Pathogens Inactivated by Low-Energy-Electron Irradiation Maintain Antigenic Properties and Induce Protective Immune Responses

Abstract

Inactivated vaccines are commonly produced by incubating pathogens with chemicals such as formaldehyde or β-propiolactone. This is a time-consuming process, the inactivation efficiency displays high variability and extensive downstream procedures are often required. Moreover, application of chemicals alters the antigenic components of the viruses or bacteria, resulting in reduced antibody specificity and therefore stimulation of a less effective immune response. An alternative method for inactivation of pathogens is ionizing radiation. It acts very fast and predominantly damages nucleic acids, conserving most of the antigenic structures. However, currently used irradiation technologies (mostly gamma-rays and high energy electrons) require large and complex shielding constructions to protect the environment from radioactivity or X-rays generated during the process. This excludes them from direct integration into biological production facilities. Here, low-energy electron irradiation (LEEI) is presented as an alternative inactivation method for pathogens in liquid solutions. LEEI can be used in normal laboratories, including good manufacturing practice (GMP)- or high biosafety level (BSL)-environments, as only minor shielding is necessary. We show that LEEI efficiently inactivates different viruses (influenza A (H3N8), porcine reproductive and respiratory syndrome virus (PRRSV), equine herpesvirus 1 (EHV-1)) and bacteria (Escherichia coli) and maintains their antigenicity. Moreover, LEEI-inactivated influenza A viruses elicit protective immune responses in animals, as analyzed by virus neutralization assays and viral load determination upon challenge. These results have implications for novel ways of developing and manufacturing inactivated vaccines with improved efficacy.

Keywords: influenza A; low-energy electron irradiation; pathogen inactivation; vaccines.

Figure 1

Characterization of the low-energy electron irradiation (LEEI) inactivated material. (A) Influenza A (H3N8) was LEEI-irradiated with the indicated doses (10 kGy, 20 kGy, and 30 kGy) or left non-irradiated (0 kGy) and RNA was isolated. Size and composition was analyzed by Agilent Bioanalyzer. The time scale on the X-axis represents the migration time in seconds, while FU represents the fluorescence intensity of the sample; (B) Median hemagglutination-units (HAU) of three independent experiments. HA-assay with LEEI-treated influenza A (H3N8)-material using chicken red blood cells in PBS. Non-irradiated (0 kGy) and irradiated (10 kGy, 30 kGy, and 50 kGy) samples were tested in triplicate. HA activity was measured as HAU and plotted as median with range; (C) Analysis of the antigen structure after inactivation by ELISA influenza A (H3N8) was inactivated by LEEI (30 kGy) or by adding formaldehyde either to a final concentration of 0.1% and incubation at 4 °C for 16 h (FA short), or to a final concentration of 0.05% followed by incubation at 37 °C for seven days (FA long). Untreated virus served as a positive control. Samples were coated on ELISA plates and probed with serum from an influenza A infected pig. Background was subtracted. Relative standard error of the mean (SEM) is indicated. p-values were determined by one-way ANOVA (*** p < 0.001); (D) LEEI inactivation is applicable to different pathogens. Porcine reproductive and respiratory syndrome virus (PRRSV) LEEI-inactivated with 30 kGy (LEEI), EHV-1 LEEI-inactivated with 10 kGy and E. coli LEEI-inactivated with 5 kGy and respective non-irradiated controls (control) were coated on ELISA plates and probed with serum from an PRRSV infected pig, serum from an EHV-1 infected horse, or a polyclonal anti-E. coli rabbit serum, respectively. Background was subtracted and ELISA results were normalized to the levels observed when active pathogens were used as antigen. Data from at least two independent experiments, each performed in triplicates are shown. Relative SEM is indicated.

Figure 2

Antibody response in mice after vaccination (five animals per group). (A) purified influenza A (H3N8) from cell culture was coated on ELISA plates and probed with sera from vaccinated (LEEI) or non-vaccinated mice (control) after first vaccination (prime) and second vaccination (boost). Respective serum dilution is indicated below; (B) virus neutralizing antibody titers were analyzed by neutralization assays. Data points represent individual animals. Sera from vaccinated (LEEI) or non-vaccinated mice (control) after first immunization (left) and second immunization (right) were incubated with influenza A (H3N8) for 1 h and then added to fresh MDCK cells. After incubation for three days, cytopathic effect (CPE) was analyzed. CPE was absent at indicated antibody titers. Median of three independent experiments is indicated.

Figure 3

Challenge of vaccinated and unvaccinated mice. (A) weight loss in mice after intranasal challenge with a sub-lethal dose of influenza A (H3N8). Light grey circles: animals vaccinated with LEEI-inactivated material (LEEI), black triangles: non-vaccinated animals (control). N = 6 animals per group. SEMs are indicated. Unpaired t-test was used to calculate statistical differences (* p < 0.05); (B) determination of viral load in lung tissue of challenged mice 3 days post-infection (d.p.i.) by quantitative real-time PCR. Two independent experiments with identical experimental setups were conducted. Circles = non-vaccinated animals (control), rectangles = animals that received formaldehyde-inactivated material with two different treatments (left—short treatment with 0.1% for 16 h at 4 °C (FA short); right—long treatment with 0.05% for seven days at 37 °C (FA long)), triangles = animals that were immunized with LEEI-inactivated material (LEEI). p-value was determined by two-way ANOVA with bonferroni post-test (*** p < 0.001). The dashed line represents the detection limit of the assay.