Optimization of the lentivirus production for the efficient transduction of primary murine macrophages

Lentiviren können im Gegensatz zu allen anderen Retroviren ihre virale DNS unabhängig vom Zellteilungsstadium der Wirtszelle in das Wirtsgenom integrieren. Dadurch können sie mitotisch inaktive Zellen transduzieren. In dieser Arbeit haben wir getestet, ob das “ViraPower™ T-REx™ Lentiviral Expression System” (Invitrogen) geeignet ist primäre Makrophagen effizient zu transduzieren. Dieses System besteht aus einem Expressionsvektor, der das zu transferierende Gen enthält, und einem Verpackungssystem aus drei Plasmiden, das für die Vektorpartikelproduktion benötigt wird. Um die Transduktionseffizienz bewerten zu können, verwendeten wir das grün fluoreszierende Protein (GFP) als Reportergen. Die Virusproduktion und die Bestimmung der viralen Titer durch Selektion Antibiotika-resistenter Zellklone wurden entsprechend dem Originalprotokoll durchgeführt. Aufgrund zu niedrigen, so erhaltenen Titerwerten wurde kein ausreichendes Verhältnis von infektiösen Viruspartikeln zu Zellen erreicht, welches zu einer erfolgreichen Transduktion der Zellen geführt hätte. In der Folge versuchten wir durch Modifikationen des Originalprotokolls die viralen Titer zu erhöhen. Diese umfassten Virusproduktion unter der Verwendung eines 2-Plasmid-Verpackungssystems, Virusproduktion im größeren Maßstab durch die Erhöhung der Anzahl der Virus produzierenden Zellen und Konzentration der viralen Überstände durch Ultrazentrifugation. All diese Ansätze führten zu einem deutlichen Titeranstieg, sodass wir in primären Zellen eine mittlere bis starke GFP Expression nachweisen konnten. Zusätzlich zur konventionellen Titration wurde die Aktivität der viralen reversen Transkriptase (RT) durch eine „product-enhanced reverse transcriptase“ (PERT) Analyse bestimmt. Die gemessenen RT Aktivitäten stimmten mit den Transduktionsergebnissen der primären Zellen überein und wir konnten zeigen, dass die PERT Analyse eine verlässliche Methode ist um die Transduktionskapazität von lentiviralen Überständen einzuschätzen.In contrast to other retroviruses, lentiviruses do not require mitosis for viral DNA integration into a host genome and thus are able to transduce non-dividing cells. In this study we evaluated the ViraPower™ T-REx™ Lentiviral Expression System (Invitrogen) in terms of efficient transduction of differentiated primary cells. This system consists of a transfer expression vector containing the gene of interest, and a three plasmid packaging system required for vector particle production. To assess the transduction efficiency of viral preparations, the green fluorescent protein (GFP) was used as a reporter gene. Virus production and titer determination by selection of antibiotic-resistant cell clones was performed as recommended, but the achieved multiplicity of infection was too low for the successful transduction of primary cells. Subsequently, we applied modifications to the original protocol in order to increase viral titers. This included the use of a two plasmid packaging system, the scale-up of virus production by the increase of the total amount of transfected cells and the concentration of viral supernatants by ultracentrifugation. All approaches resulted in a clear improvement of the titer. Consequently, the transduction of primary macrophages yielded a moderate to strong expression of GFP. In addition to the conventional titration, the reverse transcriptase activities of viral supernatants were determined by a product-enhanced reverse transcriptase (PERT) assay analysis. The measured activity values were in accordance with the transduction results of the primary cells and we could show, that the PERT assay is a reliable method to predict the transduction capacity of lentiviral supernatants

The Effects of Play Behavior, Feeding, and Time of Day on Salivary Concentrations of sIgA in Calves

The focus of animal welfare science has shifted over the last decades from efforts to avoid negative states to ways of allowing animals the experience of positive emotions. They may influence physiological processes in farmed animals, potentially providing health benefits; in addition, the physiological changes might be used as indicators of emotional states. We investigated calves’ salivary secretory immunoglobulin A (sIgA) concentrations with regard to a possible circadian rhythm and two situations that elicit positive emotions. Ten saliva samples of 14 calves were taken on two consecutive days; within the course of a day we observed a significant decline in salivary sIgA concentrations at 14:00 h. Further, we probed the animals before and after milk feeding and, contrarily to our prediction, detected lower sIgA concentrations 5 min after feeding than 15 min before. A probable explanation might be an increase in salivary flow rate caused by milk ingestion. We also took samples before and after we stimulated play behavior in calves. There was no significant difference in sIgA concentrations between samples taken before and after play. Although there was a significant correlation between the change in sIgA concentrations and the amount of play behavior shown, the correlation depended on an unexpected decrease of sIgA in animals that played little, and thus, does not support our hypothesis. In general, the data showed a large variability that might arise from different factors that are difficult to standardize in animals. Thus, the use of salivary sIgA concentrations as a marker of positive emotions in calves is not supported conclusively by the present data

Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle.

30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested-mostly from the milk-of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4).With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody's activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma

Dose and time dependency of r28M actions.

<p>Results from tumor cell viability measurements show samples normalized to the controls. Cells incubated without r28M served as controls, their number was considered as 1. Values are means ± SE. Significance levels: x: p ≤ 0.05; xx: p ≤ 0.01; xxx: p ≤ 0.001. PBMC from two different donors were used for the shown experiments. (A) CSPG4 positive (IPC-298) and negative cells (U-251 MG) were incubated with PBMC and monomeric r28M in concentrations ranging from 100–50000 ng/ml for 72 hrs in a final volume of 150 μl. (B) CSPG4 positive cells (A-375) were incubated with PBMC and 1000 ng/ml monomeric r28M for 72 hrs. 1000 ng/ml r28M were applied either immediately (day 1), immediately and with a repeated administration after 24 hrs (day 1/2), immediately and after 24 and 48 hrs (day 1/2/3) or immediately and after 48 hrs (day 1/3).</p

Specificity and time dependence of the biological activity of r28M.

<p>(A) CSPG4 positive (IPC-298) or negative cells (U-251 MG) were incubated together with PBMC and 1000 ng/ml enriched r28M fraction in a final volume of 150 μl for 24, 48 as well as 72 hrs to measure tumor cell viability. Results show samples normalized to the controls. Cells incubated without r28M served as controls and their number was considered as 1. Values are means ± SE. Significance levels: x: p ≤ 0.05; xx: p ≤ 0.01; xxx: p ≤ 0.001. (B) Plates were coated with the indicated antibodies or PBS and incubated with PBMC for 30 min, 2 hrs, 8 hrs as well as 24 hrs, respectively. Preparations were pipetted in triplicates. The supernatants of these triplicates were pooled and the IL-2 concentrations were measured by ELISA.</p

Impact of detergents on the r28M activity.

<p>The effects of the addition of the detergents Tween® 20 (A), Tween® 80 (B), saponin (C) and Na-deoxycholate (D) on the efficacy of PBMC to kill CSPG4 positive tumor cells (IPC-298) induced by 1000 ng/ml enriched r28M fraction are shown. Full grey bars: considered concentration of detergents (no positive or negative influence on the growth rate of CSPG4 positive cells and/or PBMC—see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140471#pone.0140471.s002" target="_blank">S2 Fig</a>). Dashed bars: concentration of detergents that influenced cell growth either positively or negatively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140471#pone.0140471.s002" target="_blank">S2 Fig</a>); therefore data was not included in the given statistical analysis. Results show samples normalized to the controls. Cells incubated without r28M served as controls, their number was considered as 1. Values are means ± SE. Significance levels: x: p ≤ 0.05; xx: p ≤ 0.01; xxx: p ≤ 0.001.</p

Optimized purification of r28M from plasma from transgenic cattle.

<p>The r28M (A) as well as BSA contents (B) of plasma after precipitation with ammonium sulfate (AS) or polyethylene glycol (PEG) were determined by competitive ELISAs and depicted as percentages of the bovine plasma feedstock used for purification of r28M. One representative example out of four independent experiments is shown. (C) The flow-through (FT) and eluate fraction (E) of plasma purified via Protein A or G on two different gels are shown. 500 ng of each fraction were separated by a 8% SDS-PAGE under non-reducing conditions and subsequently silver-stained. Cut FT and E sections of Protein A were on the same gel, but not on nearby slots. One representative example out of three independent experiments is shown.</p

Caspase 3 and 7 release of cells incubated with r28M or detergents.

<p>CSPG4 positive cells (IPC-298) were incubated for 24 hrs as indicated (saponin and Na-deoxycholate: [c] = 0.0067%; 1000 ng/ml r28M). Cells were lysed, cell proteins were separated by SDS-PAGE and subsequently investigated by a Western Blot with an anti-caspase 3 (35 kDa), anti-cleaved caspase 3 (17 and 19 kDa), anti-caspase 7 (35 kDa) or anti-cleaved caspase 7 (20 kDa) antibody. One representative out of 4 independent experiments is shown each.</p