Concepts for the Production of Viruses and Viral Vectors in Cell Cultures

The industrial-scale manufacturing of viruses or virus-like particles in cell culture is necessary for gene therapy and the treatment of cancer with oncolytic viruses. Complex multistep processes are required in both cases, but the low virus titers in batch cultures and the temperature sensitivity of the virus particles limit the production scale. To meet commercial and regulatory requirements, each process must be scalable and reproducible and must yield high virus titers. These requirements are met by establishing a cell culture process that matches the properties of the virus/host-cell system and by using serum-free cell culture medium. This chapter focuses on two case studies to consider the different aspects of process design, such as the reactor configuration and operational mode: the continuous production of retroviral pseudotype vectors in a retroviral packaging cell line and the production of oncolytic measles virus vectors for cancer therapy

Aeration and Shear Stress Are Critical Process Parameters for the Production of Oncolytic Measles Virus

Oncolytic Measles virus is a promising candidate for cancer treatment, but clinical studies have shown that extremely high doses (up to 1011 TCID50 per dose) are required to effect a cure. Very high titers of the virus must therefore be achieved during production to ensure an adequate supply. We have previously shown that Measles virus can be produced in Vero cells growing on a Cytodex 1 microcarrier in serum-containing medium using a stirred-tank reactor (STR). However, process optimization and further process transfer or scale up requires the identification of critical process parameters, particularly because the use of STRs increases the risk of cell damage and lower product yields due to shear stress. Using a small-scale STR (0.5 L working volume) we found that Measles virus titers are sensitive to agitator-dependent shear, with shear stress ≥0.25 N m−2 reducing the titer by more than four orders of magnitude. This effect was observed in both serum-containing and serum-free medium. At this scale, virus of titers up to 1010 TCID50 mL−1 could be achieved with an average shear stress of 0.1 N m−2. We also found that the aeration method affected the virus titer. Aeration was necessary to ensure a sufficient oxygen supply to the Vero cells, and CO2 was also needed to regulate the pH of the sodium bicarbonate buffer system. Continuous gassing with air and CO2 reduced the virus titer by four orders of magnitude compared to head-space aeration. The manufacture of oncolytic Measles virus in a STR can therefore be defined as a shear-sensitive process, but high titers can nevertheless be achieved by keeping shear stress levels below 0.25 N m−2 and by avoiding extensive gassing of the medium

A tailor-made purification strategy for oncolytic measles viruses using membrane-based processes

Cancer patients can benefit from the Measles virus, since in the early 70s a relation between cancer remission and an infection with Measles was first mentioned (Bluming, Ziegler 1971). Further studies confirmed this oncolytic activity and therefore, the Measles virus became highly interesting for the application in cancer treatment. However, for the widespread application as a therapeutic agent several bottlenecks have to be overcome in context of quantity and quality. For one therapeutic dose of oncolytic Measles viruses (OMV) at least 1011 infectious particles are needed (one vaccination contains ~103 TCID50) (Russell et al. 2014). Besides that, the impurities, such as host cell proteins (HCP) and host cell DNA (hcDNA), must be reduced to appropriate limits set by regulatory authorities. The full recovery of OMV infectivity must also addressed. This underlines the need of a tailor-made downstream processing. After we established a high titer production process, achieving OMV titers of 1011 TCID50 mL-1 (Grein et al. 2017), we are now focused on the downstream processing of OMV. For this purpose we characterized the OMV regarding process parameters used in DSP, such as stability towards ionic strength, osmolality, agglomeration and shear stress. Based on this, a clarification step was conducted, followed by the further purification with tangential flow filtration (TFF). By using polyether sulfone flat sheet membranes in concentration mode, we were able to recover the infectious virus and lowered the impurities by ~70% for hcDNA and ~80% for protein content. In the next purification step, we applied a discontinuous diafiltration and could deplete the impurities by ~95% in total. These results showed that TFF is an appropriate tool for the purification and formulation of OMV. References Bluming, Avrum Z.; Ziegler, John L. (1971): Regression of Burkitt\u27s Lymphoma in association with Measles infection. In The Lancet 298 (7715), pp. 105–106. DOI: 10.1016/S0140-6736(71)92086-1. Grein, Tanja A.; Schwebel, Felix; Kress, Marco; Loewe, Daniel; Dieken, Hauke; Salzig, Denise et al. (2017): Screening different host cell lines for the dynamic production of measles virus. In Biotechnology progress. DOI: 10.1002/btpr.2432. Russell, Stephen J.; Federspiel, Mark J.; Peng, Kah-Whye; Tong, Caili; Dingli, David; Morice, William G. et al. (2014): Remission of disseminated cancer after systemic oncolytic virotherapy. In Mayo Clinic proceedings 89 (7), pp. 926–933. DOI: 10.1016/j.mayocp.2014.04.003

Role of complement in in vitro and in vivo lung inflammatory reactions

Complement is one of the integral buttresses of the inflammatory response. In addition to host defense activities, proinflammatory properties of several complement components are described. This overview elucidates the role of complement in inflammatory reactions in vitro and in vivo, focusing on the complement activation products, C5a, and the membrane attack complex, C5b‐9. Using several approaches, the impact of these complement components in mechanisms relevant to neutrophil recruitment is emphasized. In addition, the participation of complement in endothelial superoxide generation and its essential requirement for full expression of lung injury is demonstrated, as are the involved intracellular signal transduction pathways. Understanding the mechanisms of complement‐induced proinflammatory effects may provide a basis for future therapeutic blockade of complement and/or its activation products. J. Leukoc. Biol. 64: 40–48; 1998.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142061/1/jlb0040.pd

Regulatory effects of interleukin‐11 during acute lung inflammatory injury

The role of interleukin‐11 (IL‐11) was evaluated in the IgG immune complex model of acute lung injury in rats. IL‐11 mRNA and protein were both up‐regulated during the course of this inflammatory response. Exogenously administered IL‐11 substantially reduced, in a dose‐dependent manner, the intrapulmonary accumulation of neutrophils and the lung vascular leak of albumin. These in vivo anti‐inflammatory effects of IL‐11 were associated with reduced NF‐κB activation in lung, reduced levels of tumor necrosis factor α (TNF‐α) in bronchoalveolar lavage (BAL) fluids, and diminished up‐regulation of lung vascular ICAM‐1. It is interesting that IL‐11 did not affect BAL fluid content of the CXC chemokines, macrophage inflammatory protein‐2 (MIP‐2) and cytokine‐inducible neutrophil chemoattractant (CINC); the presence of IL‐11 did not affect these chemokines. However, BAL content of C5a was reduced by IL‐11. These data indicate that IL‐11 is a regulatory cytokine in the lung and that, like other members of this family, its anti‐inflammatory properties appear to be linked to its suppression of NF‐κB activation, diminished production of TNF‐α, and reduced up‐regulation of lung vascular ICAM‐1. J. Leukoc. Biol. 66: 151–157; 1999.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141937/1/jlb0151.pd

Position-sensitive Si pad detectors for electron emission channeling experiments

Position-sensitive detector systems, initially developed for the detection of X-rays, have been adapted for their use in electron emission channeling experiments. Each detection system consists of a 30.8x30.8 mm$^{2}$ 22x22 -pad Si detector, either of 0.3 mm, 0.5 mm or 1 mm thickness, four 128$-$channel preamplifier chips, a backplane trigger circuit, a sampling analog to digital converter, a digital signal processor, and a personal computer for data display and storage. The operational principle of these detection systems is described, and characteristic features such as energy and position resolution and maximum count rate, which have been determined from tests with conversion electrons and $\beta^-\!$ -particles in the energy range 40--600 keV, are presented

Mediators of Microvascular Injury in Dermal Burn Wounds

In previous studies we have demonstrated that second-degree thermal injury of skin in rats leads to secondary effects, such as systemic complement activation, C5a-mediated activation of blood neutrophils, their adhesion-molecule-guided accumulation in lung capillaries and the development of acute pulmonary injury, largely caused by neutrophil-derived toxic oxygen metabolites. In the dermal burn wound, however, pathophysiologic events are less well understood. The injury is fully developed at four hours post-burn. To further elucidate the pathogenesis of the “late phase” dermal vascular damage, rats were depleted of neutrophils or complement by pretreatment with rabbit antibody against rat neutrophils or with cobra venom factor, respectively. In other experiments, rats were treated with blocking antibodies to IL-6, IL-1, and TNFα immediately following thermal burning or were pretreated with hydroxyl radical scavengers (dimethyl sulfoxide, dimethyl thiourea). Extravasation of 125 I-labeled bovine serum albumin into the burned skin was studied, as well as, skin myeloperoxidase levels. The studies revealed that, like in secondary lung injury, neutrophils and toxic oxygen metabolites, are required for full development of microvascular injury. In contrast, however, development of dermal vascular damage in thermally injured rats was not affected by complement depletion. Our data suggest that the development of microvascular injury in the dermal burn wound is complement-independent, involves the pro-inflammatory cytokines IL-1, TNFα and IL-6, and may result from reactive oxygen metabolites generated by neutrophils accumulating in the burn wound.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44523/1/10753_2004_Article_415307.pd

Complement C5a regulates IL-17 by affecting the crosstalk between DC and ΓΔ T cells in CLP-induced sepsis

Complement 5a (C5a) and Interleukin-17 (IL-17) are two important inflammatory mediators in sepsis. Here we studied the mechanisms underlying regulation of IL-17 by anaphylatoxin C5a. We found that C5a blockade increased the survival rate of mice following cecal ligation and puncture (CLP)-induced sepsis and decreased IL-17 expression in vivo . IL-17 was secreted mainly by ΓΔ T cells in this model. Importantly, our data suggest that C5a participates in the regulation of IL-17 secretion by ΓΔ T cells. Dendritic cells (DC) were found to act as a “bridge” between C5a and ΓΔ T cells in a mechanism involving IL-6 and transforming growth factor Β (TGF-Β). These results imply that C5a affects the crosstalk between DC and ΓΔ T cells during sepsis development, and this may result in a large production of inflammatory mediators such as IL-17.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/71378/1/1079_ftp.pd

Role of the complement in experimental sepsis

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142122/1/jlb0467.pd