Impact of defective interfering particles on virus replication and antiviral host response in cell culture-based influenza vaccine production

During the replication of influenza viruses, defective interfering particles (DIPs) can be generated. These are noninfectious deletion mutants that require coinfection with a wild-type virus but interfere with its helper virus replication. Consequently, coinfected cells mainly produce DIPs. Little is known about how such noninfectious virus particles affect the virus yield of cell culture-based influenza vaccine production. We compared infections of Madin-Darby canine kidney cells with two seed virus preparations of the influenza virus strain A/Puerto Rico/8/34 that contain different amounts of DIPs. A combination of conventional RT-PCR, RT-qPCR, and flow cytometry revealed that DI genomes indeed strongly accumulate in coinfected cells and impede the viral RNA synthesis. Additionally, cells infected at the higher DIP concentration showed a stronger antiviral response characterized by increased interferon-β expression and apoptosis induction. Furthermore, in the presence of DIPs, a significant fraction of cells did not show any productive accumulation of viral proteins at all. Together, these effects of DIPs significantly reduce the virus yield. Therefore, the accumulation of DIPs should be avoided during influenza vaccine production which can be achieved by quality controls of working seed viruses based on conventional RT-PCR. The strategy for the depletion of DIPs presented here can help to make cell culture-based vaccine production more reliable and robust.     

流感病毒在Vero细胞系与MDCK细胞系增殖条件的比较

目的研究流感病毒H1N1及其他亚型在Vero细胞系和MDCK细胞系高效增殖的最适条件,比较两种细胞系对流感病毒的敏感性差异及影响敏感性差异的条件。方法在培养好的Vero细胞系与MDCK细胞系用不同的病毒感染复数(M.O.I)、胰酶浓度、病毒吸附时间、病毒维持液血清质量浓度等条件进行流感病毒在细胞上的增殖。结果在M.O.I为0.01接种流感病毒,吸附时间为1 h,胰酶质量浓度2μg/mL,血清质量浓度为8%时,流感病毒血凝素在MDCK细胞系可获得较高的滴度。结论 MDCK细胞系是适于流感病毒培养的细胞,它作为生产新型流感病毒疫苗的主要细胞基质需要进一步的研究。     

Trypsin promotes efficient influenza vaccine production in MDCK cells by interfering with the antiviral host response

Trypsin is commonly used in Madin–Darby canine kidney (MDCK) cell culture-based influenza vaccine production to facilitate virus infection by proteolytic activation of viral haemagglutinin, which enables multi-cycle replication. In this study, we were able to demonstrate that trypsin also interferes with pathogen defence mechanisms of host cells. In particular, a trypsin concentration of 5 BAEE U/mL (4.5 μg/mL porcine trypsin) used in vaccine manufacturing strongly inhibited interferon (IFN) signalling by proteolytic degradation of secreted IFN. Consequently, absence of trypsin during infection resulted in a considerably stronger induction of IFN signalling and apoptosis, which significantly reduced virus yields. Under this condition, multi-cycle virus replication in MDCK cells was not prevented but clearly delayed. Therefore, incomplete infection can be ruled out as the reason for the lower virus titres. However, suppression of IFN signalling by overexpression of viral IFN antagonists (influenza virus PR8-NS1, rabies virus phosphoprotein) partially rescued virus titres in the absence of trypsin. In addition, virus yields could be almost restored by using the influenza strain A/WSN/33 in combination with fetal calf serum (FCS). For this strain, FCS enabled trypsin-independent fast propagation of virus infection, probably outrunning cellular defence mechanisms and apoptosis induction in the absence of trypsin. Overall, addition of trypsin provided optimal conditions for high yield vaccine production in MDCK cells by two means. On the one hand, proteolytic degradation of IFN keeps cellular defence at a low level. On the other hand, enhanced virus spreading enables viruses to replicate before the cellular response becomes fully activated.     

Validation of the safety of MDCK cells as a substrate for the production of a cell-derived influenza vaccine

Cell culture-based production methods may assist in meeting increasing demand for seasonal influenza vaccines and developing production flexibility required for addressing influenza pandemics. MDCK-33016PF cells are used in propagation of a cell-based seasonal influenza vaccine (Optaflu^®); but, like most continuous cell lines, can grow in immunocompromised mice to produce tumors. It is, therefore, essential that no residual cells remain within the vaccine, that cell lysates or DNA are not oncogenic, and that the cell substrate does not contain oncogenic viruses or oncogenic DNA. Multiple, redundant processes ensure the safety of influenza vaccines produced in MDCK-33016PF cells. The probability of a residual cell being present in a dose of vaccine is approximately 1 in 10³⁴. Residual MDCK-DNA is ≤10 ng per dose and the ß-propiolactone used to inactivate influenza virus results in reduction of detectable DNA to less than 200 base pairs (bp). Degenerate PCR and specific PCR confirm exclusion of oncogenic viruses. The manufacturing process has been validated for its capacity to remove and inactivate viruses. We conclude that the theoretical risks arising from manufacturing seasonal influenza vaccine using MDCK-33016PF cells are reduced to levels that are effectively zero by the multiple, orthogonal processes used during production.     

Growth determinants for H5N1 influenza vaccine seed viruses in MDCK cells

H5N1 influenza A viruses are exacting a growing human toll, with more than 240 fatal cases to date. In the event of an influenza pandemic caused by these viruses, embryonated chicken eggs, which are the approved substrate for human inactivated-vaccine production, will likely be in short supply because chickens will be killed by these viruses or culled to limit the worldwide spread of the infection. The Madin-Darby canine kidney (MDCK) cell line is a promising alternative candidate substrate because it supports efficient growth of influenza viruses compared to other cell lines. Here, we addressed the molecular determinants for growth of an H5N1 vaccine seed virus in MDCK cells, revealing the critical responsibility of the Tyr residue at position 360 of PB2, the considerable requirement for functional balance between hemagglutinin (HA) and neuraminidase (NA), and the partial responsibility of the Glu residue at position 55 of NS1. Based on these findings, we produced a PR8/H5N1 reassortant, optimized for this cell line, that derives all of its genes for its internal proteins from the PR8(UW) strain except for the NS gene, which derives from the PR8(Cambridge) strain; its N1 NA gene, which has a long stalk and derives from an early H5N1 strain; and its HA gene, which has an avirulent-type cleavage site sequence and is derived from a circulating H5N1 virus. Our findings demonstrate the importance and feasibility of a cell culture-based approach to producing seed viruses for inactivated H5N1 vaccines that grow robustly and in a timely, cost-efficient manner as an alternative to egg-based vaccine production.     

Cell culture-based production of defective interfering particles for influenza antiviral therapy

Defective interfering particles (DIPs) lack an essential portion of the virus genome, but retain signals for replication and packaging, and therefore, interfere with standard virus (STV) replication. Due to this property, DIPs can be potential antivirals. The influenza A virus DIP DI244, generated during propagation in chicken eggs, has been previously described as a potential candidate for influenza antiviral therapy. As a cell culture-based manufacturing process would be more suitable to fulfill large-scale production needs of an antiviral and enables full process control in closed systems, we investigated options to produce DI244 in the avian cell line AGE1.CR.pIX in chemically defined suspension culture. With a DI244 fraction of 55.8% compared to STV, the highest DI244 yield obtained from 50 million cells was 4.6 × 10⁹ vRNA copies/mL at 12 h post infection. However, other defective genomes were also detected. Since these additionally produced defective particles are non-infectious, they might be still useful in antiviral therapies. In case they would interfere with quality of the final product, we examined the impact of virus seeds and selected process parameters on DI244 yield and contamination level with other defective particles. With a DI244 fraction of 5.5%, the yield obtained was 1.7 × 10⁸ vRNA copies/mL but now without additional defective genomes. Although the DI244 yield might be decreased in this case, such controlled manufacturing conditions are not available in chicken eggs. Overall, the application of these findings can support design and optimization of a cell culture-based production process for DIPs to be used as antivirals.

     

Distributed modeling of human influenza a virus–host cell interactions during vaccine production

This contribution is concerned with population balance modeling of virus–host cell interactions during vaccine production. Replication of human influenza A virus in cultures of adherent Madin–Darby canine kidney (MDCK) cells is considered as a model system. The progress of infection can be characterized by the intracellular amount of viral nucleoprotein (NP) which is measured via flow cytometry. This allows the differentiation of the host cell population and gives rise to a distributed modeling approach. For this purpose a degree of fluorescence is introduced as an internal coordinate which is linearly linked to the intracellular amount of NP. Experimental results for different human influenza A subtypes reveal characteristic dynamic phenomena of the cell distribution like transient multimodality and reversal of propagation direction. The presented population balance model provides a reasonable explanation for these dynamic phenomena by the explicit consideration of different states of infection of individual cells. Kinetic parameters are determined from experimental data. To translate the emerging infinite dimensional parameter estimation problem to a finite dimension the parameters are assumed to depend linearly on the internal coordinate. As a result, the model is able to reproduce all characteristic dynamic phenomena of the considered process for the two examined virus strains and allows deeper insight into the underlying kinetic processes. Thus, the model is an important contribution to the understanding of the intracellular virus replication and virus spreading in cell cultures and can serve as a stepping stone for optimization in vaccine production. Biotechnol. Bioeng. 2013; 110: 2252–2266. © 2013 Wiley Periodicals, Inc.     

Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production

Background  

In cell culture-based influenza vaccine production the monitoring of virus titres and cell physiology during infection is of great importance for process characterisation and optimisation. While conventional virus quantification methods give only virus titres in the culture broth, data obtained by fluorescence labelling of intracellular virus proteins provide additional information on infection dynamics. Flow cytometry represents a valuable tool to investigate the influences of cultivation conditions and process variations on virus replication and virus yields.     

MDCK and Vero cells for influenza virus vaccine production: a one-to-one comparison up to lab-scale bioreactor cultivation

Over the last decade, adherent MDCK (Madin Darby canine kidney) and Vero cells have attracted considerable attention for production of cell culture-derived influenza vaccines. While numerous publications deal with the design and the optimization of corresponding upstream processes, one-to-one comparisons of these cell lines under comparable cultivation conditions have largely been neglected. Therefore, a direct comparison of influenza virus production with adherent MDCK and Vero cells in T-flasks, roller bottles, and lab-scale bioreactors was performed in this study. First, virus seeds had to be adapted to Vero cells by multiple passages. Glycan analysis of the hemagglutinin (HA) protein showed that for influenza A/PR/8/34 H1N1, three passages were sufficient to achieve a stable new N-glycan fingerprint, higher yields, and a faster increase to maximum HA titers. Compared to MDCK cells, virus production in serum-free medium with Vero cells was highly sensitive to trypsin concentration. Virus stability at 37 °C for different virus strains showed differences depending on medium, virus strain, and cell line. After careful adjustment of corresponding parameters, comparable productivity was obtained with both host cell lines in small-scale cultivation systems. However, using these cultivation conditions in lab-scale bioreactors (stirred tank, wave bioreactor) resulted in lower productivities for Vero cells.     

Insect larvae biofactories as a platform for influenza vaccine production

Increased production capacity is one of the most important priorities for seasonal and pandemic influenza vaccines. In the present study, we used a baculovirus-insect larvae system (considered small, living biofactories) to improve the production of recombinant influenza virus H1N1 hemagglutinin (HA). Insect larvae produced four-fold more HA protein than insect cells per biomass unit (1 g of fresh larvae weight). A single infected Trichoplusia ni larva produced up to 113 μg of soluble and easily purified recombinant HA, an amount similar to that produced by 1.2×10(8) Sf21 insect cells infected by the same baculovirus. The use of the KDEL endoplasmic reticulum retention signal fused to the HA protein further increased recombinant protein production. Larvae-derived HA was immunogenically functional in vaccinated mice, inducing the generation of hemagglutination inhibition antibodies and a protective immune response against a lethal challenge with a highly virulent virus. The productivity, scalability and cost efficiency of small, living biofactories based on insect larvae suggest a broad-based strategy for the production of recombinant subunit vaccines against seasonal or pandemic influenza as an alternative to fermentation technologies.     

Screening different host cell lines for the dynamic production of measles virus

Measles virus (MV) has a natural affinity for cancer cells and oncolytic MV preparations have therefore been investigated in several clinical trials as a potential treatment for cancer. The main bottleneck in the administration of oncolytic MV to cancer patients is the production process, because very large doses of virus particles are required for each treatment. Here, we investigated the productivity of different host cells and found that a high infection efficiency did not necessarily result in high virus yields because virus release is also dependent on the host cell. As well as producing large numbers of active MV particles, host cells must perform well in dynamic cultivation systems. In screening experiments, the highest productivity was achieved by Vero and BJAB cells, but only the Vero cells maintained their high virus productivity when transferred to a stirred tank reactor. We used dielectric spectroscopy as an online monitoring system to control the infection and harvest times, which are known to be critical process parameters. The precise control of these parameters allowed us to achieve higher virus titers with Vero cells in a stirred tank reactor than in a static cultivation system based on T‐flasks, with maximum titers of up to 10¹¹ TCID₅₀ ml^?1. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:989–997, 2017     

Production of influenza H1N1 vaccine from MDCK cells using a novel disposable packed-bed bioreactor

A process for human influenza H1N1 virus vaccine production from Madin–Darby canine kidney (MDCK) cells using a novel packed-bed bioreactor is described in this report. The mini-bioreactor was used to study the relationship between cell density and glucose consumption rate and to optimize the infection parameters of the influenza H1N1 virus (A/New Caledonia/20/99). The MDCK cell culture and virus infection were then monitored in a disposable perfusion bioreactor (AmProtein Current Perfusion Bioreactor) with proportional–integral–derivative control of pH, dissolved O₂ (DO), agitation, and temperature. During 6 days of culture, the total cell number increased from 2.0?×?10⁹ to 3.2?×?10¹⁰ cells. The maximum virus titers of 768 hemagglutinin units/100 μL and 7.8?×?10⁷ 50 % tissue culture infectious doses/mL were obtained 3 days after infection. These results demonstrate that using a disposable perfusion bioreactor for large-scale cultivation of MDCK cells, which allows for the control of DO, pH, and other conditions, is a convenient and stable platform for industrial-scale production of influenza vaccines.     

Organization of the endoplasmic reticulum in renal cell lines MDCK and LLC-PK1

The spatial organization of the endoplasmic reticulum has been studied in two renal cell lines, MDCK and LLC-PK1, which originate from the distal and proximal portions of the mammalian nephron, respectively, and which form a polarized epithelium when they reach confluence in tissue culture. The two renal cell lines, grown to confluence on either solid or permeable supports, were investigated by fluorescence microscopy, confocal microscopy, and transmission electron microscopy. Fluorescence labeling of the endoplasmic reticulum was achieved using the cationic fluorescent dye DIOC₆ (3). In order to differentiate fluorescent labeling of the endoplasmic reticulum from that of the mitochondria, cells were also labeled with rhodamine 123. For electron microscopy, the spatial organization of the endoplasmic reticulum was examined in thick sections using the long-duration osmium impregnation technique or the ferrocyanide/osmium technique. In both cell lines, the endoplasmic reticulum formed an abundant tubular network of canaliculi that frequently abutted the basolateral domain of the plasma membrane and occasionally the apical membrane. Elements of the endoplasmic reticulum were also found in close proximity to mitochondria that, as in the nephron, formed branched structures. Canaliculi appeared circular or flattened and had an inner diameter of 10–70 nm for MDCK cells and 20–90 nm for LLC-PK1 cells. Such a three-dimensional organization might facilitate the translocation of defined lipid species between the endoplasmic reticulum and the plasma membrane, and between the endoplasmic reticulum and mitochondria.     

MDCK细胞和MDCK-G1细胞感染H1N1流感病毒后病毒滴度和细胞转录组表达差异分析

目的通过分析细胞表达基因及其相关信号通路的差异等信息,探索MDCK和MDCK-G1细胞株在接种H1N1流感病毒后出现病毒滴度和细胞生长趋势差异的内在生物学特性等原因。方法通过Illumina测序平台对2株细胞进行转录组测序(RNA sequencing, RNA-seq)和测序结果生物信息学分析后,选择了17个差异基因进行实时定量聚合酶链反应(quantitative real-time polymerase chain reaction, qRT-PCR)验证。结果 MDCK和MDCK-G1细胞差异基因(differentially expressed genes, DEGs)|log_2(FoldChange)|0和padj≤0.05总数为2 786个。相比于MDCK细胞,MDCK-G1细胞有967个基因的表达显著上升,1 819个基因的表达显著下降。差异基因通过基因本体(Gene Ontology, GO)数据库富集功能注释和京都基因和基因组数据库(Kyoto Encyclopedia of Genes and Genomes, KEGG)进行通路分析发现,2株细胞在免疫调控和细胞生长周期调控上均存在差异,qRT-PCR验证的17个基因中有16个基因的表达趋势与转录组测序结果一致。结论 2株细胞的转录谱存在显著差异。     

Optimization of roller culturing of influenza virus vaccine strains in MDCK and Vero cell cultures using plant origin components

Roller culturing of MDCK and Vero cells in an experimental nutrient medium based on soy flour hydrolysate, plant material obtained using the bromelain plant enzyme, was studied. The medium supplemented with 2 or 3% fetal calf serum (FCS) had a strong growth-stimulating effect on Vero and MDCK and cells, respectively, and did not alter the cell morphology. A/Solomon Islands/03/06 (H1N1) and B/Malaysia/2506/04 influenza vaccine viruses were grown on MDCK and Vero cell cultures obtained as a result of culturing in rollers on media containing soy flour hydrolysate and FCS (2 or 3%, respectively). The titer of the viruses was high in the presence of either trypsin (2 μg/ml) or bromelain (20 μg/ml).     

Production of avian influenza virus vaccine using primary cell cultures generated from host organs

The global availability of a therapeutically effective influenza virus vaccine during a pandemic remains a major challenge for the biopharmaceutical industry. Long production time, coupled with decreased supply of embryonated chicken eggs (ECE), significantly affects the conventional vaccine production. Transformed cell lines have attained regulatory approvals for vaccine production. Based on the fact that the avian influenza virus would infect the cells derived from its natural host, the viral growth characteristics were studied on chicken embryo-derived primary cell cultures. The viral propagation was determined on avian origin primary cell cultures, transformed mammalian cell lines, and in ECE. A comparison was made between these systems by utilizing various cell culture-based assays. In-vitro substrate susceptibility and viral infection characteristics were evaluated by performing hemagglutination assay (HA), 50 % tissue culture infectious dose (TCID₅₀) and monitoring of cytopathic effects (CPE) caused by the virus. The primary cell culture developed from chicken embryos showed stable growth characteristics with no contamination. HA, TCID₅₀, and CPE exhibited that these cell systems were permissive to viral infection, yielding 2–10 times higher viral titer as compared to mammalian cell lines. Though the viral output from the ECE was equivalent to the chicken cell culture, the time period for achieving it was decreased to half. Some of the prerequisites of inactivated influenza virus vaccine production include generation of higher vial titer, independence from exogenous sources, and decrease in the production time lines. Based on the tests, it can be concluded that chicken embryo primary cell culture addresses these issues and can serve as a potential alternative for influenza virus vaccine production.     

Genetic characterization of CHO production host DG44 and derivative recombinant cell lines

The dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cell line DG44 is the dominant mammalian host for recombinant protein manufacturing, in large part because of the availability of a well-characterized genetic selection and amplification system. However, this cell line has not been studied at the cytogenetic level. Here, the first detailed karyotype analysis of DG44 and several recombinant derivative cell lines is described. In contrast to the 22 chromosomes in diploid Chinese hamster cells, DG44 has 20 chromosomes, only seven of which are normal. In addition, four Z group chromosomes, seven derivative chromosomes, and 2 marker chromosomes were identified. For all but one of the 16 DG44-derived recombinant cell lines analyzed, a single integration site was detected by fluorescence in situ hybridization regardless of the gene delivery method (calcium phosphate-DNA coprecipitation or microinjection), the topology of the DNA (circular or linear), or the integrated plasmid copy number (between 1 and 51). Chromosomal aberrations, observed in more than half of the cell lines studied, were mostly unbalanced with examples of aneuploidy, deletions, and complex rearrangements. The results demonstrate that chromosomal aberrations are frequently associated with the establishment of recombinant CHO DG44 cell lines. Noteworthy, there was no direct correlation between the stability of the genome and the stability of recombinant protein expression.