Characterization of Antigenic Properties of a Selected Gram-Positive and Gram-Negative Bacteria Following Inactivation by Low-Energy Electron Beam Irradiation (LEEI)

Abstract

In search for better pathogen inactivation methods in the development of vaccines, Low-Energy Electron Irradiation (LEEI) has shown recent promise as an effective method that could replace existing pathogen inactivation methods. Effectiveness of LEEI of pathogens has been established on viruses. While it has been used on Escherichia coli (E. coli), its effectiveness in inactivation of bacteria has not been well elucidated. This study aimed to determine the efficacy of LEEI in inactivation while maintaining antigenicity of selected Gram positive and Gram negative bacteria. A cross sectional laboratory-based study was conducted using known stock cultures of bacterial suspensions archived at -800C. Bacterial suspensions of Gram positive (Bacillus cereus) and Gram negative (Pasteurella pneumotropica) bacteria were cultured in nutrient broth and brain heart infusion media respectively after which cell concentration was adjusted to an optical density at 600nm (OD600) of 3.0 and 2.0, respectively. Irradiation was then carried with different doses ranging from 4 kGy, 8,12, 16 and 20 kGy, which were then serially diluted and plated on respective agar to determine microbial growth. Efficacy of LEEI on microbial growth was determined by calculating the colony forming units (CFU/ml). After successful irradiation, conservation of antigenicity was tested using ELISA. LEEI was effective in reducing the microbial growth for both selected bacteria with complete inactivation at 10 kGy. The results showed that the antigenicity was conserved in both bacteria with a dose-dependent decrease seen with Pasteurella pneumotropica (44.9% at 4kGy, 31.81% at 12kGy, 23.24% at 16kGy) while an increased antigenicity was realized for inactivated Bacillus cereus at doses 4kGy (125.5%), 8kGy (160.2%) and 20kGy (115.6%). This study concludes that LEEI as a proof of principle shows a potential effectivity in reducing bacterial growth post-irradiation while maintaining antigenicity. The final recommendation is that with this potential evidence of effectivity and efficacy of LEEI, immunogenicity should be analyzed through in vivo testing in mice, which finally will translate in to investigations on its protective efficacy in humans in the development of vaccines.