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Background: With increasing clinical use of NK-92 cells and their CAR-modified derivatives in cancer immunotherapy, there is a growing demand for efficient production processes of these ""off-the-shelf"" therapeutics. In order to ensure safety and prevent the occurrence of secondary tumors, (CAR-)NK-92 cell proliferation has to be inactivated before transfusion. This is commonly achieved by gamma irradiation. Recently, we showed proof of concept that low energy electron irradiation (LEEI) is a new method for NK-92 inactivation. LEEI has several advantages over gamma irradiation, including a faster reaction time, a more reproducible dose rate and much less requirements on radiation shielding. Here, LEEI was further evaluated as a promising alternative to gamma irradiation yielding cells with highly maintained cytotoxic effector function. Methods: Effectiveness and efficiency of LEEI and gamma irradiation were analyzed using NK-92 and CD123-directed CAR-NK-92 cells. LEE-irradiated cells were extensively characterized and compared to gamma-irradiated cells via flow cytometry, cytotoxicity assays, and comet assays, amongst others. Results: Our results show that both irradiation methods caused a progressive decrease in cell viability and are, therefore, suitable for inhibition of cell proliferation. Notably, the NK-mediated specific lysis of tumor cells was maintained at stable levels for three days post-irradiation, with a trend towards higher activities after LEEI treatment as compared to gamma irradiation. Both gamma irradiation as well as LEEI led to substantial DNA damage and an accumulation of irradiated cells in the G2/M cell cycle phases. In addition, transcriptomic analysis of irradiated cells revealed approximately 12-fold more differentially expressed genes two hours after gamma irradiation, compared to LEEI. Analysis of surface molecules revealed an irradiation-induced decrease in surface expression of CD56, but no changes in the levels of the activating receptors NKp46, NKG2D, or NKp30. Conclusions: The presented data show that LEEI inactivates (CAR-)NK-92 cells as efficiently as gamma irradiation, but with less impact on the overall gene expression. Due to logistic advantages, LEEI might provide a superior alternative for the manufacture of (CAR-)NK-92 cells for clinical application.

Bacterial pathogens cause severe infections worldwide in livestock and in humans, and antibiotic resistance further increases the importance of prophylactic vaccines. Inactivated bacterial vaccines (bacterins) are usually produced via incubation of the pathogen with chemicals such as formaldehyde, which is time consuming and may cause loss of immunogenicity due to the modification of structural components. We evaluated low-energy electron irradiation (LEEI) as an alternative method to generate a bacterin. Rodentibacter pneumotropicus, an invasive Gram-negative murine pathogen, was inactivated with LEEI and formaldehyde. LEEI resulted in high antigen conservation, and LPS activity was significantly better maintained when compared with formaldehyde treatment. Immunization of mice with LEEI-inactivated R. pneumotropicus elicited a strong immune response with no detectable bacterial burden upon sublethal challenge. The results of this study suggest the inactivation of bacteria with LEEI as an alternative, fast and efficient method to generate bacterial vaccines with increased efficacy.

Q-Fieber ist eine weltweit verbreitete Zoonose, die durch Coxiella (C.) burnetii verursacht wird. Dieses Bakterium wird besonders über Geburtsmaterial infizierter kleiner Wiederkäuer in großen Mengen ausgeschieden, aber auch über Milch, Kot und Urin. Der Hauptinfektionsweg für Mensch und Tier ist die Inhalation von erregerhaltigen Aerosolen. Bei der Verbreitung spielen Wind und andere meteorologische Faktoren eine Rolle. Auch Zecken wie Dermacentor spp. und Ixodes ricinus könnten eine Bedeutung bei der Übertragung von C. burnetii haben. Bei Ziegen kann eine Infektion mit C. burnetii vor allem zu Spätaborten führen. Im Gegensatz dazu scheint eine Coxiellen-Infektion bei Schafen zu weitaus geringeren Abortraten als bei Ziegen zu führen. Beim Menschen zeigen 40 % der infizierten Personen grippeähnliche Symptome wie Fieber und Kopfschmerzen. Bis zu 5 % aller Infizierten können eine Chronifizierung entwickeln, die meist mit einer Endokarditis einhergeht. In Deutschland wurden seit dem Jahr 2000 mehrere humane Q-Fieber-Kleinraumepidemien mit bis zu 331 infizierten Personen pro Ausbruch detektiert. Auslöser waren meist lammende Schafe. Aufgrund der Zunahme an Milchziegenhaltungen in Deutschland wird in Zukunft mit vermehrten Fällen auch bei dieser Tierart gerechnet. Der diagnostische Nachweis von Infektionen mit C. burnetii beim Tier erfolgt meist mit dem ELISA und/oder der PCR. Mithilfe der Multiple Loci Variable Number of Tandem Repeat Analysis (MLVA/VNTR) lassen sich Isolate genotypisieren. Das genotypische MLVA-Cluster A wurde besonders bei kleinen Wiederkäuern nachgewiesen, während das Cluster C eher mit Rindern assoziiert ist. Die antibiotische Behandlung infizierter Tiere mit Oxytetracyclinen hat sich nicht bewährt. Vielmehr senkt der Einsatz eines Impfstoffes mit inaktivierten Phase-I-Bakterien das Risiko der Ausscheidung. Aufgrund der hohen Tenazität von C. burnetii sind viele Desinfektionsmittel nicht wirksam.

Bacterial pathogens cause severe infections worldwide in livestock and in humans, and antibiotic resistance further increases the importance of prophylactic vaccines. Inactivated bacterial vaccines (bacterins) are usually produced via incubation of the pathogen with chemicals such as formaldehyde, which is time consuming and may cause loss of immunogenicity due to the modification of structural components. We evaluated low-energy electron irradiation (LEEI) as an alternative method to generate a bacterin. Rodentibacter pneumotropicus, an invasive Gram-negative murine pathogen, was inactivated with LEEI and formaldehyde. LEEI resulted in high antigen conservation, and LPS activity was significantly better maintained when compared with formaldehyde treatment. Immunization of mice with LEEI-inactivated R. pneumotropicus elicited a strong immune response with no detectable bacterial burden upon sublethal challenge. The results of this study suggest the inactivation of bacteria with LEEI as an alternative, fast and efficient method to generate bacterial vaccines with increased efficacy.