Implications of MDCK cell heterogeneity in cell-based influenza vaccine production

Influenza is a global public health issue that causes serious illness with high mortality rate. Currently, Madin-Darby canine kidney (MDCK) cell culture-based influenza vaccine production moving up to the front as an inexorable trend for the substitution of egg-based vaccine production, owing to its high degree of flexibility and scalability. However, MDCK cells are a continuous cell line and comprise a heterogeneous pool of non-clonal cells that differ in morphological as well as functional features in influenza virus production. The impurity of cell population may lead to fugacious tendency in virus production, and long-term culture may bring potential risk of unstable viral production or vaccine quality as cells in MDCK subclonal population may encounter unexpected manifestation of chromosomal rearrangement, loss of the virus susceptibility, or reduction of the virus partials packaging capability during the culture. Although many details of the influenza virus life cycle have already been unraveled, little is known about the ability of subclones in virus infection, intracellular replication, and virus release during viral vaccine production process. With the widely utilizing of omics-based approaches and progressively accumulating of omics database, transcriptome profile analysis will be a powerful strategy to explore the mechanism of cell heterogeneity, providing great significance for the development of robust virus producing cell line and robust virus production process. This work aims to explore a deeper understanding on the MDCK cell heterogeneity used in influenza virus production. For this purpose, a MDCK cell line that has been extensively used in industrial production was subcloned and examined for the influenza virus productivity. The virus productivity spread over a wide range of more than 300-fold among different clones, which revealed large variations in their ability to produce progeny viruses. The high and low producer as well as parent cell population were expanded to explore the intracellular virus propagation process, and the expression levels of all the annotated genes were quantified across the different subclones using RNA-seq. The RT-qPCR results showed that the influenza virus RNA synthesis and virus release differed dramatically among subclones during a synchronized single-cycle infection. Pathway analysis performed on the genes indicated that most of the genes are not differentially expressed, but a few key cellular metabolic pathways are differentially expressed among the subclones, especially the genes related to the virus infection, replication and release. These results spurs further hypothesis to improve our mechanistic understanding of cell line stability and virus propagation process, which will have significant impact on rationalizing cell line development of viral vaccine producing mammalian cells

Study of Resource Recovery and Epidemiology in an Anaerobic Digester

Three 4-liter packed bed anaerobic digesters were fabricated and operated at 35 degrees C, pH around 7, and hydraulic retention time (HRT) of 20, 10 and 5 days to study the resource recovery and epidemiology in a controlled ecological life support system (CELSS). A simulated wastewater, consisted of shower water, clothwash water, dishwasher water, handwash water, and urine flush water was used as the feeding solution. Under steady-state operation, chemical oxygen demand (COD), total organic carbon (TOC), pH, nitrogen, phosphorus, and potassium wer monitored in the digester input and output solutions. The volume and the CH4/CO2 ratios in the biogas produced from the anaerobic digesters were measured. The results indicate about 90 percent of TOC is converted while only 5-8 percent of N-P-K are consumed in the digester. A multi-drug resistant strain of Salmonella choleraesuis was used as the indicator bacterium in the epidemiology study. The levels of Salmonella choleraesuis in the influent and the effluent wer determined and decimal decay rate constants, k(d), were estimated. The k(d) values were greater at higher initial doses than lower doses for the same HR, and greater for batch digestion (7.89/d) than for continuous digestion (4.28, 3.82, and 3.82/d for 20, 10, and 5 d HRT, respectively)

Efficient influenza vaccine manufacturing: Single MDCK suspension cells in chemically defined medium

Facing the constant global high demand for influenza vaccines, improving production capacity is most important. For influenza vaccine production, cell culture-based processes have advantages regarding flexibility, efficiency, and safety in comparison with the traditional egg-based processes. To avoid problems related to microcarrier-based approaches and serum containing media, growth of suspension cells in chemically-defined media is favoured. In addition, such a process has advantages regarding the improvement of virus titers, the scale-up of the production process, and overall productivity in up- and downstream processing. In this study, a previously developed MDCK suspension cell line [1] was cultivated in an in-house chemically defined medium to evaluate cell growth and virus production. For the purpose of process intensification, virus adaptation and infection strategies were investigated to achieve high cell densities and to maximize virus titers. Therefore, an adapted influenza virus strain (A/PR/8/34 H1N1 RK1) was generated by a series of virus passages with low multiplicity of infection (MOI). Virus infections were carried out by supplementing 100% of fresh medium, infecting cells with a MOI of 10-3, and with trypsin addition at 72 h of cell cultivations in shake flasks and bioreactors. For scale-up, MDCK cells were cultivated in a DASGIP bioreactor system, optimizing stirring speed, time of infection, pH and DO levels both for cell growth and virus infection. Cell count, viability, main extracellular metabolites, and virus titers were measured to compare productivity between shake flasks and bioreactors. In batch culture (shake flasks and bioreactors), single MDCK cells were grown to maximum cell densities of 1.2 x107 cells/ml with cell viabilities exceeding 98% at high cell specific growth rates of 0.036 h-1. Virus adaptation to the MDCK suspension cell line led to a fast infection and stable virus titers over time. Regarding process optimization, optimal pH (cell growth: 7.00, infection: 7.20), DO (40%) and agitation speed (80 rpm) were chosen for influenza A virus production in three parallel bioreactors. Cell densities of 1.0 x107 cells/ml were achieved at time of infection (72 h) before performing a dilution step. Post infection, similar virus infection dynamics were observed in shake flasks and bioreactors. For both cultivation systems maximal HA titers of 3.6 log10(HAU/100µl) were achieved without reduction of cell-specific virus titer (12,000 virions/cell). Overall, a highly efficient and scalable upstream process was realized by cultivation of MDCK suspension cells as single cells in chemically defined medium. This is a strong basis for a promising application in large-scale influenza vaccine manufacturing and potential process intensification towards high cell density virus production. [1] Huang D. et al., PloS One 10 (2015): e0141686. doi: 10.1371/journal.pone.014168

Highly efficient influenza virus production: A MDCK-based high-cell-density process

Seasonal vaccination campaigns for influenza in developed and developing countries create a massive demand for 500 million (2015) vaccine doses every year [1]. Besides egg-based vaccine manufacturing, production platforms based on animal cell culture increasingly contribute to this overall growing market. In order to intensify cell culture-based influenza virus production, high-cell-density (HCD) cultivation of suspension cells can be applied to improve virus titer, process productivity and production costs [2]. For process optimization and evaluation of HCD conditions, cells cultivated using semi-perfusion approaches in small shakers can be used as a scale-down model for bioreactors operating in full perfusion mode [3]. In this study, a previously developed MDCK suspension cell line [4] was adapted to a new serum free medium [5] to facilitate higher growth rate, cell density and virus titer both in batch and in HCD. Therefore, MDCK cells cultivated in Smif-8 medium were slowly adapted to a new cultivation medium (Xeno™) by stepwise increasing the Xeno content. Fully adapted cells were cultivated in shaker flasks to evaluate the performance of influenza A virus production in batch and HCD. Cell densities exceeding 2∙107 cells/mL were achieved in shakers using semi-perfusion, where cell free medium was manually replaced with fresh medium. Volume and time interval of media replacement were chosen to achieve a constant cell-specific perfusion rate of 2.5 pL/(cell h). Cell cultures were infected with influenza virus (A/PR/8/34 H1N1 RKI) with trypsin addition. Cell count, viability, main metabolites and virus titer (HA-assay & TCID50) were monitored pre and post infection. Medium adaptation resulted in a MDCK suspension cell line with morphological, growth, and metabolic characteristics different from parental cells. Cells fully adapted to Xeno medium were growing to higher cell densities (1.4∙107 vs 6∙106 cells/mL) with higher specific growth rate (µmax: 0.036 vs 0.026 1/h), cells were bigger (15-16 vs 13-14 µm) and grew without aggregate formation. Due to higher cell densities at time of infection, virus titers up to 3.6 log10(HAU/100µL) were reached. In semi-perfusion, adapted MDCK cells were grown up to 6∙107 cells/mL, however, maximum virus titer and productivity were observed with 4∙107 cells/mL. In multiple harvests, very high virus titer exceeding 4 log10(HAU/100µL) and up to 9∙109 virions/mL (TCID50) were measured, which corresponded to an accumulated titer of 4.5 log10(HAU/100µL). Cell-specific virus titer was similar or higher compared to the reference batch infections, depending on perfusion and infection strategy. Overall, results in this semi-perfusion scale-down model for influenza A virus production suggest a highly efficient and productive upstream process for influenza virus production, with an up to six-fold improved space time yield compared to batch mode. [1] Palache A. et al., Vaccine 35 (2017): 4681–4686. doi: 10.1016/j.vaccine.2017.07.053 [2] Genzel Y. et al., Vaccine 32 (2014): 2770–2781. doi: 10.1016/j.vaccine.2014.02.016 [3] Vázquez-Ramírez D. et al., Vaccine (2018): article in press. doi: 10.1016/j.vaccine.2017.10.112 [4] Lohr V. et al., Vaccine 28 (2010): 6256–6264. doi: 10.1016/j.vaccine.2010.07.004 [5] Xeno™-S001S MDCK Cell Serum Free Medium (#FG0100402), Bioengine, Shanghai, Chin

Triassic collision of western Tianshan orogenic belt, China: Evidence from SHRIMP U-Pb dating of zircon from HP/UHP eclogitic rocks

A newly recognized ultrahigh-pressure (UHP) terrane in the Chinese Western Tianshan orogenic belt contains blueschists, eclogites and metapelites. This belt extends westward to the "South Tianshan" in Tajikistan, Kyrgyzstan, Kazakhstan and Uzbekistan fo

Evaluation of an identification method for the SARS-CoV-2 Delta variant based on the amplification-refractory mutation system

The Delta variant of SARS-CoV-2 dominated the COVID-19 pandemic due to its high viral replication capacity and immune evasion, causing massive outbreaks of cases, hospitalizations, and deaths. Currently, variant identification is performed mainly by sequencing. However, the high requirements for equipment and operators as well as its high cost have limited its application in underdeveloped regions. To achieve an economical and rapid method of variant identification suitable for undeveloped areas, we applied an amplification-refractory mutation system (ARMS) based on PCR for the detection of novel coronavirus variants. The results showed that this method could be finished in 90 min and detect as few as 500 copies/mL and not react with SARS-Coronavirus, influenza A H1N1(2009), and other cross-pathogens or be influenced by fresh human blood, α- interferon, and other interfering substances. In a set of double-blind trials, tests of 262 samples obtained from patients confirmed with Delta variant infection revealed that our method was able to accurately identify the Delta variant with high sensitivity and specificity. In conclusion, the ARMS-PCR method applied in Delta variant identification is rapid, sensitive, specific, economical, and suitable for undeveloped areas. In our future study, ARMS-PCR will be further applied in the identification of other variants, such as Omicron

Electrostatic-Assembly-Driven Formation of Supramolecular Rhombus Microparticles and Their Application for Fluorescent Nucleic Acid Detection

In this paper, we report on the large-scale formation of supramolecular rhombus microparticles (SRMs) driven by electrostatic assembly, carried out by direct mixing of an aqueous HAuCl4 solution and an ethanol solution of 4,4′-bipyridine at room temperature. We further demonstrate their use as an effective fluorescent sensing platform for nucleic acid detection with a high selectivity down to single-base mismatch. The general concept used in this approach is based on adsorption of the fluorescently labeled single-stranded DNA (ssDNA) probe by SRM, which is accompanied by substantial fluorescence quenching. In the following assay, specific hybridization with its target to form double-stranded DNA (dsDNA) results in desorption of ssDNA from SRM surface and subsequent fluorescence recovery

Rectangular Coordination Polymer Nanoplates: Large-Scale, Rapid Synthesis and Their Application as a Fluorescent Sensing Platform for DNA Detection

In this paper, we report on the large-scale, rapid synthesis of uniform rectangular coordination polymer nanoplates (RCPNs) assembled from Cu(II) and 4,4′-bipyridine for the first time. We further demonstrate that such RCPNs can be used as a very effective fluorescent sensing platform for multiple DNA detection with a detection limit as low as 30 pM and a high selectivity down to single-base mismatch. The DNA detection is accomplished by the following two steps: (1) RCPN binds dye-labeled single-stranded DNA (ssDNA) probe, which brings dye and RCPN into close proximity, leading to fluorescence quenching; (2) Specific hybridization of the probe with its target generates a double-stranded DNA (dsDNA) which detaches from RCPN, leading to fluorescence recovery. It suggests that this sensing system can well discriminate complementary and mismatched DNA sequences. The exact mechanism of fluorescence quenching involved is elucidated experimentally and its use in a human blood serum system is also demonstrated successfully