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Gamma-ray irradiation, using the cobalt-60 isotope, is the most common radiation modality used for medical device and biopharmaceutical products sterilization. Although X-ray and electron-beam (e-beam) sterilization technologies are mature and have been in use for decades, impediments remain to switching to these sterilization modalities because of lack of data on the resulting radiation effects for the associated polymers, as well as a lack of education for manufacturers and regulators on the viability of these sterilization alternatives. For this study, the compatibility of ethylene vinyl acetate (EVA) multilayer films with different ionizing radiation sterilization (X-ray, e-beam, and gamma irradiation) is determined by measuring chemical and physical film properties using high performance liquid chromatography, differential scanning calorimetry, Fourier-Transform InfraRed spectroscopy (FTIR), surface energy measurement, and electron spin resonance techniques. The results indicate that the three irradiation modalities induce no differences in thermal properties in the investigated dose range. Gamma and X-Ray irradiations generate the same level of reactive species in the EVA multilayer film, whereas e-beam generates a reduced quantity of reactive species.

Electron beam (eBeam) inactivation of pathogens is a commercially proven technology in multiple industries. While commonly used in a variety of decontamination processes, this technology can be considered relatively new to the pharmaceutical industry. Rotavirus is the leading cause of severe gastroenteritis among infants, children, and at-risk adults. Infections are more severe in developing countries where access to health care, clean food, and water is limited. Passive immunization using orally administered egg yolk antibodies (chicken IgY) is proven for prophylaxis and therapy of viral diarrhea, owing to the stability of avian IgY in the harsh gut environment. Since preservation of viral antigenicity is critical for successful antibody production, the aim of this study was to demonstrate the effective use of electron beam irradiation as a method of pathogen inactivation to produce rotavirus-specific neutralizing egg yolk antibodies. White leghorn hens were immunized with the eBeam-inactivated viruses every 2 weeks until serum antibody titers peaked. The relative antigenicity of eBeam-inactivated Wa G1P[8] human rotavirus (HRV) was compared to live virus, thermally, and chemically inactivated virus preparations. Using a sandwich ELISA (with antibodies against recombinant VP8 for capture and detection of HRV), the live virus was as expected, most immunoreactive. The eBeam-inactivated HRV’s antigenicity was better preserved when compared to thermally and chemically inactivated viruses. Additionally, both egg yolk antibodies and serum-derived IgY were effective at neutralizing HRV in vitro. Electron beam inactivation is a suitable method for the inactivation of HRV and other enteric viruses for use in both passive and active immunization strategies.

Given the current pandemic the world is struggling with, there is an urgent need to continually improve vaccine technologies. Ionizing radiation technology has a long history in the development of vaccines, dating back to the mid-20th century. Ionizing radiation technology is a highly versatile technology that has a variety of commercial applications around the world. This brief review summarizes the core technology, the overall effects of ionizing radiation on bacterial cells and reviews vaccine development efforts using ionizing technologies, namely gamma radiation, electron beam, and X-rays.

Ionizing radiation such as Electron beam (EB) and gamma irradiation inactivate microbial cells preventing their multiplication. These cells, however, are structurally intact and appear to have residual metabolic activity. We were interested in understanding the metabolic pathways that were still functional in EB-inactivated cells. Therefore, the primary objective of this study was to compare the metabolites accumulating in EB-inactivated pathogens E. coli 026:H11 and S. Typhimurium immediately after EB inactivation and 24 h post inactivation. Defined aliquots (109 CFU/mL) of E. coli O26-H11 (TW 1597) and S. Typhimurium (ATCC 13311) suspended in phosphate-buffered saline were exposed to lethal EB doses of 3 kGy and 2 kGy, respectively. Complete inactivation (inability of cells to multiply) was confirmed by traditional plating methods. An untargeted analysis of the primary metabolites accumulating in un-irradiated (control) cells, EB-inactivated cells immediately after irradiation, and EB-inactivated cells that were incubated at room temperature for 24 h post EB inactivation was performed using gas chromatography/mass spectrometry. A total of 349 different metabolites were detected in the EB-inactivated S. Typhimurium and E. coli O26:H11 cells, out of which, only 50% were identifiable. In S. Typhimurium, 98 metabolites were expressed at statistically different concentrations (P < 0.05) between the three treatment groups. In E. coli O26:H11, 63 metabolites were expressed at statistically different concentrations (P < 0.05) between the three treatment groups. In both these pathogens, the β-alanine, alanine, aspartate, and glutamate metabolic pathways were significantly impacted (P < 0.01). Furthermore, the metabolomic changes in EB-inactivated cells were amplified significantly after 24 h storage at room temperature in phosphate-buffered saline. These results suggest that EB-inactivated cells are very metabolically active and, therefore, the term Metabolically Active yet Non-culturable is an apt term describing EB-inactivated bacterial cells.

Reports in the literature suggest that bacteria exposed to lethal doses of ionizing radiation, i.e., electron beams, are unable to replicate yet they remain metabolically active. To investigate this phenomenon further, we electron beam irradiated Escherichia coli cells to a lethal dose and measured their membrane integrity, metabolic activity, ATP levels and overall cellular functionality via bacteriophage infection. We also visualized the DNA double-strand breaks in the cells. We used non-irradiated (live) and heat-killed cells as positive and negative controls, respectively. Our results show that the membrane integrity of E. coli cells is maintained and that the cells remain metabolically active up to 9 days post-irradiation when stored at 4°C. The ATP levels in lethally irradiated cells are similar to non-irradiated control cells. We also visualized extensive DNA damage within the cells and confirmed their cellular functionality based on their ability to propagate bacteriophages for up to 9 days post-irradiation. Overall, our findings indicate that lethally irradiated E. coli cells resemble live non-irradiated cells more closely than heat-killed (dead) cells.