Immunization with Genetically Modified Trypanosomes Provides Protection against Transmissible Spongiform Encephalopathies

Abstract

Transmissible spongiform encephalopathies are incurable neurodegenerative diseases, associated with the conversion of the physiological prion protein to its disease-associated counterpart. Even though immunization against transmissible spongiform encephalopathies has shown great potential, immune tolerance effects impede the use of active immunization protocols for successful prophylaxis. In this study, we evaluate the use of trypanosomes as biological platforms for the presentation of a prion antigenic peptide to the host immune system. Using the engineered trypanosomes in an immunization protocol without the use of adjuvants led to the development of a humoral immune response against the prion protein in wild type mice, without the appearance of adverse reactions. The immune reaction elicited with this protocol displayed in vitro therapeutic potential and was further evaluated in a bioassay where immunized mice were partially protected in a representative murine model of prion diseases. Further studies are underway to better characterize the immune reaction and optimize the immunization protocol.

Keywords: active immunization; neuroprophylaxis; prion diseases; trypanosomes immunization.

Figure 1

Flow cytometry to identify trypanosomes expressing the engineered variable surface glycoprotein (VSG). Following selection, stably transfected (PrP-tryp) and control (untransfected) T. brucei clones were stained with an anti-FLAG and an anti-PrP (6H4) antibody. A T. brucei clone with robust expression of the surface tag and the PrP peptide was identified.

Figure 2

Schematic representation of the immunization and the bioassay. Female BALB/c mice were immunized with T. brucei expressing PrP-VSG (N = 11) or WT-VSG (N = 15). Serum was harvested at regular time points throughout the immunization and the possible protective role of the immunization was evaluated in a bioassay using a murine scrapie strain.

Figure 3

Characterization of the immune sera. Immune serum was used as the primary antibody in Western blots to detect recombinant murine PrP (a) or PrP in brain homogenates from terminally ill Rocky Mountains Laboratory (RML) mice (b). The immune serum recognizes recombinant PrP, as well as total PrP (PrP^C and PrP^Sc) and PrP^Sc in brain homogenates. (c) The immune serum also recognized PrP expressed on the surface of DT-40 cells stably transfected to express PrP. PrP-tryp: serum from a mouse immunized with PrP-VSG expressing trypanosomes; Control: serum from a mouse immunized with WT-VSG expressing trypanosomes; 6H4: monoclonal anti-PrP antibody. Red boxes indicate the di-, mono- and unglycosylated forms of PrP. Data from a representative PrP-tryp and a control serum (second bleed) are depicted in all three panels.

Figure 4

Immunization provides partial protection in the murine animal model. (a) Representative Western blotting of splenic homogenates enriched in PrP^Sc from PrP-tryp immunized and control mice, using 6H4. PrP-tryp immunized mice accumulate less splenic PrP^Sc at the early stages of disease (a total of four PrP-tryp immunized and four control immunized mice were sacrificed at early timepoints). (b) Survival curves indicate that PrP-tryp-immunized (N = 7) mice survive significantly longer than control (Ν = 11) mice (left panel, Log-rank (Mantel-Cox) test, p < 0.0001). Immunization seems to result in different levels of protection, as two distinct subgroups with different survival curves (Log-rank (Mantel-Cox) test, p = 0.0177) can be identified (right panel).