Experimental Assessment of Possible Factors Associated with Tick-Borne Encephalitis Vaccine Failure

Abstract

Currently the only effective measure against tick-borne encephalitis (TBE) is vaccination. Despite the high efficacy of approved vaccines against TBE, rare cases of vaccine failures are well documented. Both host- and virus-related factors can account for such failures. In this work, we studied the influence of mouse strain and sex and the effects of cyclophosphamide-induced immunosuppression on the efficacy of an inactivated TBE vaccine. We also investigated how an increased proportion of non-infectious particles in the challenge TBE virus would affect the protectivity of the vaccine. The vaccine efficacy was assessed by mortality, morbidity, levels of viral RNA in the brain of surviving mice, and neutralizing antibody (NAb) titers against the vaccine strain and the challenge virus. Two-dose vaccination protected most animals against TBE symptoms and death, and protectivity depended on strain and sex of mice. Immunosuppression decreased the vaccine efficacy in a dose-dependent manner and changed the vaccine-induced NAb spectrum. The vaccination protected mice against TBE virus neuroinvasion and persistence. However, viral RNA was detected in the brain of some asymptomatic animals at 21 and 42 dpi. Challenge with TBE virus enriched with non-infectious particles led to lower NAb titers in vaccinated mice after the challenge but did not affect the protective efficacy.

Keywords: TBEV; cyclophosphamide; flavivirus; immunosuppression; mouse model; neuroinvasion; non-infectious virus particles; structural heterogeneity; tick-borne encephalitis; vaccine failure.

Figure 1

Survival and weight changes in BALB/c and ICR mice vaccinated with TBE vaccine Tick-E-Vac after challenge with 300 PFU/animal of TBEV strain Vasilchenko. (a,b) female BALB/c mice (n = 20 per group), (e,f) female ICR mice (n = 15 per group), (c,d) male BALB/c mice (n = 20 per group), (g,h) male ICR mice (n = 16 for vaccine group, n = 15 for control group). Animals were monitored for mortality (a,c,e,g), weight loss (b,d,f,h), and disease symptoms (data not shown) for 42 days after challenge. Error bars represent SEM. * All brain samples from dead mice were considered RNA-positive. Statistical significance for mortality data was determined by log rank test (**** p < 0.0001).

Figure 2

Survival and weight changes in BALB/c mice with and without low-dose Cy immunosupression. Mice were vaccinated with TBE vaccine Tick-E-Vac and challenged with 100 LD₅₀ of TBEV strain Vasilchenko. (a) Survival curves for vaccinated and control mice. (b) Weight changes after the virus challenge. Error bars represent SEM. Group Vac-Vac was vaccinated twice without Cy treatment (green); group Cy-Vac-Vac received Cy before the first vaccine dose (blue); group Vac-Vac-Cy received Cy after the vaccination and immediately before the challenge (pink); Virus—a control non-vaccinated group (violet). In all vaccinated groups, the vaccine was i/m administered twice (50 mkl/mouse) with a two-week interval; the virus challenge was carried out two weeks after the second vaccination. Cy was i/p administered twice at doses 60mg/kg and 30 mg/kg with a one-day interval. For all groups, N = 15. Data only for mice surviving till 21 dpi was used for weight curves. * All brain samples from dead mice were considered RNA-positive. Statistical significance for mortality data was determined by log rank test (**** p < 0.0001).

Figure 3

Seroconversion and levels of neutralizing antibodies (NAb) against TBEV vaccine strain Sofjin (&Sof) and the challenge strain Vasilchenko (&Vas) in sera of Cy-treated mice (low-dose scheme). (a) Seroconversion rate after the first vaccine dose and (b) after the second vaccine dose. NAb titers were measured in sera of mice (c) 10 days after the first vaccine dose, (d) 10 days after the second vaccine dose, and (e) one day after the challenge with Vas TBEV strain. The control group Vac-Vac did not receive Cy treatment (green); Cy-Vac-Vac group was treated with Cy before the first vaccine dose (blue); Vac-Vac-Cy was treated with Cy after two-dose vaccination and immediately before the challenge (pink). N = 7–8 mice per group. In (a–d) Vac-Vac group includes the data for Vac-Vac and Vac-Vac-Cy groups, since at this stage of the experiment they received identical treatment. * p < 0.05; ** p < 0.01; **** p < 0.0001.in Mann-Whitney test. Box plots indicate median and range.

Figure 4

Survival and weight changes in BALB/c mice with and without high-dose Cy immunosupression. Mice were vaccinated with TBE vaccine Tick-E-Vac and challenged with 100 LD₅₀ of TBEV strain Vasilchenko. (a) Survival curves for vaccinated and control mice. (b) Weight changes after the virus challenge. Error bars represent SEM. Vac-Vac group was used as a control without Cy treatment (green); Cy-Vac-Vac received Cy treatment before the first vaccine dose (blue); Cy-Vac-Cy-Vac was treated with Cy twice: before the first and the second vaccine doses (red); Vac-Cy-Vac received Cy treatment before the second vaccine dose (black) and Vac was vaccinated only once (orange). N = 12–15 animals per group. Data only for mice surviving till 21 dpi was used for weight curves. * All brain samples from dead mice were considered RNA-positive. Statistical significance for mortality data was determined by log rank test (*** p < 0.001; **** p < 0.0001).

Figure 5

Seroconversion and levels of neutralizing antibodies against TBEV strains Sofjin (&Sof) and Vasilchenko (&Vas) in sera of Cy-treated mice (high-dose scheme). (a) Seroconversion rate after the first vaccine dose and (b) the second vaccine dose. Nab titer (c) after the first vaccine dose, (d) the second vaccine dose, and (e) one day after the challenge with 100 LD₅₀ TBEV strain Vasilchenko: Vac-Vac group was used as a control without Cy treatment (green); Cy-Vac-Vac received Cy treatment before the first vaccine dose (blue); Cy-Vac-Cy-Vac was treated with Cy twice: before the first and the second vaccine doses (red); Vac-Cy-Vac was treated with Cy before the second vaccine dose (black); and Vac was vaccinated only once (orange). In (a) and (c) Vac-Vac group includes the data for Vac-Vac, Vac-Cy-Vac and Vac groups, Cy-Vac-Cy-Vac includes the data for Cy-Vac-Vac group since at this stage of the experiment they received identical treatment. N = 7–8 animals per group. * p < 0.05; ** p < 0.01; *** p < 0.001 obtained by Mann-Whitney test. Box plots indicate median and range.

Figure 6

NAb titers in vaccinated BALB/c mice against two TBEV strains Sofjin and EK-328 (a) after the second vaccine dose (b) on day 2 after the virus challenge with TBEV EK-328 strain (green) (n = 7–8) (Vac-Vac) and TBEV EK-328 strain heated at 37 °C for 48 h (Vac-Vac-heat), red) (n = 7–8). The virus infection dose was equalized to 30 LD₅₀ per mouse for both groups. *** p < 0.001 obtained by Mann-Whitney test. Box plots indicate median and range.

Figure 7

The values of log (GCP/PFU) for the virus samples of four TBEV strains: EK-328 (blue), Absettarov (Abs, green), Sofjin (Sof, yellow) and 256 (orange) after the reproduction (a) in PEK cell culture (CF) and in ICR suckling mouse brains (MB) after i/c inoculation. (b) GCP/PFU ratio of Absettarov TBEV strain after 7 days in Ixodes ricinus ticks. The experiment was carried out in 3–5 biological replicates. Error bars indicate SD. * p < 0.05; ** p < 0.01 obtained by Mann-Whitney test.