Project Leader:

Dr. Scott Napper

Project Leader:

Dr. Andrew Potter

Project Leader:

Dr. Philip Griebel

Overview:
The biological function of prion protein, and the mechanisms by which the infectious form of the protein cause disease, remain elusive despite intensive research efforts. Effective treatments and preventative therapies also remain out of reach. VIDO is working to reveal how natural prions develop into disease-causing forms, and how those infectious prions cause disease. To develop vaccines against prion diseases, the research will focus on an antibody-binding site on the surface of infectious prions in a variety of mammals. The conservation of this site among species makes it an ideal target for potential prevention and treatment of BSE, CWD, and other prion diseases. The project will use a patented vaccine system proven effective when vaccinating against a naturally occurring protein produced by the host, making it an excellent candidate for use in a prion vaccine.

VIDO is a member of .. , a Network of Centres of Excellence.

Background:
Transmissible spongiform encephalopathies (TSEs) represent the first characterized example of an infectious disease that is mediated exclusively by a protein. Prion diseases affect a range of mammals, including humans. Dr. Stanley Prusiner published a theory in 1982 about the cause of these unusual diseases, and coined the term “prion” from “proteinaceous infectious particle.”

Now endorsed by the Nobel Prize committee, the presumed cause of prion diseases is that a natural protein becomes malformed and then goes on to “infect” normal proteins of the same type, causing them to take on this unnatural shape. When the abnormal proteins accumulate, they cause a breakdown in brain matter, giving the tissue the “spongy” appearance seen in post-mortem examinations.

Although this novel mechanism of infection was proposed more than 20 years ago, fundamental questions about the protein and disease remain. This project aims to address some of these questions from two directions.

Comparative and Global Analysis of Prion Protein Physiology and Disease Pathogenesis
What is the function of cellular prion (PrPC)? What are the cellular mechanisms underlying disease progression and pathology? These questions quite likely have related answers. Efforts to define a biological role for PrPC on the basis of the cellular consequences of its absence have proven unsuccessful. Therefore, we will apply an alternate strategy through monitoring the cellular consequences of prion activation.

Immunoprophylaxis of Bovine Spongiform Encephalopathy
Prion diseases in a variety of mammalian species are characterized by the conversion of the normal cellular prion protein (PrPC) to an abnormal infectious form (PrPSc). A variety of assays have indicated an antibody-binding protein sequence (YYR) is found on the molecular surface of the abnormal form of the prion protein. Antibodies to this site react with PrPSc but not the normal form PrPC. We have a unique advantage in the search for a vaccine against prion diseases with our development of a patented vaccine carrier and adjuvant system that has been used previously to induce sustained, high-titre antibody responses to recombinant peptide antigens and self-proteins.

Objectives:
1. Develop vaccine candidates against infectious prions, focusing on the specificity, safety, and efficacy of a YYR-motif-specific vaccine in ruminants:

a) Construct, express, and purify these recombinant vaccine antigens and peptides
b) Carry out immunization studies in sheep to identify the optimal dose and formulation of recombinant protein required to consistently induce YYR-motif-specific antibody responses
c) Extend vaccine studies from sheep to cattle, focusing on vaccination protocols that induce sustained antibody responses; analyze the specificity of YYR motif antibody responses

2. Describe the cellular events associated with both pathological and endogenous models of stimulation by infectious prions:

a) Compare physiologically relevant cell types representing peripheral and neural phases of the disease
b) Examine species-specific responses of sheep and cattle for functional differences between scrapie and BSE
c) Examine intra-species variable responses within sheep with different susceptibilities to scrapie

Progress:
1) Genetic Screening of Sheep Flock: Within sheep populations there are natural genetic polymorphisms that confer differential susceptibilities to scrapie. In particular, three codons, 136, 154 and 171, have been linked to host susceptibility to scrapie; the A136R154Q171 allele confers susceptibility (OviSus) while the A136R154R171 allele is associated with resistance (OviRes). We genotyped 54 members of a sheep flock maintained by the University of Saskatchewan to identify scrapie-susceptible animals. The results demonstrated that this flock is an excellent source of both prion-resistant and -susceptible sheep. A breeding program for sheep with the susceptible (ARQ/ARQ) prion protein (PrP) genotype has been established and the first lambs will be available in March, 2007. These animals will be used for the proposed vaccine trials and to address issues regarding vaccine safety.

Construction of Recombinant Leukotoxin Vaccines: The genes for these four synthetic PrPSc epitopes were commercially synthesized and supplied in generic vectors. From these vectors, the appropriate fragments were sub-cloned as C-terminal additions to a proprietary epitope delivery system. The integrity of these vectors has been confirmed through sequencing.

Expression/Purification of Recombinant Lkt-PrPSc Epitope Vaccines: Chimeric epitope expression vectors were transformed into a BL21(DE3) Escherichia coli followed by growth and induction by standard protocols. The recombinant proteins are produced as inclusion bodies, facilitating purification as well as increased efficiency of elicited immune responses. The presence of the incorporated epitopes on the carrier proteins was confirmed through Western blot analysis, employing a monoclonal antibody specific to the engineered poly-histidine groups. A reactive band of the appropriate molecular weight was observed for each SN construct and the control did not produce a reactive species. The solubilized protein from isolated inclusion bodies was approximately 85% pure, which is of sufficient purity for immunization trials.

Immunization of Sheep: Sheep (n = 3) have received a primary immunization with 200 μg of purified protein for each of the four constructs (SN1-4). A secondary and tertiary immunization will be given at six-week intervals. Serum will be collected at regular intervals to monitor peptide epitope-specific antibody responses. These responses will be monitored using ELISAs being developed with synthetic peptides that are identical in sequence to the original SN1-4 constructs. Antibody responses to both the carrier protein and the peptides will be monitored following each immunization.

Cloning of Full Length PrPC: We have completed cloning and sequencing of the complete ORF of the ovine ARQ prion gene. This gene is being ligated with the carrier gene to create a chimeric PrPC protein. This vaccine construct will be used to induce PrPC–specific antibodies in ARQ/ARQ sheep. This model system will allow us to directly assess any potential risks, which may result if there is epitope spreading from the YYR-specific epitopes resulting in autoreactive PrPC–specific antibodies.

Establishment of Cell Lines: We developed alloreactive, CD4+ ovine T cell lines from three sheep and these T cell lines were used to develop flow cytometric methods to detect PrPC expressed on the cell surface. Two monoclonal antibodies (SAF-32 and 6H4) were purchased and used to confirm expression of detectable levels of PrPC. Equivalent levels of cell labelling were observed when the monoclonal antibodies were used either individually or to co-label PrPC on the same cell population. These T cell lines will now be used to develop a flow cytometric assay for the detection of autoreactive PrPC–specific antibodies in sheep serum following immunization with either chimeric LktA- PrPC or the LktA-PrPSc chimeric epitopes. Similar bovine T cell lines are also being developed for the same application when the BSE vaccine technology is transferred from sheep to cattle.

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