High titers of mucosal and systemic anti-PrP antibodies abrogate oral prion infection in mucosal-vaccinated mice

Abstract

Significant outbreaks of prion disease linked to oral exposure of the prion agent have occurred in animal and human populations. These disorders are associated with a conformational change of a normal protein, PrP(C) (C for cellular), to a toxic and infectious form, PrP(Sc) (Sc for scrapie). None of the prionoses currently have an effective treatment. Some forms of prion disease are thought to be spread by oral ingestion of PrP(Sc), such as chronic wasting disease and variant Creutzfeldt-Jakob disease. Attempts to obtain an active immunization in wild-type animals have been hampered by auto-tolerance to PrP and potential toxicity. Previously, we demonstrated that it is possible to overcome tolerance and obtain a specific anti-PrP antibody response by oral inoculation of the PrP protein expressed in an attenuated Salmonella vector. This past study showed that 30% of vaccinated animals were free of disease more than 350 days post-challenge. In the current study we have both optimized the vaccination protocol and divided the vaccinated mice into low and high immune responder groups prior to oral challenge with PrP(Sc) scrapie strain 139A. These methodological refinements led to a significantly improved therapeutic response. 100% of mice with a high mucosal anti-PrP titer immunoglobulin (Ig) A and a high systemic IgG titer, prior to challenge, remained without symptoms of PrP infection at 400 days (log-rank test P<0.0001 versus sham controls). The brains from these surviving clinically asymptomatic mice were free of PrP(Sc) infection by Western blot and histological examination. These promising findings suggest that effective mucosal vaccination is a feasible and useful method for overcoming tolerance to PrP and preventing prion infection via an oral route.

Figure 1

Shows the plasma IgG titers in the 5 groups (the 4 vaccinated animals group plus controls) at T0, T1 and TF. Two-way ANOVA analysis showed that the difference between the groups was significant (p<0.0001). Bonferroni post-tests showed that the T1 values for the 4 vaccinated groups differed from their respective T0 values significantly (p<0.001). The plasma IgG values dropped at TF; however, they remained significantly above the T0 values (p<0.001). (* p<0.001; ns: not significant)

Figure 2

Shows the gut IgA titers in the 5 groups (the 4 vaccinated animals group plus controls) at T0, T1 and TF. Two-way ANOVA analysis showed that the difference between the groups was significant (p<0.0001). Bonferroni post-tests showed that the T1 values for the 2 vaccinated groups with a high T1 IgA titer differed from their respective T0 values significantly (p<0.001). The group with a high IgG and low IgA at T1 also differed significantly from the T0 IgA titer (p<0.05). ). At TF the two vaccinated groups with a high IgA at T1 differed significantly from their prior T0 value. (* p<0.001; + p<0.05; ns: not significant)

Figure 3

Shows the Kaplan and Meier survival curve of the different groups. At 400 days post-inoculation 100% of the animals in the high IgG, high IgA group (n=14) were free from clinical symptoms (solid red line in graph, p<0.0001 using the logrank test [GraphPad Prism, version 4; GraphPad Inc., San Diego CA]). By 205 days post-oral challenge with scrapie strain 139A all the animals in the control groups (n=20, solid blue line in graph) and in the low IgG, low IgA group (n=14, solid black line in graph) had shown clinical signs of prion infection which were confirmed by Western blotting. The mice in the low IgG, high IgA group (n=10, yellow short, long dashed line in graph) had a slightly longer survival (median survival was 198 days versus 194 for the control group), but this difference was not statistically significant. In the high IgG, low IgA group 4 out of the 12 mice (33%) were without clinical symptoms of infection at 400 days (green, short dashed line in graph, p=0.02 versus control group).

Figure 4

A shows a representative section through the dentate gyrus of the hippocampus showing the characteristic spongiform change of prion infection in a control animal (see arrows). In a clinically asymptomatic animal (figure 4B) there is an absence of pathology. Scale bar = 100 microns.

Figure 5

Shows a Western blot of proteinase K treated brain homogenates developed using anti-PrP 6D11 (Spinner et al., 2007). Each sample consisted of exactly 20 µg of brain homogenate which was incubated with 1 µg of proteinase K for 1 hr at 37°C, prior to loading onto gels. In lanes 1 and 2 brains from representative control mice were used. In lanes 3 and 4 brains from representative animals which were clinically sick and showed spongiform change on histological examination from the high IgA, low IgG group were used. There is no apparent difference in the level of the PrP^Sc bands in lanes 1 and 2 versus 3 and 4. In lanes 5 through 8 representative brains from mice sacrificed 400 days post 139A challenge from the high IgG, high IgA group were used. These animals had no clinical signs of prion infection and no PrP^Sc was detectable in their brains. The position of molecular weight markers is shown on the left side of the figure.