TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES
 Immediately following Blood Product Safety  

Corporate Support:
Corporate Donor:

Sponsoring Publications:
American Journal of Hematology
IBPN
Intervirology

Sponsoring Association:
PPTA

Scientific Advisors
Dr. Nicola Boschetti, ZLB Bioplasma AG (Switzerland)
Dr. Larisa Cervenakova, American Red Cross
Dr. William N. Drohan, Clearant, Inc.
Dr. Bernard Horowitz, Horowitz Consultants, LLC
Dr. Thomas J. Lynch, Clearant, Inc.
Dr. Paul R. McCurdy, Consultant
Dr. Kathryn Martin Remington, Bayer Corporation
Dr. Hannelore Willkommen, Paul-Ehrlich-Institut (Germany)

Transmissible Spongiform EncephalopathiesSpeakers
Dr. Gerald Baron, National Institutes of Health
Dr. Paul W. Brown, National Institutes of Health
Dr. Neil Cashman, Caprion Pharmaceuticals
Dr. Larisa Cervenakova, American Red Cross
Dr. Andrzej Drukier, BioTraces, Inc.
Dr. Benoit Flan, LFB (France)
Dr. Luisa Gregori, VA Medical Center-Baltimore
Dr. Fiona Houston, Institute for Animal Health (UK)
Dr. Corinne Ida Lasmézas, Atomic Energy Commission, Service de Neurovirologie (France)
Dr. P. K. Nandi, Institut National de la Recherche Agronomique (France)
Dr. Alex J. Räber, Prionics, AG (Switzerland)
Dr. Robert Rohwer, VA Medical Center-Baltimore
Dr. Jiri G. Safar, University of California-San Francisco
Dr. Christopher J. Stenland, Bayer Corporation
Dr. Man-Sun Sy, Case Western Reserve University School of Medicine
Dr. Jaroslav Vostal, U.S. Food and Drug Administration
Dr. Stuart Wilson, Microsens Biotechnology (UK)

Session Topics Include:
Prion Science
Screening
Removal or Inactivation

 

PROGRAM

Tuesday, February 11

4:45-5:45pm Early Registration and Poster and Exhibit Setup

Wednesday, February 12

8:00am Registration, Poster and Exhibit Viewing, and Light Continental Breakfast

 

PRION SCIENCE

9:00 Welcome by Conference Chairpersons
Dr. Larisa Cervenakova, American Red CrossDr. Paul W. Brown, National Institute of Neurological Disorders and Stroke, National Institutes of Health

9:15 Effect of Membrane Association on Prion Protein Interactions
Dr. Gerald Baron, Visiting Fellow, Rocky Mountain Labs, National Institutes of Health
The prion protein (PrP) is a cell-surface glycoprotein that is bound to membranes via a glycolipid (GPI) anchor. Recent studies have revealed that PrP can also associate with membranes via an alternative method independent of the GPI anchor. The effects of these various types of membrane association on the ability of cellular PrP (PrP-sen) to interact with the pathological isoform (PrP-res) will be discussed.

9:45 Unusual Property of Prion Protein Unfolding in Solution
Dr. P. K. Nandi, Institut National de la Recherche Agronomique
In order to understand the unusual property of prion protein unfolding in nucleic acid solution, we have carried out structural studies of the prion protein in neutral salt solutions. We have observed that Hofmeister salts (Na2SO4, NaCl, Na2HPO4, etc.), which enhance folding (stabilize structure) of every protein studied in the literature for more than one hundred years, unfold PrPC probably by a new mechanism involving favorable ion-amide dipole interaction in the glycine rich unstructured N-terminal region of the protein and induce unfolding of the prion protein a-helices situated at protein's distant C-terminal end. The relevance of these observations on the prion protein structural destabilization involving protein helix 1 (the most hydrophilic of 10000 helices studied from the protein data bank) by nucleic acid would be discussed to explain PrPC structural conversion and amyloid formation in nucleic acid solution.

10:15 Transmission of Prion Diseases by Blood Transfusion in Sheep
Dr. Fiona Houston, Institute for Animal Health
We have developed a model for the experimental study of transmission of TSEs by blood transfusion in sheep. Using as donors sheep experimentally infected with bovine spongiform encephalopathy (BSE) or with natural scrapie, we have found significant transmission rates to susceptible recipient sheep at both pre-clinical and clinical time points.

10:45 Poster and Exhibit Viewing, Refreshment Break

11:15 Low Levels of Infectivity in Blood of Mice Infected with vCJD and Non-vCJD Strains of TSE
Dr. Larisa Cervenakova
Extensive report on an experimental study of vCJD agent transmissibility through blood components in mice will be presented. The results of the study with vCJD agent will be compared to the results of the other blood-infectivity studies in rodents. Our results indicate that vCJD and non-vCJD strains of human TSE have similar blood-related properties (incubation period, distribution, and infectivity levels in blood components) in mice.

11:45 Transmission of Bovine Spongiform Encephalopathy to the Nonhuman Primate Species Cynomolgus Macaque
Dr. Corinne Ida Lasmézas, Prion Research Group, Atomic Energy Commission, Service de Neurovirologie
We are working on the transmission of bovine spongiform encephalopathy to the nonhuman primate species cynomolgus macaque, which today represents the closest model available for the study of human vCJD. We are studying the primate-to-primate transmission of the BSE agent to examine the risk of iatrogenic transmission of the disease through subclinical vCJD carriers. We are investigating the distribution of PrPres in different organs, the efficiency of the intravenous route of infection compared to other routes, and the infectivity of different blood components at the preclinical and clinical stage of the disease.

12:15 Update on the Baxter Monkey TSE Study
Dr. Paul W. Brown
This presentation will update delegates on the new data gleaned from the Baxter monkey TSE transmission study.

12:30 Lunch (on your own)

 

SCREENING

2:00 Comments by Session Chairpersons
Dr. Larisa Cervenakova, Dr. Paul W. Brown

2:10 Establishment of Antemortem Tests for TSE Diagnosis Based on the Detection of the Abnormal Form of the Prion Protein in Body Fluids
Dr. Alex J. Räber, Director of Research, Prionics, AG
Prionics AG's recent work focuses on the establishment of antemortem tests for TSE diagnosis based on the detection of the abnormal form of the prion protein in body fluids. In vivo testing for TSEs should allow screening of cattle for subclinical cases of BSE as well as assessing the risk of transmitting prions via blood or blood-derived products.

2:40 Application of a Sensitive Western Blot Assay to Measure PrPSc Partitioning in Plasma Fractionation
Dr. Christopher J. Stenland, Bayer Corporation
A Western blot assay was developed that is specific, sensitive, and reproducible for the detection of PrPres, a surrogate marker for TSE infectivity. Our work is based on the established relationship of TSE infectivity and the presence of PrPSc/res. Several plasma fractionation steps from manufacturing were scaled down and spiked with brain homogenates from TSE infected hamsters, and the purification steps were performed. The partitioning of PrPSc in the Cohn-Oncley fractionation trunk and product specific steps were monitored by Western blot, and to varying degrees each step was shown to clear PrPSc. Follow-up bioassays were performed, and the data from these studies demonstrated the TSE infectivity tracked with PrPSc partitioning. The relevance of the hamster agent was investigated by performing selected fractionation steps with human vCJD and sheep scrapie brain homogenate as the spiking source.

3:10 Recent Developments in Testing for TSE in Animals and Humans
Dr. Man-Sun Sy, Professor of Pathology, Case Western Reserve University School of Medicine
The only reliable in vitro test for TSE is based on the resistance of the TSE agent to proteinase K (PK) digestion in vitro. This immunoblotting assay is time consuming and technically demanding. Furthermore, proteinase K digestion is difficult to control because it depends on the concentrations of the proteinase K resistant species in the brain homogenates and the amounts of PK used in the digestion. We have developed a capture ELISA assay that can distinguish TSE brain homogenates from non-TSE brain homogenates with a very high degree of sensitivity and specificity. This assay provides significant advantages over the currently available assays.

3:40 Poster and Exhibit Viewing, Refreshment Break

4:10 Demonstration of the Development and Application of a PrPres-Specific Ligand for Postmortem and Ultrahigh-Sensitivity Antemortem Screening
Dr. Stuart Wilson, Scientific Director, Microsens Biotechnology
We will present our work on the development of a PrPres-specific ligand. We have developed the ligand into different affinity formats including microtiter plate ELISA and bead-capture approaches. Data will also be presented that show that the capture ligand can replace the usual proteinase K digestion step in postmortem screening and results in an assay that is more robust and less prone to false positives. The bead-capture approach has been developed for antemortem screening and can be used for screening larger volumes of blood or urine. In conjunction with our novel ultrahigh-sensitivity detection label, we will present data that show that this approach has potential for blood screening.

4:40 An Epitope Selective for the Pathogenic Prion Protein
Drs. Neil R. Cashman and Wen-Quan Zou, Centre for Research in Neurodegenerative Diseases, University of Toronto

5:10 MultiPhoton Detection (MPD) Technique with Immunoassay (IA)
Dr. Andrzej Drukier, President and Chief Executive Officer, BioTraces, Inc.
IA/MPD techniques, developed by BioTraces, enable more sensitive and less expensive testing for a variety of biomolecules, including prions.

5:40 Panel Discussion with All Afternoon Speakers

6:00-7:00 Networking Reception

Thursday, February 13

8:00am Poster and Exhibit Viewing and Light Continental Breakfast

 

SCREENING

8:30 Comments by Session Chairpersons
Dr. Paul W. Brown and Dr. Larisa Cervenakova

8:40 Conformation-Dependent Immunoassays (CDI): An Update
Dr. Jiri G. Safar, University of California-San Francisco
Prions can be detected in vivo by bioassays in susceptible laboratory animals or by in vitro methods. For many years, detection of the proteolytic-resistant fragment of PrPSc by Western blot or immunohistochemistry represented the only means of identifying prions in tissue samples in vitro. Reliance on the complete enzymatic hydrolysis of ubiquitous and immunoreactive PrPC, however, has limited both the specificity and sensitivity of these assays. As a result, the assays were positive only when the titer of prions was high and detection of low levels of PrPSc has been difficult. To address these problems, we developed a rapid, specific, and highly sensitive method for the detection of PrPSc using a conformation-dependent immunoassay (CDI). Using the CDI, we were able to discriminate between PrPSc molecules from eight different prion strains propagated in Syrian hamsters (Safar, Wille et al. 1998). Because the CDI quantifies PrP isoforms by following antibody binding to both the native (N) and denatured (D) forms of PrP, it may be performed independently of proteolytic treatments. Subsequently, we applied the CDI in a sandwich protocol using high-affinity recombinant antibody fragments (recFab) to examine chronic wasting disease (CWD) and bovine spongiform encephalopathy (BSE) prions. As previously accomplished with Syrian hamster-adapted prion strains, the CDI discriminated between PrPSc conformers from BSE-infected cattle and Tg(BoPrP) mice as well as from CWD-infected deer and elk. In two-dimensional plots of D – N values and D/N ratios, BSE and CWD prions mapped in distinct areas, arguing that BSE and CWD prions are conformationally unrelated. The CDI also measured PrPSc in bovine brainstems with a sensitivity similar to that of end-point titrations in Tg(BoPrP) mice, which are ~10,000´ more sensitive to BSE prions than wild-type mice (Safar, Scott et al. 2002). The CDI can detect potentially large in vivo concentrations of protease-sensitive (s) PrPSc molecules. sPrPSc accumulates along with protease-resistant (r) PrPSc throughout the asymptomatic stage of prion infection. In hamster brains, sPrPSc was detected much earlier than rPrPSc and the fraction of accumulated sPrPSc differed by strain. When this sPrPSc fraction was plotted as a function of the incubation time, a linear relationship was found. Combined with the time course of prion infection in Syrian hamsters, Tg(MHu2M) and Tg(SHaPrP) mice, these results argue that differences in incubation times among strains may arise from distinct rates of PrPSc clearance rather than from different rates of PrPSc formation. This suggests that accelerating clearance may be an important strategy in treating prion diseases and warrants the revision of the definition of an infectious unit with respect to the number of PrPSc molecules. Clearly, the position of sPrPSc within the reaction coordinates of PrPC to PrPSc conversion and its role in prion infection require further investigation.

9:10 Assessment of Prion Partitioning in Plasma Protein Purification Using a Highly Sensitive Conformation Dependent Immunoassay (CDI) and Multiple Spike Approach
Dr. Henry Baron, Aventis Behring
Because of continued uncertainty as to the presence and, if present, the biophysical properties of the theoretical prion contaminant in plasma, we have assessed the partitioning of different biophysical forms of prion infectivity, including purified, non-membrane-bound pathogenic prion protein, in the processes used for plasma protein purification. A highly sensitive conformation dependent immunoassay (CDI) was used to measure prion levels in these studies. Our data show that different prion spiking agents can partition differently for different production steps, that many production steps have robust prion removal capacity for different spike forms, and that hamster brain-derived prion preparations relevantly mimic the partitioning of human prion preparations. An improved, sandwich CDI with enhanced sensitivity for human prions also will be described.

 

REMOVAL OR INACTIVATION

9:40 Expression of Cellular Prion Protein (PrPc) on Blood and Endothelial Cells
Dr. Jaroslav Vostal, Laboratory of Cellular Hematology, Division of Hematology, CBER, U.S. Food and Drug Administration
Protease resistant prion protein (PrPres) is thought to be the infectious particle of transmissible spongiform encephalopathy diseases (TSE) that propagates by physically interacting with cellular prion protein (PrPc) and imparting on it the altered, protease resistant conformation. The physical interaction with PrPc suggests that cells which express PrPc could bind and transport PrPres. We investigated the expression of PrPc on blood cells of animals that have been used in experimental TSE infections and normal humans. There are major differences in PrPc expression on different blood cell types in rodents, ungulates and primates. Unexpectedly, there are also large differences between primates and humans. The variability in PrPc expression on blood cells may contribute to the inconsistencies between TSE infectivity by blood components in different experimental animals and negative epidemiological studies in humans. Endothelial cells that line blood vessels may also contribute to the propagation and distribution of TSE infectivity through the organism. We investigated PrPc expression on endothelial cells and the release of PrPc on microvesicles. Such microvesicles could carry PrPres and TSE infectivity through the organism and may account for infectivity found in plasma. Co-authors: Paul Brown, MD*, Karel Holada, PhD, and Jan Simak, PhD

10:10 TSE Leukoreduction and TSE Removal by Nanofilters
Dr. Luisa Gregori, Head, Biochemistry Group, Molecular Neurovirology Lab, VA Medical Center-Baltimore
Universal leukoreduction of blood donations has been suggested and in some countries implemented, as a precautionary measure against risk of human exposure to TSE infectivity from blood and blood products. However, the effectiveness of this procedure in removing TSE infectivity has never been fully demonstrated. We conducted two validation studies with two in-line leukotrap systems, one for whole blood leukofiltration and the other for platelet-red cell leukofiltration. The preliminary results of these titrations will be presented.

10:40 Poster and Exhibit Viewing, Refreshment Break

11:00 Measurements of the TSE Agent Operational Size by the Planova Nanofilters
Dr. Robert Rohwer, Director of Molecular & Neurovirology, VA Medical Center-Baltimore
Nanofiltration is often the method of choice for viral clearance in biologicals and manufactured products. Removal of TSE infectivity by nanofiltration is complicated by the size heterogeneity of TSE infectivity in the tissue homogenates used to challenge the filters. We have used tandem serial filtration to investigate the size distribution and operational unit size of TSE infectivity in order to identify the maximum pore size capable of removing all TSE infectivity. Our measurement of the TSE agent's unit size is, to our knowledge, the most accurate yet reported for the agent. The overall implications of our results for any technology based on size exclusion separation of the TSE agent will be discussed.

11:30 Nanofiltration for Prion Removal for Factor VIII
Dr. Benoit Flan, Pharmacist, LFB
Data demonstrating the effectiveness of nanofiltration for removal of prions from Factor VIII will be discussed.

12:00 Inactivation of Scrapie Utilizing High Pressure
Dr. Paul W. Brown
Short (1- to 5-minute) pulses of high pressure (7,000-12,000 atmospheres) at starting temperatures of 90°C significantly reduce (3-6 log10 LD50) infectivity in 263K hamster-adapted scrapie-spiked meat products. Further tests under varying pressure-temperature conditions, and using additional kinds of tissues (including plasma products), are in progress.

12:30 Panel Discussion with All Morning Speakers

12:45 Close of Conference

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Hotel Information
Washington Marriott Hotel
1221 22nd Street, N.W.
Washington, DC 20037
T: 202-872-1500
F: 202-872-1424
Room Rate: $175 S/D
Cut-off Date: January 16, 2003
Please call the hotel directly to make your room reservation. Identify yourself as a Cambridge Healthtech Institute conference attendee to receive the reduced room rate. Reservations made after the cut-off date or after the group room block has been filled (whichever comes first) will be accepted on a space-and-rate-availability basis. Rooms are limited, so please book early.

Travel Information
Special Zone and Discount Fares have been established for this conference with United Airlines. Please call United Airlines Meeting Reservation Desk at 800-521-4041 and reference ID #579YS.

Call for Posters
Cambridge Healthtech Institute encourages attendees to gain further exposure by presenting their work in the poster sessions. Please fill out the registration form, with the poster title and primary author. To ensure inclusion in the conference CD, a one-page summary must be submitted and registration must be paid in full by January 10, 2002.   Click here for poster instructions

Call for Sponsors and Exhibitors
This multi-conference meeting provides excellent venues for companies wishing to target the leading researchers active in developing and implementing the latest advances and new strategies for the inactivation and removal of infectious agents in whole blood, cord blood, component concentrates, and plasma. CHI strongly encourages any company with new techniques, services, or products related to blood product safety and screening to consider sponsoring and/or exhibiting at this event. For additional information please contact Deborah Brooks (617) 630-1312, via email at dbrooks@healthtech.com or Angela Parsons (617) 630-1367, via email at aparsons@healthtech.com.

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