The EB66® cell line for yellow fever vaccine production at high cell concentrations

The global threat of the emerging yellow fever disease can be effectively countered by vaccination. First vaccines against yellow fever have been developed in embryonated chicken eggs in the 1930s and this production platform remained almost unchanged until today. However, recent outbreaks revealed vaccine supply shortages due to limiting options to ramp up production. Here, we present a cell culture-based process using EB66® cells for production of a live, attenuated yellow fever vaccine (YFV; WHO 17D-213/77 strain). The duck embryo-derived EB66® suspension cell line showed good growth performance in batch mode achieving up to 1.8 × 107 cells/mL and doubling times of less than 17 h in shake flasks in the chemically-defined CDM4Avian medium at 37°C. The seed virus material was adapted by five serial passages to the cell substrate, which resulted in an 8-fold increase in virus titer to 1.3 × 108 PFU/mL (infectious virions per mL). Changes in process temperature and cell disruption to facilitate virus release did not improve final virus titers. In a next step, the process was transferred into benchtop bioreactors equipped with an alternating tangential flow filtration unit (ATF2) operating at a working volume of 700 mL. An on-line conductivity probe was implemented, which enabled cell growth monitoring in real-time. This setup allowed to achieve high cell densities of up to 9.5 × 107 cells/mL resulting in a further increase of YFV titers up to 7.3 × 108 PFU/mL. Based on an input of 4.7 log infectious units per dose, raw virus material equivalent to 10 Mio vaccine doses was produced in less than two weeks operation time. Taken together, EB66® suspension cells can grow to very high cell densities in perfusion systems. Present process intensification clearly demonstrated the potential to produce millions of YFV vaccine doses from small scale cultures in a controllable and scalable manner

The EB66® cell line for yellow fever vaccine production at high cell concentrations

The global threat of the emerging yellow fever disease can be effectively countered by vaccination. First vaccines against yellow fever have been developed in embryonated chicken eggs in the 1930s and this production platform remained almost unchanged until today. However, recent outbreaks revealed vaccine supply shortages due to limiting options to ramp up production. Here, we present a cell culture-based process using EB66® cells for production of a live, attenuated yellow fever vaccine (YFV; WHO 17D-213/77 strain). The duck embryo-derived EB66® suspension cell line showed good growth performance in batch mode achieving up to 1.8 × 107 cells/mL and doubling times of less than 17 h in shake flasks in the chemically-defined CDM4Avian medium at 37°C. The seed virus material was adapted by five serial passages to the cell substrate, which resulted in an 8-fold increase in virus titer to 1.3 × 108 PFU/mL (infectious virions per mL). Changes in process temperature and cell disruption to facilitate virus release did not improve final virus titers. In a next step, the process was transferred into benchtop bioreactors equipped with an alternating tangential flow filtration unit (ATF2) operating at a working volume of 700 mL. An on-line conductivity probe was implemented, which enabled cell growth monitoring in real-time. This setup allowed to achieve high cell densities of up to 9.5 × 107 cells/mL resulting in a further increase of YFV titers up to 7.3 × 108 PFU/mL. Based on an input of 4.7 log infectious units per dose, raw virus material equivalent to 10 Mio vaccine doses was produced in less than two weeks operation time. Taken together, EB66® suspension cells can grow to very high cell densities in perfusion systems. Present process intensification clearly demonstrated the potential to produce millions of YFV vaccine doses from small scale cultures in a controllable and scalable manner

Flavivirus production in perfusion processes using the EB66® cell line

The outbreak of mosquito-borne yellow fever virus (YFV) in Angola 2016 rapidly spread to urban regions and other countries. Vaccination campaigns were subsequently intensified, but the increased vaccine demand led to depleted stockpiles. Current yellow fever vaccine manufacturing processes rely on embryonated chicken eggs, which are strongly limited with respect to flexible capacity increase in emergencies. The global vaccine demand is estimated by the WHO to 1.38 billion doses needed to eliminate epidemics. Thus, an urgent need for an improved production platform is needed, ideally transferable to new vaccine developments against emerging flaviviruses, such as Zika virus. Here we present a cell culture-based YFV 17D and Zika virus (ZIKV) production process using the EB66® cell line. The avian EB66® suspension cell line grew fast and stable in chemically defined medium to cell concentrations of 1.8 × 107 cells/mL in shake flasks and batch mode. Seed virus was prepared from Vero-derived YFV and ZIKV material over five passages in EB66® cells. Thereby, infectious virus titers successfully increased by one log unit and maximum titers of 1.4×108 PFU/mL (infectious virions per mL) and 8.0×107 PFU/mL were obtained two days post infection for YFV and ZIKV, respectively. The process was intensified using perfusion bioreactors to increase cell concentrations. Therefore, EB66® cells were cultivated in 1 L benchtop bioreactors equipped with an alternating tangential flow filtration (ATF2) perfusion unit. Perfusion rates were adjusted to maintain glutamine concentrations above 1 mM and cells grew up to 9.5×107 cells/mL. A maximum YFV titer of 7.3 × 108 PFU/mL was achieved. The cell-specific virus yield (CSVY) was 8 PFU/cell, similar to shake flask experiments. For ZIKV production, another approach aimed at the use of on-line capacitance sensors to control cell-specific perfusion rates (CSPRs) based on cell concentrations. This automated system was set to a CSPR of 0.017 and 0.034 nL/cell/day leading to maximum cell concentrations of 8.9×107 cells/mL and 1.6×108 cells/mL. ZIKV titers peaked after three to four days post infection with 2.6×109 PFU/mL and 1.0×1010 PFU/mL, respectively. CSVYs increased from 5 PFU/cell (shake flask experiments) to 30 PFU/cell and even above 64 PFU/cell in this set-up. The increased CSPR resulted in an improved volumetric productivity by factor three compared to the lower CSPR. Further process intensification was achieved by direct cell inoculation to the ZIKV production bioreactor. A 15 mL cryo-bag was thawed with 8.5×108 cells and cell viabilities of 90% after inoculation quickly increased over the cultivation period. Taken together, EB66® suspension cells can be grown to concentrations exceeding 1.5×108 cells/mL in perfusion bioreactors, and cells are highly permissive for YFV and ZIKV. YFV production using perfusion systems generated virus material equivalent to 10 Mio vaccine doses (4.7 log infectious units per dose) in less than two weeks operation time. With the use of on-line sensors to adjust CSPRs meeting cellular nutrient demands, ZIKV titers exceeding 1.0×1010 PFU/mL were obtained for the first time. Direct cryo-bag inoculation shortened the seed train phase, and virus production was initiated with full flexibility. This is a powerful demonstration on how next generation flavivirus vaccine production can be realized

TRIM32 Regulates Skeletal Muscle Stem Cell Differentiation and Is Necessary for Normal Adult Muscle Regeneration

Limb girdle muscular dystrophy type 2H (LGMD2H) is an inherited autosomal recessive disease of skeletal muscle caused by a mutation in the TRIM32 gene. Currently its pathogenesis is entirely unclear. Typically the regeneration process of adult skeletal muscle during growth or following injury is controlled by a tissue specific stem cell population termed satellite cells. Given that TRIM32 regulates the fate of mammalian neural progenitor cells through controlling their differentiation, we asked whether TRIM32 could also be essential for the regulation of myogenic stem cells. Here we demonstrate for the first time that TRIM32 is expressed in the skeletal muscle stem cell lineage of adult mice, and that in the absence of TRIM32, myogenic differentiation is disrupted. Moreover, we show that the ubiquitin ligase TRIM32 controls this process through the regulation of c-Myc, a similar mechanism to that previously observed in neural progenitors. Importantly we show that loss of TRIM32 function induces a LGMD2H-like phenotype and strongly affects muscle regeneration in vivo. Our studies implicate that the loss of TRIM32 results in dysfunctional muscle stem cells which could contribute to the development of LGMD2H

JAM-A is a novel surface marker for NG2-Glia in the adult mouse brain.

BACKGROUND: Junctional adhesion molecule-A (JAM-A) is an adhesive protein expressed in various cell types. JAM-A localizes to the tight junctions between contacting endothelial and epithelial cells, where it contributes to cell-cell adhesion and to the control of paracellular permeability. RESULTS: So far, the expression pattern of JAM-A has not been described in detail for the different cell types of the adult brain. Here we show that a subset of proliferating cells in the adult mouse brain express JAM-A. We further clarify that these cells belong to the lineage of NG2-glia cells. Although these mitotic NG2-glia cells express JAM-A, the protein never shows a polarized subcellular distribution. Also non-mitotic NG2-glia cells express JAM-A in a non-polarized pattern on their surface. CONCLUSIONS: Our data show that JAM-A is a novel surface marker for NG2-glia cells of the adult brain

Spatial Interplay between PIASy and FIP200 in the Regulation of Signal Transduction and Transcriptional Activity▿

The members of the protein inhibitor of activated STAT (PIAS) family of proteins are implicated in fundamental cellular processes, including transcriptional regulation, either through action as E3 SUMO ligases or through SUMO-independent effects. We report here the identification of FIP200 (focal adhesion kinase family-interacting protein of 200 kDa) as a new PIASy-interacting protein. We show that the interaction depends on the integrity of the RING finger of PIASy and the carboxy terminus of FIP200. Both in vitro and in vivo sumoylation assays failed to reveal any sumoylation of FIP200, suggesting that FIP200 is not a bona fide SUMO substrate. Immunofluorescence microscopy and subcellular fractionation, either upon forced PIASy expression or in the absence of PIASy, revealed that interaction with PIASy redistributes FIP200 from the cytoplasm to the nucleus, correlating with abrogation of FIP200 regulation of TSC/S6K signaling. Conversely, FIP200 enhances the transcriptional activation of the p21 promoter by PIASy whereas PIASy transcription activity is severely reduced upon FIP200 depletion by RNA interference. Chromatin immunoprecipitation analysis demonstrates that endogenous PIASy and FIP200 are corecruited to the p21 promoter. Altogether, these results provide the first evidence for the existence of a close—spatially controlled—mode of regulation of FIP200 and PIASy nucleocytoplasmic functions

Mass spectrometry imaging differentiates chromophobe renal cell carcinoma and renal oncocytoma with high accuracy

Background: While subtyping of the majority of malignant chromophobe renal cell carcinoma (cRCC) and benign renal oncocytoma (rO) is possible on morphology alone, additional histochemical, immunohistochemical or molecular investigations are required in a subset of cases. As currently used histochemical and immunohistological stains as well as genetic aberrations show considerable overlap in both tumors, additional techniques are required for differential diagnostics. Mass spectrometry imaging (MSI) combining the detection of multiple peptides with information about their localization in tissue may be a suitable technology to overcome this diagnostic challenge. Patients and Methods: Formalin-fixed paraffin embedded (FFPE) tissue specimens from cRCC (n=71) and rO (n=64) were analyzed by MSI. Data were classified by linear discriminant analysis (LDA), classification and regression trees (CART), k-nearest neighbors (KNN), support vector machine (SVM), and random forest (RF) algorithm with internal cross validation and visualized by t-distributed stochastic neighbor embedding (t-SNE). Most important variables for classification were identified and the classification algorithm was optimized. Results: Applying different machine learning algorithms on all m/z peaks, classification accuracy between cRCC and rO was 85%, 82%, 84%, 77% and 64% for RF, SVM, KNN, CART and LDA. Under the assumption that a reduction of m/z peaks would lead to improved classification accuracy, m/z peaks were ranked based on their variable importance. Reduction to six most important m/z peaks resulted in improved accuracy of 89%, 85%, 85% and 85% for RF, SVM, KNN, and LDA and remained at the level of 77% for CART. t-SNE showed clear separation of cRCC and rO after algorithm improvement. Conclusion: In summary, we acquired MSI data on FFPE tissue specimens of cRCC and rO, performed classification and detected most relevant biomarkers for the differential diagnosis of both diseases. MSI data might be a useful adjunct method in the differential diagnosis of cRCC and rO

TERT promoter mutations are frequent in cutaneous basal cell carcinoma and squamous cell carcinoma.

Activating mutations in the TERT promoter were recently identified in up to 71% of cutaneous melanoma. Subsequent studies found TERT promoter mutations in a wide array of other major human cancers. TERT promoter mutations lead to increased expression of telomerase, which maintains telomere length and genomic stability, thereby allowing cancer cells to continuously divide, avoiding senescence or apoptosis. TERT promoter mutations in cutaneous melanoma often show UV-signatures. Non-melanoma skin cancer, including basal cell carcinoma and squamous cell carcinoma, are very frequent malignancies in individuals of European descent. We investigated the presence of TERT promoter mutations in 32 basal cell carcinomas and 34 cutaneous squamous cell carcinomas using conventional Sanger sequencing. TERT promoter mutations were identified in 18 (56%) basal cell carcinomas and in 17 (50%) cutaneous squamous cell carcinomas. The recurrent mutations identified in our cohort were identical to those previously described in cutaneous melanoma, and showed a UV-signature (C>T or CC>TT) in line with a causative role for UV exposure in these common cutaneous malignancies. Our study shows that TERT promoter mutations with UV-signatures are frequent in non-melanoma skin cancer, being present in around 50% of basal and squamous cell carcinomas and suggests that increased expression of telomerase plays an important role in the pathogenesis of these tumors