Production of Modified Vaccinia Ankara Virus by Intensified Cell Cultures: A Comparison of Platform Technologies for Viral Vector Production

Modified Vaccinia Ankara (MVA) virus is a promising vector for vaccination against various challenging pathogens or the treatment of some types of cancers, requiring a high amount of virions per dose for vaccination and gene therapy. Upstream process intensification combining perfusion technologies, the avian suspension cell line AGE1.CR.pIX and the virus strain MVA-CR19 is an option to obtain very high MVA yields. Here the authors compare different options for cell retention in perfusion mode using conventional stirred-tank bioreactors. Furthermore, the authors study hollow-fiber bioreactors and an orbital-shaken bioreactor in perfusion mode, both available for single-use. Productivity for the virus strain MVA-CR19 is compared to results from batch and continuous production reported in literature. The results demonstrate that cell retention devices are only required to maximize cell concentration but not for continuous harvesting. Using a stirred-tank bioreactor, a perfusion strategy with working volume expansion after virus infection results in the highest yields. Overall, infectious MVA virus titers of 2.1–16.5 × 10⁹ virions/mL are achieved in these intensified processes. Taken together, the study shows a novel perspective on high-yield MVA virus production in conventional bioreactor systems linked to various cell retention devices and addresses options for process intensification including fully single-use perfusion platforms.     

Tanshinone biosynthesis in Salvia miltiorrhiza and production in plant tissue cultures

Salvia miltiorrhiza Bunge (Lamiaceae) root, generally called Danshen, is an important herb in Chinese medicine widely used for treatment of cardiovascular diseases. Diterpenoid tanshinons are major bioactive constituents of Danshen with notable pharmacological activities and the potential as new drug candidates against some important human diseases. The importance of Danshen for traditional and modern medicines has motivated the research interest over two decades in the biosynthesis and biotechnological production of tanshinones. Although diterpenes in plants are presumably derived from the non-mevalonate (MVA) pathway, tanshinone biosynthesis in S. miltiorrhiza may also depend on the MVA pathway based on some key enzymes and genes detected in the early steps of these pathways. Plant tissue cultures are the major biotechnological processes for rapid production of tanshinones and other bioactive compounds in the herb. Various in vitro cultures of S. miltiorrhiza have been established, including cell suspension, adventitious root, and hairy root cultures, which can accumulate the major tanshinones as in the plant roots. Tanshinone production in cell and hairy root cultures has been dramatically enhanced with various strategies, including medium optimization, elicitor stimulation, and nutrient feeding operations. This review will summarize the above developments and also provide our views on future trends.     

Expanding the Repertoire of Modified Vaccinia Ankara-Based Vaccine Vectors via Genetic Complementation Strategies

Background

Modified Vaccinia virus Ankara (MVA) is a safe, highly attenuated orthopoxvirus that is being developed as a recombinant vaccine vector for immunization against a number of infectious diseases and cancers. However, the expression by MVA vectors of large numbers of poxvirus antigens, which display immunodominance over vectored antigens-of-interest for the priming of T cell responses, and the induction of vector-neutralizing antibodies, which curtail the efficacy of subsequent booster immunizations, remain as significant impediments to the overall utility of such vaccines. Thus, genetic approaches that enable the derivation of MVA vectors that are antigenically less complex may allow for rational improvement of MVA-based vaccines.

Principal Findings

We have developed a genetic complementation system that enables the deletion of essential viral genes from the MVA genome, thereby allowing us to generate MVA vaccine vectors that are antigenically less complex. Using this system, we deleted the essential uracil-DNA-glycosylase (udg) gene from MVA and propagated this otherwise replication-defective variant on a complementing cell line that constitutively expresses the poxvirus udg gene and that was derived from a newly identified continuous cell line that is permissive for growth of wild type MVA. The resulting virus, MVAΔudg, does not replicate its DNA genome or express late viral gene products during infection of non-complementing cells in culture. As proof-of-concept for immunological ‘focusing’, we demonstrate that immunization of mice with MVAΔudg elicits CD8+ T cell responses that are directed against a restricted repertoire of vector antigens, as compared to immunization with parental MVA. Immunization of rhesus macaques with MVAΔudg-gag, a udg ⁻ recombinant virus that expresses an HIV subtype-B consensus gag transgene, elicited significantly higher frequencies of Gag-specific CD8 and CD4 T cells following both primary (2–4-fold) and booster (2-fold) immunizations as compared to the udg ⁺ control virus MVA-gag, as determined by intracellular cytokine assay. In contrast, levels of HIV Gag-specific antibodies were elicited similarly in macaques following immunization with MVAΔudg-gag and MVA-gag. Furthermore, both udg ⁻ and udg ⁺ MVA vectors induced comparatively similar titers of MVA-specific neutralizing antibody responses following immunization of mice (over a 4-log range: 10⁴–10⁸ PFU) and rhesus macaques. These results suggest that the generation of MVA-specific neutralizing antibody responses are largely driven by input MVA antigens, rather than those that are synthesized de novo during infection, and that the processes governing the generation of antiviral antibody responses are more readily saturated by viral antigen than are those that elicit CD8+ T cell responses.

Significance

Our identification of a spontaneously-immortalized (but not transformed) chicken embryo fibroblast cell line (DF-1) that is fully permissive for MVA growth and that can be engineered to stably express MVA genes provides the basis for a genetic system for MVA. DF-1 cells (and derivatives thereof) constitute viable alternatives, for the manufacture of MVA-based vaccines, to primary CEFs – the conventional cell substrate for MVA vaccines that is not amenable to genetic complementation strategies due to these cells'' finite lifespan in culture. The establishment of a genetic system for MVA, as illustrated here to allow udg deletion, enables the generation of novel replication-defective MVA mutants and expands the repertoire of genetic viral variants that can now be explored as improved vaccine vectors.     

Agrobacterium-mediated stable transformation of cell suspension cultures of barley (Hordeum vulgare)

Barley (Hordeum vulgare L. cvs. Igri and Dissa) cell suspension cultures, which had been initiated from immature embryo-derived (IED) and microspore-derived (MSD) callus, were co-cultivated with various Agrobacterium tumefaciens strains. The T-DNA vectors contained visually-detectable marker genes (C1/Lc orgusA-intron), as reporters of transient T-DNA transfer, and also drug resistance genes (hph or bar) to facilitate selection of stably transformed cell lines. A set of normal binary vectors in a super-virulent Agrobacterium strain [EHA101(pBECKS)] and also a super-binary vector [LBA4404(pTOK233)] were used in this study. Cells of the suspension cultures which received T-DNA were able to proliferate under selection regimes and a number of hygromycin- or phosphinothricin-resistant barley callus lines were isolated which expressed a co-transferred gusA gene. To ensure homogeneity of the cell lines, prolonged tissue culture regimes were used but these resulted in a loss of the capacity to regenerate plants from the transgenic callus lines. The frequency of recovery of transformed callus lines ranged from 0.3% to 2.9%. Southern blot analyses of the transformed callus lines confirmed the presence of the marker genes and demonstrated them to be associated with DNA which was distinct from that of the original Agrobacterium plasmid. Furthermore, independent transgenic lines showed diverse patterns of hybridising bands. These data suggest that the T-DNA fragment was stably maintained through integration into the genomes of the barley cell lines. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cellular characteristics of head and neck cancer stem cells in type IV collagen-coated adherent cultures

Although head and neck squamous carcinoma cancer stem cells (HNSC-CSCs) can be enriched in serum-free suspension cultures, it is difficult to stably expand HNSC-CSC lines in suspension due to spontaneous apoptosis and differentiation. Here, we investigated whether HNSC-CSCs can be expanded without loss of stem cell properties by adherent culture methods. Cell culture plates were coated with type IV collagen, laminin, or fibronectin. We examined cancer stem cell traits of adherent HNSC-CSCs grown on these plates using immunocytochemistry for stem cell marker expression and analyses of chemo-resistance and xenograft tumorigenicity. We also assessed the growth rate, apoptosis rate, and gene transduction efficiency of adherent and suspended HNSC-CSCs. HNSC-CSCs grew much faster on type IV collagen-coated plates than in suspension. Adherent HNSC-CSCs expressed putative stem cell markers (OCT4 and CD44) and were chemo-resistant to various cytotoxic drugs (cisplatin, fluorouracil, paclitaxel, and docetaxel). Adherent HNSC-CSCs at the limiting dilution (1000 cells) produced tumors in nude mice. Adherent HNSC-CSCs also showed less spontaneous apoptotic cell death and were more competent to lentiviral transduction than suspended HNSC-CSCs. In conclusion, compared to suspension cultures, adherence on type IV collagen-coated culture plates provides better experimental conditions for HNSC-CSC expansion, which should facilitate various refined cellular studies.     

Isolation of stem-like cells from human MG-63 osteosarcoma cells using limiting dilution in combination with vincristine selection

To isolate stem-like cells from the human MG-63 osteosarcoma cell line, different subpopulations of MG-63 cells were cloned by limiting dilution and passaged to obtain different sublines. The subline with highest clonogenicity was identified using a proliferation assay, cell-cycle analysis, and soft-agar colony-forming assay. The sublines were further selected in serum-free medium containing 20 ng/ml vincristine to identify cells that could form suspended sarcospheres. Identified cells were then characterized based on morphology, cell surface markers, adipogenic and osteogenic differentiation, and tumorigenicity in nude mice. A total of 19 holoclones that could be stably passaged were obtained. Sublines A1, A3, and D1 were markedly different from other sublines and the parental cell line. Subline D1 not only had a higher colony-forming efficiency and formed larger colonies, but also possessed a shorter latency of tumorigenesis in vivo. After subline D1 was cultured in suspension in medium containing vincristine, a highly enriched subpopulation of cells that could form sarcospheres and be stably passaged were obtained. These cells, designated as MG-63-M expressed multiple markers of multipotent or embryonic stem cells and possessed the capacity for self-renewal, multilineage differentiation, and significant multi-drug resistance. Thus, our results suggest that a subpopulation of stem-like cells can be isolated from human MG-63 osteosarcoma cell line.     

Expression of a human anti-rabies virus monoclonal antibody in tobacco cell culture

A Nicotiana tabacum cv. Xanthi cell culture was initiated from a transgenic plant expressing a human anti-rabies virus monoclonal antibody. Within 3 months, plant cell suspension cultures were established and recombinant protein expression was examined. The antibody was stably produced during culture growth. ELISA, protein G purification, Western blotting, and neutralization assay confirmed that the antibody was fully processed, with association of light and heavy-chains, and that it was able to bind and neutralize rabies virus. Quantification of antibody production in plant cell suspension culture revealed 30 microg/g of cell dry weight for the highest-producing culture (0.5 mg/L), 3 times higher than from the original transgenic plant. The same production level was observed 3 months after cell culture initiation. Plant cell suspension cultures were successfully grown in a new disposable plastic bioreactor, with a growth rate and production level similar to that of cultures in Erlenmeyer flasks.     

甲羟戊酸途径限速步骤研究及其在产番茄红素重组大肠杆菌中的应用

甲羟戊酸途径(MVA途径)被引入重组大肠杆菌中,能够提高重组大肠杆菌中萜类化合物的合成能力。但因重组大肠杆菌中萜类化合物合成途径中间产物积累,导致细胞生长和萜类化合物合成受到限制。本研究在稳定表达MVA途径以及优化2-甲基-D-赤藻糖醇-4-磷酸途径(MEP途径)、番茄红素合成途径关键基因表达的重组大肠杆菌LYC103中,用质粒高表达MVA途径和番茄红素合成途径关键基因,挖掘该途径的限速步骤。结果表明,ispA、crtE、mvaK1、idi和mvaD基因过表达后,细胞生长没有明显变化,番茄红素产量依次提高了13.5%、16.5%、17.95%、33.7%和61.1%,说明这几个基因可能是合成番茄红素的限速步骤。mvaK1、mvaK2、mvaD三个基因在同一操纵子上,用mRNA稳定区(RNA stabilizing region)进行启动子文库(mRSL)调控mvaK1,相当于对3个基因同时调控。用高效基因组编辑技术(CAGO)对mvaK1基因的mRNA稳定区进行启动子文库的调控,得到菌株LYC104。番茄红素产量与对照菌株LYC103相比增加了2倍,细胞生长提高了32%。然后,利用CRISPR-Cas9技术在染色体lacZ位点整合idi基因,得到LYC105菌株。与出发菌株LYC103相比,细胞生长提高了147%,番茄红素产量增加了2.28倍。本研究在染色体上具有完整MVA途径的基础上,利用质粒高表达单个基因挖掘限速步骤,用同源重组方法整合限速基因、解除限速,为代谢工程构建高产菌株提供新策略。     

Differential interaction of branch-specific inhibitors of isoprenoid biosynthesis with cell cycle progression in tobacco BY-2 cells

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGR), which catalyzes the formation of mevalonic acid (MVA), can be specifically blocked by mevinolin. Inhibition of HMGR in vivo leads to an arrest in cell cycle progression in tobacco ( Nicotiana tabacum L. Bright Yellow 2) cells. As MVA in plants is the common precursor of a myriad of isoprenoid products synthesized in the cytosol and mitochondria, it is difficult to identify among such MVA-dependent molecules those whose lack may lead to cell cycle arrest. In an attempt to do so, branch-specific inhibitors of the cytosolic isoprenoid pathway downstream from MVA were used to study their capacity to block cell cycle progression. The effects of squalestatin (sterol biosynthesis inhibitor), chaetomellic acid A and patulin (protein prenyltransferase (PT) inhibitors) and tunicamycin (inhibitor of dolichol-dependent protein glycosyl transferase, thus mimicking the effect of an absence of dolichol) were compared to those induced by mevinolin. In this way, squalestatin and chaetomellic acid were identified as behaving like true cell cycle inhibitors, in that they led to a specific arrest in the cell cycle. However, they did not exactly mimic the mevinolin-induced effects. Patulin proved to be of high general toxicity, which suggests that it may affect other reactions besides blockage of protein isoprenylation. Finally, tunicamycin efficiently blocked growth of cell suspension cultures, but did not arrest the cells in a specific phase of the cell cycle. Results are discussed in the context of a better understanding of the essential implication of isoprenoids in plant cell cycle progression.     

Comparison of plant-based expression platforms for the heterologous production of geraniol

We compared the ability of different plant-based expression platforms to produce geraniol, a key metabolite in the monoterpenoid branch of the terpenoid indole alkaloid biosynthesis pathway. A geraniol synthase gene isolated from Valeriana officinalis (VoGES) was stably expressed in different tobacco systems. Intact plants were grown in vitro and in the greenhouse and were used to generate cell suspension and hairy root cultures. VoGES was also transiently expressed in N. benthamiana. The highest geraniol content was produced by intact transgenic plants grown in vitro (48 μg/g fresh weight, fw), followed by the transient expression system (27 μg/g fw), transgenic plants under hydroponic conditions in the greenhouse and cell suspension cultures (16 μg/g fw), and finally hairy root cultures (9 μg/g fw). Differences in biomass production and the duration of cultivation resulted in a spectrum of geraniol productivities. Cell suspension cultures achieved a geraniol production rate of 1.8 μg/g fresh biomass per day, whereas transient expression produced 5.9 μg/g fresh biomass per day (if cultivation prior to agroinfiltration is ignored) or 0.5 μg/g fresh biomass per day (if cultivation prior to agroinfiltration is included). The superior productivity, strict process control and simple handling procedures available for transgenic cell suspension cultures suggest that cells are the most promising system for further optimization and ultimately for the scaled-up production of geraniol.     

20-hydroxyecdysone accumulation and regulation in Ajuga lobata D. Don suspension culture

Suspension culture of Ajuga lobata D. Don cells provides a method of synthesis of the phytoecdysteroid 20-hydroxyecdysone (20E) which can regulate the molting process of larvae. We characterized the culture conditions to optimize 20E production. Growth of A. lobata D. Don cells fits the logistic equation curve with a growth cycle of 19 days. Medium conductivity was negatively correlated with dry cell weight and 20E accumulation, thus could be used to determine the optimal time for cell harvest. Continuous subculture reduced 20E synthesis, but supplementing medium with 20E precursors mevalonic (MVA), α-Pinene, and nitric oxide (NO) can significantly promote cell growth and influence 20E accumulation. Combination of α-Pinene, MVA, and SNP significantly elevated 20E accumulation, thus may synergistically enhance 20E synthesis in A. lobata D. Don. The optimal concentrations of α-Pinene, MVA, and NO donor SNP in suspension culture were 50 μL L^?1, 10 mg L^?1, and 80 μmol L^?1.     

Functional heterogeneity and heritability in CHO cell populations

In this study, we address the hypothesis that it is possible to exploit genetic/functional variation in parental Chinese hamster ovary (CHO) cell populations to isolate clonal derivatives that exhibit superior, heritable attributes for biomanufacturing—new parental cell lines which are inherently more “fit for purpose.” One‐hundred and ninety‐nine CHOK1SV clones were isolated from a donor CHOK1SV parental population by limiting dilution cloning and microplate image analysis, followed by primary analysis of variation in cell‐specific proliferation rate during extended deep‐well microplate suspension culture of individual clones to accelerate genetic drift in isolated cultures. A subset of 100 clones were comparatively evaluated for transient production of a recombinant monoclonal antibody (Mab) and green fluorescent protein following transfection of a plasmid vector encoding both genes. The heritability of both cell‐specific proliferation rate and Mab production was further assessed using a subset of 23 clones varying in functional capability that were subjected to cell culture regimes involving both cryopreservation and extended sub‐culture. These data showed that whilst differences in transient Mab production capability were not heritable per se, clones exhibiting heritable variation in specific proliferation rate, endocytotic transfectability and N‐glycan processing were identified. Finally, for clonal populations most “evolved” by extended sub‐culture in vitro we investigated the relationship between cellular protein biomass content, specific proliferation rate and cell surface N‐glycosylation. Rapid‐specific proliferation rate was inversely correlated to CHO cell size and protein content, and positively correlated to cell surface glycan content, although substantial clone‐specific variation in ability to accumulate cell biomass was evident. Taken together, our data reveal the dynamic nature of the CHO cell functional genome and the potential to evolve and isolate CHO cell variants with improved functional properties in vitro. Biotechnol. Bioeng. 2013; 110: 260–274. © 2012 Wiley Periodicals, Inc.