Low-Energy Electron Irradiation of Tick-Borne Encephalitis Virus Provides a Protective Inactivated Vaccine

Abstract

Tick-borne encephalitis virus (TBEV) is a zoonotic flavivirus which is endemic in many European and Asian countries. Humans can get infected with TBEV usually via ticks, and possible symptoms of the infection range from fever to severe neurological complications such as encephalitis. Vaccines to protect against TBEV-induced disease are widely used and most of them consist of whole viruses, which are inactivated by formaldehyde. Although this production process is well established, it has several drawbacks, including the usage of hazardous chemicals, the long inactivation times required and the potential modification of antigens by formaldehyde. As an alternative to chemical treatment, low-energy electron irradiation (LEEI) is known to efficiently inactivate pathogens by predominantly damaging nucleic acids. In contrast to other methods of ionizing radiation, LEEI does not require substantial shielding constructions and can be used in standard laboratories. Here, we have analyzed the potential of LEEI to generate a TBEV vaccine and immunized mice with three doses of irradiated or chemically inactivated TBEV. LEEI-inactivated TBEV induced binding antibodies of higher titer compared to the formaldehyde-inactivated virus. This was also observed for the avidity of the antibodies measured after the second dose. After viral challenge, the mice immunized with LEEI- or formaldehyde-inactivated TBEV were completely protected from disease and had no detectable virus in the central nervous system. Taken together, the results indicate that LEEI could be an alternative to chemical inactivation for the production of a TBEV vaccine.

Keywords: irradiation; tick-borne encephalitis virus; vaccine; virus inactivation; zoonosis.

Figure 1

Confirmation of inactivation of LEEI-TBEV samples used for mice immunization. Virus inactivation was performed using a LEEI dose of 20 kGy. Samples were transferred in triplicates to BHK-21 cells and after 3 days the supernatant was passaged onto fresh cells, followed by another passage three days later. Negative control= mock-infected cells; positive control= active TBEV undergoing the LEEI process without irradiation; P0 samples collected at 1 hour post-inoculation; P1= supernatant collected 3 days after inoculation; P2= supernatant collected 3 days after the first passage; P3= supernatant collected 3 days after the second passage. Collected cell culture supernatants were analyzed for TBEV RNA by RT-qPCR. Shown are mean values of triplicates ± standard deviations. The dotted line indicates the limit of detection (100 viral RNA copies).

Figure 2

Antigenicity of inactivated TBEV samples. Virus inactivation process was performed either with 20 kGy LEEI or with 0.05% formaldehyde (FA). The ELISA was performed with serum from two TBEV immune individuals. Untreated: virus before inactivation; 0 kGy: virus undergoing the LEEI-process without irradiation; blank: dilution buffer. Shown are mean values of three measurements in duplicates ± standard deviations. Statistical analysis was performed using Kruskal-Wallis test and Dunn’s test of multiple comparisons. No statistically significant differences were detected among the groups.

Figure 3

Humoral immune responses after immunization with different inactivated TBEV vaccines. BALB/c mice (n=8) were immunized three times with TBEV inactivated either with 20 kGy LEEI (squares) or 0.05% FA (dots). Sham-immunized mice (triangles) received only buffer and adjuvant. (A) scheme of the immunization experiment. (B, C) Binding antibodies (B) and antibody avidity (C) were analyzed in IgG-ELISAs with untreated purified TBEV virions as coating antigen. For avidity measurement, the IgG-ELISA was performed with and without an additional urea wash step after the antibody binding. Both signals were compared in order to calculate the relative antibody avidity. Each data point represents an individual mouse of the same experiment. Data derive from two independent ELISA-assays, each serum sample measured in duplicates per run. Mean values of the groups ± standard error of the mean (SEM) are indicated. (D) Neutralizing activities of mouse sera were measured in focus reduction neutralization tests. The dotted line represents the lower detection limit (FRNT50 = 20). Shown are the neutralizing titers of individual mice and the geometric mean of each group. Data derive from two independent FRNT50 assays. All data were tested for normal distribution by a Shapiro-Wilk test. Statistical analysis of normally distributed avidity data was performed by an unpaired t-test. Binding and neutralizing antibodies were analyzed using an unpaired Mann-Whitney U-test (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

Figure 4

Protective efficacy of inactivated TBEV vaccines against viral-challenge in mice. Immunized mice were infected two weeks after the third immunization with active TBEV. Mice were monitored daily for 14 days post-infection for body weight (A), and clinical score (B). Mice were euthanized upon reaching humane endpoints (marked by a cross) or latest at day 14 post-infection. Body weight and cumulative clinical score are presented as means ± standard errors. After euthanasia, brains and spinal cords were collected and homogenized for viral RNA load analysis using RT-qPCR (C) and quantification of infectious virus by a focus-forming assay (D). Shown are viral RNA copy numbers and infectious viral titers of individual mice, geometric means of the groups, and viral reduction compared to the sham-immunized group. Dotted lines indicate limit of detection (= 100 viral genome copies in C and 10 FFU/ml in D). Statistical evaluation of all data was performed using Kruskal-Wallis test and Dunn’s pairwise multiple comparison test. The analysis of weight loss and clinical score was performed for each day post-infection and the statistically significant differences are indicated at the corresponding days (∗p < 0.05; ∗∗p < 0.01).