus infection. In contrast, the VSV production decreased when the adherent culture was infected at higher cell density. Additional process development revealed that the maximum cell density in batch culture was doubled, reaching 6x106 cells/mL, when the culture medium was replaced during the process of batch culture, which indicates potential for further increases in product titer