Characterization of the avian designer cells AGE1.CR and AGE1.CR.plX considering growth, metabolism and production of influenza virus and Modified Vaccinia Virus Ankara (MVA)

Magdeburg, Univ., Fak. für Verfahrens- und Systemtechnik, Diss., 2014von Verena Loh

Metabolic and Kinetic analyses of influenza production in perfusion HEK293 cell culture

<p>Abstract</p> <p>Background</p> <p>Cell culture-based production of influenza vaccine remains an attractive alternative to egg-based production. Short response time and high production yields are the key success factors for the broader adoption of cell culture technology for industrial manufacturing of pandemic and seasonal influenza vaccines. Recently, HEK293SF cells have been successfully used to produce influenza viruses, achieving hemagglutinin (HA) and infectious viral particle (IVP) titers in the highest ranges reported to date. In the same study, it was suggested that beyond 4 × 10⁶cells/mL, viral production was limited by a lack of nutrients or an accumulation of toxic products.</p> <p>Results</p> <p>To further improve viral titers at high cell densities, perfusion culture mode was evaluated. Productivities of both perfusion and batch culture modes were compared at an infection cell density of 6 × 10⁶cells/mL. The metabolism, including glycolysis, glutaminolysis and amino acids utilization as well as physiological indicators such as viability and apoptosis were extensively documented for the two modes of culture before and after viral infection to identify potential metabolic limitations. A 3 L bioreactor with a perfusion rate of 0.5 vol/day allowed us to reach maximal titers of 3.3 × 10¹¹IVP/mL and 4.0 logHA units/mL, corresponding to a total production of 1.0 × 10¹⁵IVP and 7.8 logHA units after 3 days post-infection. Overall, perfusion mode titers were higher by almost one order of magnitude over the batch culture mode of production. This improvement was associated with an activation of the cell metabolism as seen by a 1.5-fold and 4-fold higher consumption rates of glucose and glutamine respectively. A shift in the viral production kinetics was also observed leading to an accumulation of more viable cells with a higher specific production and causing an increase in the total volumetric production of infectious influenza particles.</p> <p>Conclusions</p> <p>These results confirm that the HEK293SF cell is an excellent substrate for high yield production of influenza virus. Furthermore, there is great potential in further improving the production yields through better control of the cell culture environment and viral production kinetics. Once accomplished, this cell line can be promoted as an industrial platform for cost-effective manufacturing of the influenza seasonal vaccine as well as for periods of peak demand during pandemics.</p

Zebra finches and Dutch adults exhibit the same cue weighting bias in vowel perception

Vocal tract resonances, called formants, are the most important parameters in human speech production and perception. They encode linguistic meaning and have been shown to be perceived by a wide range of species. Songbirds are also sensitive to different formant patterns in human speech. They can categorize words differing only in their vowels based on the formant patterns independent of speaker identity in a way comparable to humans. These results indicate that speech perception mechanisms are more similar between songbirds and humans than realized before. One of the major questions regarding formant perception concerns the weighting of different formants in the speech signal (“acoustic cue weighting”) and whether this process is unique to humans. Using an operant Go/NoGo design, we trained zebra finches to discriminate syllables, whose vowels differed in their first three formants. When subsequently tested with novel vowels, similar in either their first formant or their second and third formants to the familiar vowels, similarity in the higher formants was weighted much more strongly than similarity in the lower formant. Thus, zebra finches indeed exhibit a cue weighting bias. Interestingly, we also found that Dutch speakers when tested with the same paradigm exhibit the same cue weighting bias. This, together with earlier findings, supports the hypothesis that human speech evolution might have exploited general properties of the vertebrate auditory system

The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis

Lohr V, Haedicke O, Genzel Y, et al. The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis. BMC Biotechnology. 2014;14(1): 72.Background: In human vaccine manufacturing some pathogens such as Modified Vaccinia Virus Ankara, measles, mumps virus as well as influenza viruses are still produced on primary material derived from embryonated chicken eggs. Processes depending on primary cell culture, however, are difficult to adapt to modern vaccine production. Therefore, we derived previously a continuous suspension cell line, AGE1.CR.pIX, from muscovy duck and established chemically-defined media for virus propagation. Results: To better understand vaccine production processes, we developed a stoichiometric model of the central metabolism of AGE1.CR.pIX cells and applied flux variability and metabolic flux analysis. Results were compared to literature dealing with mammalian and insect cell culture metabolism focusing on the question whether cultured avian cells differ in metabolism. Qualitatively, the observed flux distribution of this avian cell line was similar to distributions found for mammalian cell lines (e.g. CHO, MDCK cells). In particular, glucose was catabolized inefficiently and glycolysis and TCA cycle seem to be only weakly connected. Conclusions: A distinguishing feature of the avian cell line is that glutaminolysis plays only a minor role in energy generation and production of precursors, resulting in low extracellular ammonia concentrations. This metabolic flux study is the first for a continuous avian cell line. It provides a basis for further metabolic analyses to exploit the biotechnological potential of avian and vertebrate cell lines and to develop specific optimized cell culture processes, e.g. vaccine production processes

Cell-free DNA profiling of metastatic prostate cancer reveals microsatellite instability, structural rearrangements and clonal hematopoiesis.

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.BACKGROUND: There are multiple existing and emerging therapeutic avenues for metastatic prostate cancer, with a common denominator, which is the need for predictive biomarkers. Circulating tumor DNA (ctDNA) has the potential to cost-efficiently accelerate precision medicine trials to improve clinical efficacy and diminish costs and toxicity. However, comprehensive ctDNA profiling in metastatic prostate cancer to date has been limited. METHODS: A combination of targeted and low-pass whole genome sequencing was performed on plasma cell-free DNA and matched white blood cell germline DNA in 364 blood samples from 217 metastatic prostate cancer patients. RESULTS: ctDNA was detected in 85.9% of baseline samples, correlated to line of therapy and was mirrored by circulating tumor cell enumeration of synchronous blood samples. Comprehensive profiling of the androgen receptor (AR) revealed a continuous increase in the fraction of patients with intra-AR structural variation, from 15.4% during first-line metastatic castration-resistant prostate cancer therapy to 45.2% in fourth line, indicating a continuous evolution of AR during the course of the disease. Patients displayed frequent alterations in DNA repair deficiency genes (18.0%). Additionally, the microsatellite instability phenotype was identified in 3.81% of eligible samples (≥ 0.1 ctDNA fraction). Sequencing of non-repetitive intronic and exonic regions of PTEN, RB1, and TP53 detected biallelic inactivation in 47.5%, 20.3%, and 44.1% of samples with ≥ 0.2 ctDNA fraction, respectively. Only one patient carried a clonal high-impact variant without a detectable second hit. Intronic high-impact structural variation was twice as common as exonic mutations in PTEN and RB1. Finally, 14.6% of patients presented false positive variants due to clonal hematopoiesis, commonly ignored in commercially available assays. CONCLUSIONS: ctDNA profiles appear to mirror the genomic landscape of metastatic prostate cancer tissue and may cost-efficiently provide somatic information in clinical trials designed to identify predictive biomarkers. However, intronic sequencing of the interrogated tumor suppressors challenges the ubiquitous focus on coding regions and is vital, together with profiling of synchronous white blood cells, to minimize erroneous assignments which in turn may confound results and impede true associations in clinical trials.The Belgian Foundation Against Cancer (grant number C/2014/227); Kom op tegen Kanker (Stand up to Cancer), the Flemish Cancer Society (grant number 00000000116000000206); Royal College of Surgeons/Cancer Research UK (C19198/A1533); The Cancer Research Funds of Radiumhemmet, through the PCM program at KI (grant number 163012); The Erling-Persson family foundation (grant number 4-2689-2016); the Swedish Research Council (grant number K2010-70X-20430-04-3), and the Swedish Cancer Foundation (grant number 09-0677)