Advancing Prion Science: Guidance for the National Prion Research Program (2004)

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Assessment of Strategies to - ~ Prevent and Treat Transmissible Spongiform Encepha/opathies The prospects for successfully treating an established prion disease are so disheartening at present that the most effective strategy for man-aging the threat of transmissible spongiform encephalopathies (TSEs) is to avoid preventable exposure to the infectious agents. This chap- ter begins, then, by describing the strategies and policies adopted by the United States to prevent human and animal exposure to the agent of bovine spongiform encephalopathy (BSE) through food and feed. Next, the chap- ter describes means of preventing human and animal exposure to the agent of chronic wasting disease (CWD) in food and the environment in the United States. We then discuss the challenges of preventing exposure to TSE agents by inactivating them in blood, blood derivatives, and tissue, as well as on surfaces and in the environment; this section also addresses the potential for vaccination as a preventative strategy. The final section of the chapter re- views the therapeutic agents used to date in attempts to treat TSEs. The development of a successful therapy will require a level of innovation and effort as exceptional as that needed for the development of antemortem diagnostics, described in Chapter 4. MEASURES TO PREVENT THE BSE AGENT FROM ENTERING THE U.S. FOOD CHAIN The United States has built a multilayered preventive barrier during the past 15 years against the introduction of the BSE agent into the U.S. animal 160

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 161 and human food chains.) This barrier has three components (PL 107-9 Fed- eral Inter-agency Working Group, 20031: 1. Prevent the agent of BSE from entering the United States and infect- ing U.S. cattle. 2. If the agent of BSE penetrates U.S. borders and infects cattle, prevent the amplification of the agent throughout the U.S. cattle herd. 3. Prevent the exposure of U.S. residents to the agent of BSE through food and other products that come either partially or completely from cattle. According to a 3-year risk assessment by Harvard and Tuskegee Uni- versities, this trilayer barrier keeps animals and humans in the United States at very low risk of exposure to the BSE agent despite imperfect compli- ance with and enforcement of certain prevention strategies. "If BSE were somehow to arise in the U.S.," the study concludes, "few additional ani- mals would be infected, little infectivity would be available for human ex- posure, and the disease would be eradicated" (HCRA and TUCCE, 2001 :97-981. Although U.S. policies toward BSE effectively safeguard animal and human health, their effectiveness in protecting the U.S. economy is less cer- tain. According to a recent congressionally mandated analysis by the U.S. Department of Agriculture's (USDA) Economic Research Service, the iden- tification of just a single case of BSE in the United States could be more costly to this country than the BSE outbreak has been to the United King- dom to date (Mathews and Perry, 20031. The analysis does not give a dollar amount for the potential U.S. cost, but for perspective, farmers alone in the United Kingdom lost an estimated $700 million (Mathews and Perry, 2003), not to mention the losses to the beef processing and related industries. The authors of the USDA analysis based their prediction in part on the fact that the U.S. population is 5 times that of the United Kingdom, the U.S. beef sector is 10 times greater, and U.S. beef exports far exceed the amount of beef exported from the United Kingdom before the BSE outbreak. The very low risk that a case of BSE would enter the U.S. food chain and spread to other cattle would not mitigate the predicted financial im- pacts of a BSE case in the United States, the analysts forecasted, especially if the cow were a native-born animal (Mathews and Perry, 20031. Domestic consumption of beef products would likely decrease,2 U.S. renderers would iEDITORS' NOTE: After this report was completed, the first U.S. case of BSE was identi- fied in Washington State and was announced to the public on December 23, 2003. 2By contrast, the USDA Foreign Agricultural Service reports anecdotal evidence that Cana- dians responded to the BSE outbreak there by consuming more beef, not less (Myles, 2003).

162 ADVANCING PRION SCIENCE have to find new ways to use or dispose of cattle offal3 and other edible waste if these materials were banned from use in animal feed, and countries with BSE policies similar to those of the United States would stop importing U.S. beef and ruminant products. In addition, such industries as cosmetics, pharmaceuticals, and medical supplies that use livestock by-products or rendered products might need to find alternative sources of these materials for a period of time. Later in this chapter, we discuss the impact of the single Canadian case of BSE on that country's beef, cattle, and related in- dustries. This section describes the policies behind each layer of the United States' preventive barrier against the infectious agent of BSE (with the exception of surveillance, which is discussed in Chapter 61. Table 7-1 provides a chrono- logical overview of the implementation of many of these policies. In the discussion that follows, we note salient criticisms regarding certain policies and describe how federal agencies have responded to those criticisms. While policy recommendations and cost-benefit analyses are beyond the scope of this committee's mandate, we recommend research that would further strengthen the present safeguards against BSE. Restrictions on Imports It is widely believed that the exportation of BSE-infected cattle and cattle-derived products from the United Kingdom spread the infectious agent of BSE to countries in Europe and beyond. Beginning in 1989, therefore, USDA's Animal and Plant Health Inspection Service (APHIS) banned the importation of live ruminants4 and most ruminant products from all na- tions that had identified a case of BSE (USDA APHIS, 200331. Twenty- three countries fall into that category as of this writing (Office International des Epizooties, 20031. The Harvard/Tuskeegee risk assessment cites the import ban as one of the most effective tools for keeping the agent of BSE out of the United States (HCRA and TUCCE, 20011. USDA expanded the ban in 1997 to all of Europe, regardless of whether a country had reported a case of BSE (USDA APHIS, 200331. The ban was further expanded the following year to include any country "at risk" for BSE, meaning countries that conduct inadequate surveillance for BSE or regulate imports related to BSE in a less restrictive manner than does the United States (USDA APHIS, 19991. Subsequently, after the European Union 30ffal is the parts of butchered animals not processed into human food. These parts gener- ally include blood, internal organs, legs, heads, and spinal cords (Harlan, 2003). 4Ruminants are hoofed, even-toed, usually horned animals that characteristically have a four-chambered stomach and chew their cud.

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 163 (KU) announced in 2000 that feed made in EU countries from the offal of nonruminants may have been contaminated with the agent of BSE, USDA prohibited the import of all rendered protein and rendering wastes originat- ing from or processed in the KU, regardless of the animal species (USDA APHIS, 2003d). A 2002 report by the U.S. General Accounting Office (GAO) on U.S. vuinerabilities to BSE asserts that the United States lacks sufficient capacity to inspect all cattle imports, a weakness in the enforcement of the import bans (GAO, 2002~. USDA responded with a description of several efforts under way to remedy this problem: · USDA proposed using a portion of its fiscal year 2003 budget to increase the number of its inspectors at ports of entry from 2,500 to 4,000 people (USDA and Department of Health and Human Services EDHHS], 2002~. · To "strengthen coordination and documentation" among agencies that inspect products at U.S. ports of entry, USDA noted that it had ob- tained funds through the 2002 Defense Appropriations Act to integrate its computer technologies with those of the other relevant agencies (USDA, 2002:1~. · USDA would invest in new detection systems, such as x-ray equip- ment (USDA and DHHS, 2002~. Feed Ban While the import bans described above attempt to keep BSE out of the United States, restrictions on the ingredients of feed products intended for ruminants aim to prevent BSE from spreading in the United States should it be introduced through imported goods, through a spontaneous case of BSE in a U.S. cow, or through other means. In 1997, the U.S. Food and Drug Administration (FDA) prohibited the use of most mammalian protein in animal feed intended for ruminants (FDA CFSAN, 19971. This prohibition is often termed simply the feed ban. There is a consensus among scientists that cattle can contract BSE by eating animal feed made from the offal of scrapie-infected sheep or of BSE- infected cattle. This opinion stems largely from epidemiological work by Anderson and colleagues (1996), who conclude that the widespread con- sumption of cattle feed contaminated with the infectious agents of scrapie and BSE was the most likely cause of the BSE epidemic in the United King- dom. The Harvard/Tuskegee risk assessment indicates that the feed ban is one of the most important elements of the U.S. barrier against BSE. In fact, the authors conclude, the effectiveness of the feed ban influences the risk of

164 ADVANCING PRION SCIENCE TABLE 7-1 Measures Taken by the United States to Prevent the Introduction, Spread, and Consumption of the Infectious Agent of BSE Date Measure Taken 1987 BSE made a reportable disease. 1989 Ban on importation of live ruminants and most ruminant products from BSE- reporting countries instituted. 1990 USDA's Animal and Plant Health Inspection Service (APHIS) launches active surveillance for BSE and a BSE-education program.a 1992 U.S. Food and Drug Administration (FDA) recommends that manufacturers of dietary supplements avoid materials that could contain BSE or scrapie . . . . 1ntectlvlty. 1993 Nonambulatory cattle added to targets of BSE surveillance. FDA requests that most bovine source materialsb used in the manufacture of regulated products come from scrapie-free countries. 1994 FDA requests that bovine-derived materials for animals, cosmetics, or dietary supplements come from BSE-free countries. 1997 Ban on importation of live ruminants and most ruminant products from all of Europe instituted. FDA bans the feeding of most mammalian proteins to ruminants. FDA requests that bovine gelatin from countries reporting BSE not be used in certain products.c 1998 Ban on importation of ruminants and ruminant-derived products from countries at risk for BSE instituted.d Vermont quarantines two flocks of imported sheep possibly exposed to the BSE agent from contaminated feed in Europe. 1999 2000 USDA proposes a new rule to intensify scrapie-eradication efforts. Ban on import of rendered animal protein products originating from or processed in Europe instituted. BSE surveillance more than doubles from 1,300 to nearly 2,700 cattle brains tested. USDA seizes Vermont sheep after four die with an atypical TSE of foreign origin. BSE to the United States more than any other factor. They note: "A single breach of the feed ban can introduce . . . cattle to a substantial amount of BSE infectivity" (HCRA and TUCCE, 2001:97~. Challenges of Enforcing the Feed Ban Before and during 2001,FDA had serious problems with monitoring and enforcing compliance with the feed ban. A significant percentage of animal rendering plants and feed mills failed to meet at least one major requirement from 1998 through 2000, according to an FDA report on the

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs TABLE 7-1 Continued 165 Date Measure Taken 2001 Precautions enacted to protect safe, edible ruminant products from contamination while passing through countries reporting or at risk for BSE en route to the United States. 2002 Food Safety and Inspection Service (FSIS) issues a directive for routine inspection of advanced meat recovery (AMR) systems and for regulatory actions if spinal cord is detected in beef products produced by AMR. 2003 USDA APHIS solicits public comment on ways to control the risk that dead and nonambulatory ruminants could facilitate the spread of BSE.e USDA APHIS closes a loopholef to prevent the importation of live ruminants from Canada immediately after the May 20 announcement of a BSE-positive cow in Canada. On August 8, USDA lifts part of the import ban on Canada by allowing the importation of hunter-harvested wild ruminant products intended for personal use and accepting applications for import permits for a number of ruminant products." aThe education provided involves teaching veterinarians, farmers, and others who work with cattle to recognize the clinical signs of BSE. bExcluding gelatin. CIncludes injectable, implantable or ophthalmic products. Also, FDA asked that manufacturers take special precautions when using gelatin for oral and topical use. An at-risk country is one that conducts inadequate surveillance for BSE or that regulates imports related to BSE in a less restrictive manner than does the United States (USDA APHIS, 1999). eSource: USDA APHIS (2003b). fThe original rule had exempted certain regions, including Canada, under certain circumstances, from the requirement to obtain a permit to import live ruminants into the United States (USDA APHIS, 2003a). The import ban automatically applies to ruminant meat, ruminant meat products, and ruminant by-products from Canada as of May 20. "Source: USDA (2003c). SOURCES: Adapted from Brown et al. (2001) and USDA APHIS (2003d). 9,947 inspections of the rendering plants, feed mills, and related businesses5 conducted during those years (FDA CVM, 2001a). For instance, the report states that 28 percent of the inspected rendering plants lacked a system to prevent commingling of mammalian protein with other materials, and 20 percent of the inspected, FDA-licensed feed mills did not place a required 5These include ruminant feeders (operations that feed and care for ruminants), and protein blenders (GAO, 2000; FDA, 2001b).

166 ADVANCING PRION SCIENCE caution label on animal feed containing mammalian protein. However, these data do not capture the true rate of compliance because, according to the report, state and FDA officials had not inspected 30 to 40 percent of all U.S. renderers and feed mills (FDA CVM, 2001a). The Harvard/Tuskegee risk assessment addressed these shortcomings and incorporated them into its analysis. Subsequently, FDA boosted its ef- forts by inspecting more firms that handled mammalian protein and by reinspecting more of the firms previously found to be out of compliance (FDA CVM, 2001b). FDA's Center for Veterinary Medicine (CVM) gave businesses easy access to the checklist used by inspectors to determine com- pliance with the feed ban by placing a link to the checklist on the center's Web site (FDA CVM, 2001c). CVM also hired a contractor to restructure the database used to manage the information reported by the state officials and FDA field officers who conducted inspections for compliance with the feed ban (FDA CVM, 2001b). Nevertheless, GAO's 2002 report sharply criticized FDA for its poor enforcement of the feed ban and for its "severely flawed" database, and recommended a number of ways in which the agency could improve compliance rates (GAO, 20021. FDA continued to improve its methods of enforcement. Feed mills that used mixed-species meat and bone meal came under increasing scrutiny because of the risk that mammalian protein could contaminate feed des- tined for ruminants (personal communication, D. Harlan, Excel Food Solu- tions Company, March 25, 20031. Some firms decided to stop using mam- malian proteins altogether. By March 2002, CVM reported, the compliance rate for 2,153 U.S. firms handling materials prohibited for use in ruminant feed had reached 95 percent (FDA CVM, 20021. The next month, CVM began using a new database to better manage the information on nation- wide inspections and enforcement activities related to the feed ban. While criticisms of enforcement of the feed ban had subsided by 2003, they had not been altogether eliminated. At least one major U.S. rendering firm, Darling International Inc. of Irving, Texas, stated in February 2003 that FDA should take "more vigorous enforcement actions against viola- tors" of the feed ban (Ransweiler, 2003:21. At the same time, industries affected by the feed ban have taken steps to monitor themselves and to make changes that reduce the risk of transmitting the BSE agent to rumi- nants. For instance, in 2001 the American Protein Producers Association and the American Feed Industry Association began to hire outside auditors to conduct inspections of plants and mills (Ransweiler, 20031. A number of firms subject to the feed ban stopped using mixed-species meat and bone meal (Harlan, 20031. Some producers voluntarily stopped feeding mamma- lian-derived meat and bone meal to all their livestock, reducing the risk that ruminants on a farm would accidentally be given the banned feed. The

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 167 combination of stronger FDA enforcement and self-monitoring by industry bolsters the effectiveness of the feed ban. Tools to Detect Mammalian Protein in Animal Feed An additional way to prevent ruminants from eating feed containing mammalian protein is for ruminant producers to test their purchased feed for the banned material. At least one test that detects some banned material has been on the U.S. market since 2002: Agri-Screen~ for Ruminant Feed, manufactured by Neogen Corp. of Lansing, Michigan (Neogen Corp., 2002a). According to Neogen, its product enables feediots, dairies, market- ers of feed products, regulators, and auditors to verify that ruminant feed and feed supplements do not contain ruminant muscle proteins, a marker for the presence of ruminant tissue. Since the product does not detect mam- malian muscle protein in general, however, the test cannot verify whether a sample of feed made for ruminants is in compliance with the feed ban. The company describes Agri-Screen as a single-step, lateral-flow immunochromatographic assay (Neogen Corp., 2002b). It consists of an absorbent strip with a reagent area containing color-tagged antibodies that are specific to heat-stable ruminant muscle protein, and a control area far- ther upstream. Like a pregnancy test, the strip wicks the extract through the reagent and control areas. Within about 10 minutes, a colored line always forms in the control zone, but a second line forms in the reagent zone only if the feed sample contains ruminant muscle protein. Although the com- pany claims the test can detect ruminant muscle protein present in concen- trations as low as 1 percent of a sample, an industry source who has used the test says its lower limit in practice ranges from 1 to 5 percent (personal communication, D. Harlan, Excel Food Solutions Company, May 20031. Feed tests such as Agri-Screen provide a worthwhile, additional line of defense against the introduction of BSE into the food chain. It would be even better to have a test that could detect mammalian protein, not just ruminant protein. The development of such products for an affordable price should be encouraged, as should their use by farmers. Additional Ways to Prevent the BSE Agent from Entering Ruminant Feed Policy recommendations are beyond the charge of this committee. How- ever, it is worth noting that additional precautions beyond those in the FDA feed ban would further reduce the risk of amplification of the BSE agent should the disease arise in the United States. For instance, FDA could pro- hibit the use of mammalian protein in feed for all animals, not just rumi-

168 ADVANCING PRION SCIENCE nants. The United Kingdom instituted such a policy6 in 1996, and the EU7 followed in 2001 (Brown et al., 2001~. This more stringent measure makes sense in countries that, unlike the United States, have diagnosed cases of BSE or are at high risk for the disease. At least one challenge posed by the potential prohibition of mammalian protein in animal feed is to find alternative means of eliminating the 3.6 billion pounds of ruminant meat and bone meal left over from meat pro- cessing each year (Harlan, 2003~. Present U.S. research into alternative fu- els may offer at least a partial solution. Some scientists in this field are developing an experimental industrial boiler powered by agricultural by- products, including meat and bone meal, blood meal, and tallow (The En- ergy Institute, 2002~. Although the boiler may be a more efficient disposal mechanism than incineration, it would yield less energy than the amount required to create the meat and bone meal from producing feed, to raising animals, to processing parts into by-products (personal communication, D. Cliver, University of California, Davis, Tune 21, 2003~. The potential prohi- bition of mammalian protein in all animal feed also would challenge animal producers to find alternative affordable sources of nutrients for their live- stock. How Beef Processors Prevent the BSE Agent from Entering the Food Chain The beef processing industry and USDA have developed procedures and regulations to prevent tissue infected with the BSE agent from entering the food chain should a cow infected with the BSE agent go undetected on the farm and be sent to slaughter. These measures are not foolproof, how- ever. The sheer number of cattle involved U.S. beef processors slaughtered more than 36 million cattle in 2002 (see Figure 7-1) makes BSE detection at the slaughterhouse a formidable task. Beef processors reduce the risk of BSE-infected tissue entering the food chain by focusing on three aspects of their operation: procurement, antemortem inspection, and the removal of central nervous system (CNS) tissue. Specifically, the United Kingdom bans mammalian meat and bone meal from all animal feed and fertilizer (Brown et al., 2001). 7The EU prohibits the use of most animal protein in feed for any farmed animal species. Exempted proteins include those in milk, blood, and gelatin (Brown et al., 2001).

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 35 30 25 In o 20 . _ 15 10 5 o Calves Fed Cattle Mature Cattle 3% 80% 17% 169 Dairy > 8 yr 1% FIGURE 7-1 The 36.75 million U.S. cattle slaughtered in 2002 comprised calves (3 percent); fed cattle (80 percent); and mature cattle (17 percent), which included a small percentage of dairy cattle older than age 8. Fed cattle are 1 to 2 years old. Mature cattle are more than 2 years old. SOURCE: Harlan (2003~. Procurement Controls Some processors have limited their risk of exposure to BSE through company policies that specify the kinds of cattle that will or will not be purchased. For instance, a processor may decline to purchase nonambula- tory cattle (Harlan, 2003), that is, cattle that cannot rise from a recumbent position. These so-called downers can no longer stand because they are ill, and BSE is always suspect as the cause of illness in downer cattle. As noted in Chapter 6, nonambulatory cattle are the targets of active U.S. surveil- lance for BSE. Other procurement controls include buying only cattle of North Ameri- can origin (Harlan, 2003), although, as noted in Table 7-1, the United States banned the importation of cattle from Canada as of May 2003 because a case of BSE was discovered in Alberta (USDA and FDA, 20031. Buyers may also request that the producer certify the BSE-free status of an animal, and such certification is provided frequently. However, the committee sees little value in this sort of certification at present because only a neuropathologi- cal exam can establish the BSE-free status of a cow. An antemortem test would make the BSE-free certification of live cattle meaningful.

170 Antemortem Inspection ADVANCING PRION SCIENCE Officials from USDA's Food Safety and Inspection Service (FSIS) in- spect all incoming cattle at all U.S. slaughterhouses for signs of neurological disease (USDA and FDA, 2003~. In general, if an animal shows such signs, it is condemned, and its meat may not be used for human consumption (HCRA and TUCCE, 2001~. However, if the signs are not pronounced or typical, an inspector may designate the animal as suspect but not condemned (personal communication, D. Cliver, University of California, Davis, July 2003~. After FSIS notifies USDA Veterinary Services of the suspect animal, laboratory staff at one of USDA's 15 National Veterinary Service Laborato- ries analyze the animal's brain tissue for evidence of BSE or some other TSE. FSIS tracks instances of antemortem or postmortem condemnation due to signs of disease (HCRA and TUCCE, 2001~. As mentioned earlier, no case of BSE has been identified in the United States to date. However, antemortem inspections can identify only clinical cases; they will not identify infected cattle during the incubation period, which lasts 2 to 8 years. The Harvard/Tuskegee risk assessment could not determine with certainty what percentage of clinical BSE cases inspectors might miss. In fact, the authors note that this was one of the most impor- tant sources of uncertainty behind the study's estimates of human exposure to the BSE agent. Condemned animals are rendered or incinerated (HCRA and TUCCE, 2001~. Rendered by-products could be turned into feed for nonruminant animals or an ingredient for cosmetics, among other products. Removal of CNS Tissue from Slaughtered Cattle BSE infectivity becomes concentrated in CNS tissue during the later stages of the disease. Therefore, in case a BSE-infected animal should fail to be detected by antemortem inspection, meat processors can reduce the risk of human consumption of the BSE agent by removing all CNS tissue from cattle. The air-injection captive bolt pistol, a too! used to render cattle uncon- scious before slaughter, has been implicated in the inadvertent spread of CNS tissue to the blood and thereby to the heart, lungs, and liver. The pistol would thrust a bolt under high pressure into the skull of an animal to render it unconscious (TSE BSE Ad Hoc Group,2001). Projecting a volume of air into the cranial cavity at high speed would displace small but visible pieces of brain into the bloodstream. Most U.S. meat processors have not used air-injection stunning devices since at least 2000 (HCRA and TUCCE, 2001~. FSIS plans to complete a direct final rule in 2003 prohibiting the use of those devices (USDA, 2003b). From the standpoint of BSE risk, a safer

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 171 too! is the nonpenetrating captive bolt stunner, which literally knocks the animal unconscious without penetrating the skull. Once the animal has been slaughtered and exsanguinated, the body is split open. At some plants, workers vacuum the spinal cord out of the spi- nal canal, then visually check for any remaining spinal cord and remove what is found using a hand-held scraper. However, this method does not remove dorsal root ganglia, which are directly in contact with the spinal column and would likely contain BSE infectivity if the spinal cord did. Other plants use a saw that removes the intact spinal column. Some processors conduct hourly quality checks to verify the complete removal of the spinal cord. In the 1990s, the beef processing industry embraced a new technology called advanced meat recovery (AMR). The technology mechanically recov- ers from bones meat that otherwise would be rendered or discarded, in- creasing the amount of meat obtained from and thus the value of each animal. The use of AMR has created controversy, however. Opponents have asserted that the technology slices residual spinal cord tissue off neck bones and backbones, and that beef processors have mixed that CNS tissue with muscle tissue to sell as meat. As early as 1997, USDA FSIS issued a directive explicitly reminding beef processors that "product that contains spinal cord does not come within the definition of 'meat' in . . . the regulations" (FSIS, 19971. The directive instructed inspectors to determine whether processors were com- pletely removing spinal cord from neck and back bones before sending those bones to AMR. If a processor did not appear to be doing so, the inspector was to send a quarter-pound (113.4 gram) sample of finished product to an FSIS laboratory for verification. The GAO (2002) report mentioned earlier asserted that USDA had not rigorously enforced this directive, that inspec- tors tested AMR-derived products too infrequently, and that FSIS did find spinal cord tissue in a significant number of samples taken. In 2002, FSIS surveyed 34 beef processing plants that use AMR systems and found that approximately 35 percent of the final product samples tested positive for spinal cord and associated tissues (FSIS, 2003b). Consequently, FSIS issued a directive in December 2002 with new instructions for the "routine" sampling of beef products produced by AMR and new enforce- ment actions should spinal cord be found in those samples (FSIS, 2002, 2003:11. On the day the directive took effect March 3, 2003 FSIS an- nounced that the survey had provided enough data to support the creation of a new rule on AMR systems (FSIS, 2003b). This rule will include specifi- cations for the removal of CNS and associated tissues. Meanwhile, FSIS determined that the amount of noncompliance with existing rules regarding AMR and spinal cord tissue demanded that the 2002 directive be substan- tially rewritten "to reiterate that establishments whose AMR system repeat-

72 ADVANCING PRION SCIENCE edly fails to produce product that is free of spinal cord will not be allowed to produce AMR meat from beef vertebrae, and that product containing spinal cord tissue will not be allowed to enter commerce labeled as meat" (FSIS, 2003a). An effective, inexpensive, and convenient test for verifying the absence of CNS tissue in a beef product is the Ridascreen~ glial fibrillary acidic protein (GFAP) test developed by Schmidt and colleagues (personal com- munication, D. Cliver, University of California-Davis, March 20, 2003; Schmidt et al., 19991. GFAP is the most abundant protein in the glial fila- ments of differentiated astrocytes, which appear only in CNS tissue. The test, an enzyme-linked immunosorbent assay, can detect as little as 1.0 ng of CNS tissue in a sample (Schmidt et al., 19991. According to an industry source whose company uses the test as back-up verification for spinal-cord removal, the assay reliably detects CNS tissue concentrations as low as 0.1 percent of raw meat (Harlan, 20031. This sensitivity is sufficient to keep BSE risk materials out of the food supply in BSE-free countries such as the United States (personal communication, D. Cliver, University of California- Davis, March 20, 20031. An independent study found that the Ridascreen~ Risk Material 10/5 test is 10 times more sensitive than another CNS-tissue assay, the ScheBo~ Brainostic™ kit, which uses immunoblotting to detect a different CNS-spe- cific antigen (Hajmeer et al., 20031. However, the Ridascreen~ test cannot distinguish between GFAP from cattle and GFAP from pigs. This means the test would yield a positive result if a meat product containing tissue from both cattle and pigs included porcine CNS tissue. Recommendation 7.1: Fund research to improve rapid, accurate, and affordable screening assays for central nervous system (CNS) tissue such that the assays can specifically identify CNS material from cattle in processed meat products. [Priority 218 Rendering of Ruminant Tissue and the Potential Spread of the BSE Agent Many animal rendering plants use discarded tissue from multiple sources to manufacture a variety of edible products for animals and hu- mans and inedible products for people. These products include meat-and- bone meal (MBM), poultry feed, gelatin, and cosmetics ingredients. The raw materials that are rendered in the United States include certain animals 8The committee denotes each recommendation as priority level 1, 2, or 3 based on the criteria and process described in the Introduction.

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 173 that were condemned during slaughter surveillance, leftover cattle tissue from beef processors (Harlan, 2003), and plate waste (HCRA and TUCCE, 2001). According to the Harvard/Tuskegee risk assessment, the rendering of dead-on-farm and downer cattle is one of the most likely routes by which the BSE agent would enter the animal feed chain (HCRA and TUCCE, 20011. Present U.S. policies permit the rendering of these at-risk cattle for further processing into animal feed. By contrast, scrapie-suspect sheep and cattle condemned because of neurological signs are incinerated (personal communication, L. Detwiler, independent consultant, August 20031. USDA APHIS acknowledges that if BSE or scrapie were present in the rendered tissue of dead-on-farm cattle, downer cattle, or sheep, and if that rendered material were used in cattle feed (deliberately or accidentally in violation of the feed ban), healthy cattle could contract BSE (USDA APHIS, 2003b). In January 2003, USDA APHIS wrote, "EW]e believe that dead stock and downer animals represent the most significant potential pathway that has not been addressed in previous efforts to reduce BSE risks" (USDA APHIS, 2003b:27041. Another possible source of TSE infectivity in rendered material is the offal of scrapie-infected sheep or BSE-infected cattle that are presymptom- atic. Other countries Canada most recently have dealt with this issue by requiring that ruminant tissues known to harbor TSE infectivity be excluded from the human food and feed chains. This so-called specified risk material (SRM) includes the brain, skull, eyes, tonsils, vertebral column, dorsal root ganglia, and distal ileum (CFIA and Health Canada, 2003; Government of Canada, 2003b). A review of U.S. policy on SRM would be appropriate. Plate waste represents a third potential source of TSE infectivity in ren- dered material. Plate waste consists of food products that were inspected by FSIS or an equivalent state agency, cooked, and presented for humans to eat (e.g., leftovers from meals served to restaurant patrons) (HCRA and TUCCE,2001). Such material may contain CNS tissue from sheep or cattle. The Harvard/Tuskegee risk assessment considered plate waste to be a low- risk source of BSE infectivity, primarily because about 90 percent of all plate waste consists of bakery products and because it is "extremely un- likely" that plate waste would contain high-risk animal tissues (HCRA and TUCCE, 2001:32~. Yet USDA APHIS expressed support for an unsuccess- ful effort by FDA to amend regulations such that plate waste would be prohibited from ruminant feed (Medley, 19971. The committee believes such a prohibition should be reconsidered. The rendering processes at U.S. plants would not eliminate BSE intec- tivity. In most cases, the raw materials are heated for a specified time at a temperature and pressure that would reduce the infectivity of the BSE agent if it were present by 1 to 2 logs (Harlan, 2003; USDA APHIS,

174 ADVANCING PRION SCIENCE 2003b). Fewer than 5 percent of rendered animals undergo processing that would reduce BSE infectivity by 3 logs (USDA APHIS, 2003b). It is note- worthy, however, that part of a respected hypothesis on the origin of the BSE outbreak in the United Kingdom is that changes made in the 1970s and 1980s to rendering processes may have permitted the scrapie agent to retain its infectivity, whereas before those changes were made, rendering processes had inactivated the agent (Wilesmith et al., 19881. Later in this chapter, we discuss the few successful methods of inactivating the TSE agent and rec- ommend research to develop new approaches to this problem. Renderers, like beef processors, may reduce the risk of spreading the BSE agent by accepting tissue only from ambulatory cattle of U.S. origin that have passed antemortem inspection at the abattoir. In addition, some rendering plants and beef processing operations are run by the same com- pany, and these companies may render only tissues from their own beef processors to control the quality of BSE risk-reduction through the entire process. In addition, some rendering plants may divert CNS tissues from mature cattle (Harlan, 20031. However, the extent to which U.S. renderers take these voluntary risk-reduction measures is unclear. In early 2003, USDA APHIS issued an advance notice of proposed rulemaking indicating that it wants to develop a new regulation to reduce the BSE risk posed by dead-on-farm cattle in the context of rendering (USDA APHIS, 2003b). A Case Study: One BSE-Positive Canadian Cow To demonstrate the impact of a single case of BSE on a country and its major trading partners, we review here the BSE case discovered in northern Alberta, Canada. On May 20, 2003, Canadian agricultural officials an- nounced that a native 8-year-old black Angus beef cow from a farm in Wanham, Alberta, sent for slaughter in January 2003 had been condemned during antemortem inspection and tested positive for BSE (Government of Canada and Government of Alberta, 20031. An inspector had noticed that the animal was unusually thin, and he suspected the cow had pneumonia (Krause and Blakeslee, 2003a). Immediately after the announcement, the United States and several other countries prohibited the importation of Ca- nadian live cattle, beef, beef products, and cattle by-products (USDA and FDA, 20031. This ban was obviously a blow to Canada's cattle, beef, and rendering industries, especially in the loss of trade to the United States, which had until then received 80 percent of Canada's exported beef and nearly all its exported live cattle (Agriculture and Agri-Food Canada, 20031. In 2002, Canadan farm cash receipts for the export of beef and cattle to- taled about $4 billion (U.S. $2.96 billion) (Agriculture and Agri-Food

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 175 Canada, 2003), almost 63 percent of the total outputs of those industries (Myles, 20031. Those exports accounted for less than 1 percent of the country's 2002 gross domestic product, however (Central Intelligence Agency, 20031. U.S. stock markets reacted immediately to the case. Fast-food ham- burger chains were particularly affected. McDonald's stock fell by 6.7 per- cent, Wendy's International by 6.6 percent, and Tyson's Food by 4.9 per- cent (Day, 20031. Futures and commodity markets in beef, cattle, and feed products were affected in the United States, Canada, and elsewhere. Because the BSE-positive cow, dubbed the index case, was condemned during antemortem inspection, Canadian policies prevented its meat from entering the human or ruminant food chains (Evans, 20031. The carcass was rendered into meat and bonemeal, and possibly tallow as well. The Canadian Food Inspection Agency (CFIA) is tracing the path of this ren- dered material. The agency has reported that at least some of the material was used in the production of certain brands of dry dog food manufactured in Alberta (Government of Canada, 2003a). Figure 7-2 illustrates the path under investigation. Some of these pet-food products crossed the U.S. bor- der. A week after the incident, FDA published a warning that a pet-food product sold by Pet Pantry in the United States might contain rendered material from the infected cow. The company reportedly planned to re- trieve the product from its customers (Carlisle, 20031. The BSE-positive cow had come from a commercial herd of 150 cattle established 3 years earlier (Evans, 20031. CFIA depopulated the case herd and tested the cattle for BSE; all the results were negative (USDA and FDA, 20031. The cow's farm of origin was unclear, however. Epidemiologists identified two potential farms of origin, one in Saskatchewan and one in Alberta. Consequently, CFIA quarantined 18 Canadian farms that were the potential source farm or were at risk for BSE (Evans, 20031. Tests on the brain tissue of a sampling of cattle from those herds yielded negative results (CFIA, 2003a). As of June 4, 2003, approximately 1,500 cattle had been killed, and most had been tested for BSE, with negative results (CFIA, 2003a). Within 2 i/2 weeks after the case of BSE had been announced, CFIA lifted the quarantines on the farm where the BSE-infected cow had com- mingled in a pasture, and on three herds containing offspring from the case herd (CFIA, 2003b). A Montana ranch purchased 5 bulls from one of the possible birth herds of the index case. These 5 were among 24 bulls that left the farm between 1997 and 2002 (USDA and FDA, 20031. During a technical brief- 9Total output is measured as all beef produced plus the beef equivalent of all live cattle destined for slaughter.

176 ADVANCING PRION SCIENCE Case Herd Alberta Abattoir Alberta Rendering Alberta Lo | Feed Mills (8)1 | Farms (2) | | Pet Food (2) 200 Trace-out Inspections _ . . I D 1 BC Farm | D Tested negative; removed from quarantine as of June 16, 2003 D 2 BC Farms | FIGURE 7-2 The path of the Canadian forward trace of rendered tissue from the BSE-positive cow found in Alberta. This diagram reflects findings as of Tune 16, 2003. Reprinted with permission from the Canadian Food Inspection Agency (2003). SOURCE: CFIA (2003b). ing on the BSE investigation, the deputy administrator of Veterinary Ser- vices at USDA APHIS said, "We think it is very unlikely that any five of those bulls were infected" (USDA, 2003a:51. Of the 24 animals, 22 have been traced to abattoirs in Minnesota, Nebraska, Texas, and South Da- kota. One bull was sent to a location in Wyoming, and another was slaugh- tered for personal consumption (USDA and FDA, 20031. In August 2003, USDA partially lifted the import ban by allowing sev- eral types of ruminant products from Canada back into the United States.

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 177 These include boneless sheep or goat meat from animals under 12 months of age, boneless bovine meat from cattle under 30 months of age, boneless veal from calves that were 36 weeks or younger at slaughter, fresh or frozen bovine liver, vaccines for veterinary medicine for nonruminant use, and pet products and feed ingredients that contain processed animal protein and tallow of nonruminant sources when produced in facilities with dedicated manufacturing lines (USDA, 2003c). Even with this change in U.S. policy, however, USDA's Foreign Agricultural Service predicts that Canada will export 25 percent less beef in 2003 than it did the year before (Myles, 20031. The long-term economic effects of the Canadian BSE case remain un- clear. It is difficult to predict how long the United States' and Canada's other trading partners will keep their import bans partially or fully in effect. However, the short-term impacts are evident. According to the Canadian Beef Export Federation, each day after the case was announced, Canada's beef industry lost U.S. $8 million (Krause and Blakeslee, 2003b). Layoffs inevitably followed. For example, one beef processor and exporter to the United States laid off 75 to 100 employees just a day after the announce- ment (Olsen et al., 20031. Another consequence of the case of BSE in Canada was the decision by CFIA to ban SRMs from the rendering process to help prevent the inadvertent spread of the BSE agent (CFIA and Health Canada, 2003; Government of Canada, 2003b). The EU took the same step in 2000 (Brown, et al., 20011. The lessons learned from the impact of this single BSE-infected cow, discovered among the 3 million Canadian cattle slaughtered annually, are sobering. If the United States experienced a similar case of BSE, U.S. trading partners would impose an import ban on this country. The ban would have less of an effect on the U.S. beef industry than has been the case for the equivalent Canadian industries because the United States exports only about 9 percent of its beef production (Van Eenoo et al., 2000), while, as men- tioned earlier, Canada had exported almost 63 percent of its beef and live beef cattlei° before the discovery of the BSE case (Myles, 20031. Neverthe- less, the United States is the worId's second-largest beef exporter, with those exports, including variety beef (internal organs), totaling $3.2 billion annu- ally (Canfax, 2003; U.S. Meat Export Federation, 20031. If large cattle ranches were involved in a U.S case of BSE, the costs associated with quarantine, culling, destruction, and testing of cattle could be enormous. The case would require extensive investigations into its lin- eage and movement, as well as traces of all rendered beef by-products of suspected cases. Given the relatively high proportion of cattle, beef, beef i°On a beef-equivalent basis (Myles, 2003).

178 ADVANCING PRION SCIENCE products, and beef by-products exported from the United States, import bans on those products would shake financial and commodity markets worldwide. Within the United States, a case of BSE would lead to a short- term oversupply of the banned products, causing their prices to fall domes- tically. Layoffs would likely follow. Retail sales of all beef end-products might plummet if consumer confidence faltered. These likely effects underscore the importance of preventing even one case of BSE in the United States. Plans for Responding to a BSE Outbreak in the United States The Harvard/Tuskegee risk assessment concluded that if the BSE agent were to enter the United States and infect a cow, the trilayer barrier de- scribed in this chapter would very likely prevent the agent from entering the human food supply. Nonetheless, as the Canadian BSE case demonstrated, the potential consequences of an outbreak on the economy, on animal and human health, and on public confidence would demand a swift, coordi- nated, transparent response. For that reason, the federal government has developed plans for how to respond to a confirmation of BSE in the United States. USDA would have primary responsibility for managing a BSE outbreak. BSE Emergency Disease Guidelines (APHIS and FSIS, 2002), informally called the BSE Red Book, describes in detail the laboratory and field activi- ties to be performed in an emergency. In addition, a joint working group composed of representatives from APHIS and FSIS have a BSE Response Plan (APHIS and FSIS, 2002) to coordinate communication with the public, with other federal agencies, and among the departments within USDA that would carry out the laboratory and field activities. Similarly the FDA has prepared a BSE Response Plan much like that of USDA (personal communi- cation, D. Asher, FDA, August 2003~. MEASURES TO PREVENT THE CWD AGENT FROM ENTERING THE U.S. FOOD CHAIN The principal question regarding CWD and human food is whether the infectious agent of CWD can cause a TSE in humans. As described in Chap- ter 3, the answer is that no one knows. Without a definitive answer, the scientific consensus is to proceed on the assumption that tissue infected with the agent of CWD could theoretically be fatal to hu mans. Therefore, venison processors and consumers should avoid CWD-contaminated meat at present. The committee notes a number of precautions in the discussion that follows. When it comes to processing cervid meat, however, the wide range of practices, the lack of regulation, and the many opportunities for

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 179 spreading the CWD agent suggest the need to research and document the human risks of contact with the infectious agent. Methods to Avoid Contact with the Agent of CWD As a first step, hunters should be aware of known CWD-endemic areas. States can help hunters locate these areas through surveillance. Colorado and Wyoming, for example, conduct excellent surveillance of both wild and captive cervids, providing good guidance for hunters from a food-safety standpoint. As described in Chapter 6, however, the lack of adequate na- tionwide surveillance for CWD means that neither scientists nor hunters of wild venison know all the locations where the disease presently occurs. Moreover, CWD is emerging in new locations with increasing frequency as the disease appears to spread. For example, Utah reported a new case of CWD in April 2003 near the town of Moab, more than 100 miles south of the first case of the disease identified in the state (Knowles, 2003~. As a second precaution, hunters could be trained to recognize clinically ill cervids. As is the case with other TSEs, however, a significant amount of infectivity develops in CWD-positive animals before the onset of clinical disease. The brain, spinal cord, lymph nodes, and spleen of a CWD-positive cervid would contain infectivity at various stages of the disease. In addition, given that the disease is transmitted laterally, some material shed by the animal (saliva, feces, urine, or hair, for instance) is likely to contain infectiv- ity as well. To avoid contact with and eating of CWD-infected tissue, processors theoretically could test cervids with one of three USDA-approved rapid post- mortem diagnostics for CWD: an enzyme-linked immunosorbent assay (ELISA) from Bio-Rad Laboratories Inc. of Hercules, California (USDA APHIS, 2002~; the Dot Blot ELISA from VMRD Inc. of Pullman, Washing- ton (USDA APHIS,2003c; VMRD Inc., 2003~; and the IDEXX Her3Chek'~' CWD Antigen Test Kit from IDEXX Laboratories Inc. of Westbrook, Maine (IDEXX Laboratories, 2003) (see also Chapter 4~. All three tests can detect PrP in peripheral lymphoid tissues (personal communication, Rick Hill, USDA APHIS Center for Veterinary Biologics, November 25, 2003~. The Bio-Rad test is approved for use on the tissues of white-tailed deer, mule deer and elk, the VMRD test for use on the tissues of white-tailed and mule deer, and the IDEXX test for use on white-tailed deer tissue (personal com- munication, Rick Hill, USDA APHIS Center for Veterinary Biologics, No- vember 25, 2003~. As of November 2003, however, USDA had licensed the tests for use exclusively by approved veterinary laboratories for surveillance purposes only. It would be desirable to have a USDA-approved test for CWD in brain tissue designed for use at any cervid-processing facility. The easier such a test was, the more likely a processor would be to use it.

180 ADVANCING PRION SCIENCE Processing of Cervids There is little documentation on or uniformity to cervid processing in the United States. The committee gleaned most of its information on the subject from Dr. Warrie Means, an associate professor of animal science at the University of Wyoming, Laramie (Means, 20031. He is the source of information on cervid processing in this chapter unless otherwise noted. An estimated 4,510 meat processing plants in the United States process game meat (Means, 2003. This figure is based on the assumptions that about one-third of the country's 7,500 to 8,000 small FSIS- or state- inspected plants process venison, and that about 85 percent of the country's 2,200 custom processors do the same. The latter type of establishment gen- erally is not licensed or inspected by USDA and is not necessarily inspected by the state, either. Commercial Processing of Cervid Meat The processing of cervids takes one of two paths, depending on whether the animal is harvested commercially or for sport. A flow chart for each path appears in Figures 7-3 and 7-4. Commercial game processors work with deer harvested specifically for commercial sale. Edible tissue is fabricated into cuts or processed into such items as sausage, jerky, and snack sticks, and is sold to restaurants and individuals. Inedible tissue is sent to renderers or landfi~Is. From a food-safety standpoint, the commercial processing of venison is probably safer than any other method. Most game processors use a tech- nique called boneless fabrication, which minimizes contact with the brain, spinal cord, and lymph nodes if the animal was not quartered first. Based on the practices in Wisconsin and Wyoming, these commercial establish- ments also process an average of 115 to 260 head of beef per year, or 1 to 3 percent of all cattle slaughtered annually in the United States. Thus, CWD- infected tissue could potentially cross-contaminate a small percentage of U.S. beef tissue or equipment at processing sites. The responses of deer and elk processors to the risks posed by CWD have ranged from apathy to serious concern. Not surprisingly, the actions taken by various establishments to reduce the risk of CWD infectivity range from minimal to dramatic, as outlined in Box 7-1. Only about half a dozen plants are taking the most extreme measures listed at the bottom of the box (personal communication, W. Means, University of Wyoming, March 26, iiDr. Means cited a personal communication with the American Association of Meat Pro- cessors for the figures in this paragraph.

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs FIGURE 7-3 Commercial processing of cervid tissues in the United States. SOURCE: Adapted from Means (2003~. 181 FIGURE 7-4 Paths taken by hunter-harvested cervid tissues during processing in the United States. SOURCE: Adapted from Means (2003~.

182 ADVANCING PRION SCIENCE 2003~. However, there has been a noticeable increase in the number of cervids being tested for CWD. Hunter Harvesting of Cervids Hunters may process cervids to various degrees in the field, then com- plete the fabrication at home or give the animal to a custom processor to fabricate into cuts of meat and/or processed meat, such as sausage. Most hunters "field dress" the animal by removing its internal organs. Some hunt- ers further process the animal, depending on its size, the hunter's skill, and the distance the hunter (and his or her horse) will have to carry the animal. The traditional method of field processing is to remove the head and quar- ter the animal. In the process, the hunter bisects the spinal cord, exposing CNS tissue. An alternative, infrequently used method called field butcher- ing does not bisect the spinal cord and is recommended in areas known to have CWD. Obtaining the trophy antlers that hunters often prize exposes the brain. The tools used for this purpose in the field may include a chain

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 183 saw or battery-powered saw (both of which scatter tissue into the air), a handsaw, or a hatchet. When the field work is done, the cervid tissues left behind sometimes include the spinal column, the head, and the spleen. These organs could theoretically contaminate the environment or cause TSE in a scavenger ani- mal that ate them if the slain cervid were infected with the CWD agent. As noted, hunters complete the fabrication of the meat into cuts or other products either at home or at a custom processor. These processors are typically very small establishments. Nevertheless, when a hunter arrives to pick up his or her butchered and processed venison, it is not necessarily the meat from the animal he or she killed, but rather an amount equal to what the hunter dropped off. Therefore, even if the hunter was very careful in the handling of risky tissues, the safety of his or her meat depends also on how other hunters handled their cervids, as well as how the processing , . . . taco sty Is run. Regulation of Live Cervids, Venison, and Venison Products There is relatively little regulation of live cervids, venison, and venison products in the United States. Each state is responsible for the regulation of such products produced and sold within its borders. To the committee's knowledge, few states regulate the industry; however, some states require venison to be inspected before being sold. USDA regulates interstate commerce in live cervids, cattle, sheep, swine, goats, and horses, as well as meat and poultry, while FDA regulates inter- state commerce in cervid meat and meat products. Although beef, pork, and mutton must be inspected by USDA to be sold across state lines, FDA does not require such inpection of venison. Federal regulations simply re- quire that the venison come from an approved source when intended for sale in retail stores and restaurants. Approved sources are licensed food establishments, federally inspected food plants, and state-inspected meat plants (Deer Farmer's Information Network, 20031. Conclusion Regarding the Prevention of CWD Transmission There is a dearth of scientific information on cervid processing, despite the opportunities for contamination by the CWD agent during cervid pro- cessing and the theoretical possibility that the infectious agent might be transmissible to humans. Recommendation 7.2: Fund risk assessments that characterize the exposure of hunters, cervid processing establishments, and consum- ers to the infectious agent of chronic wasting disease. [Priority 31

184 ADVANCING PRION SCIENCE PREVENTING TSE TRANSMISSION THROUGH BLOOD, BLOOD DERIVATIVES, AND TRANSPLANTED TISSUES In addition to measures to prevent TSE agents from entering the food chain, steps must be taken to prevent transmission of such agents by other means. This section reviews measures to prevent transmission of TSE agents through blood products, blood derivatives, and transplanted tissues. Preventing TSE Transmission Through Blood Products Regulatory bodies throughout the world, including FDA, have taken steps to prevent the potential transmission of TSEs among humans through blood, blood components, and blood derivatives (Foster, 20001. FDA has instituted a number of policies, listed in Table 7-2, to prevent the infec- tious agents of human TSEs from entering U.S. supplies of these biological products. FDA keeps the blood collection and processing industry informed of its policies through "guidance to industry" documents that convey the infor- mation necessary to comply with policy decisions (FDA, 20021. The center within FDA that regulates biological products is the Center for Biologics Evaluation and Research (CBER). CBER has a TSE Scientific Advisory Com- mittee and a Blood Products Advisory Committee, composed of national experts in the field (with community representation), that provide the scien- tific underpinnings for new regulatory policy regarding TSEs. In addition to developing preventive policies regarding deferral of high- risk donors, FDA provides guidance to industry and evaluates manufactur- ing processes to ensure the production of pure, safe, and effective products. Studies show that the processes involved in manufacturing blood deriva- tives, such as precipitation, filtration, and chromatography, remove between 1 and 6 logs of TSE infectivity, or an average of 3 to 4 logs (Brown et al., 19991. This reduction of potential infectivity is highly desirable, but its ad- equacy will remain unclear until scientists develop an estimate of the size of an infectious dose of the TSE agent in human blood. Meanwhile, the blood industry is evaluating methods for inactivating the TSE agent. Scientists have invested much effort in pursuing the goal of inactivating TSE agents in blood. An important challenge is to inactivate these prions within living tissue, such as blood, without exacting collateral damage on other tissues. Most of this effort has focused on ways to inactivate or re- move prions from blood and, in particular, blood derivatives. This focus is reasonable because existing tests are unable to detect small amounts of prions that may be circulating in blood, and it is therefore impossible at present to identify asymptomatic, TSE-infected blood donors. Gamma radiation has been used experimentally to inactivate hamster scrapie prions added to human albumin. A dose of 50 kGy (100 reds = 1

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs TABLE 7-2 Measures Taken in Response to Concern That TSE Agents May Be Transmissible via Human Blood Products 185 Date Event 1987 (Nov.) FDA defers recipients of human pituitary-derived growth hormone from donating blood. 1995 (Aug.) FDA extends donor deferral to those with a family history of CJD and to recipients of aura mater. Plasma derivatives prepared by using a donation that would now be excluded must be withdrawn. 1996 FDA recommends withdrawal of blood plasma and plasma products made from pooled blood donations to which persons who later died of CJD have contributed. 1996 (Dec.) FDA clarifies which donors are at risk of developing CJD. 1998 (Sept.) FDA recommends that plasma derivatives manufactured from a donor who develops vCJD be withdrawn, but not plasma derivatives involving donors with other forms of CJD. 1999 (Aug.) FDA extends donor deferral to those who spent 6 months or more in the United Kingdom between 1980 and 1996. 1999 (Nov.) FDA revises donor deferral to include recipients of bovine insulin unless the product was not manufactured since 1980 from cattle in the United Kindom. 2000 FDA holds public discussion regarding the possible risk associated with vaccines produced with bovine-derived materials from countries experiencing BSE. 2002 (Nov.) FDA extends donor deferral to those who have spent 3 months or more in the United Kingdom between 1980 and 1996, and those who have spent 5 years or more in France or any of 29 other European countries between 1980 and the present.a aPage (2002). SOURCE: adapted from Foster (2000: Table 1). Gray tGy] = absorbed radiation dose) was used in the experiment. This is a massive dose that would, if applied to food, kill all microorganisms present, including viruses. In the experiment this dose caused an estimated reduction of 1.5 loglO ID50 of the scrapie prion level based on a delay in onset of clinical symptoms in a hamster infective assay. The radiation did not ap- pear to cause excessive damage to the albumin, and it delayed clinical end points in hamsters inoculated intracerebrally (i.c.) with the albumin, but it failed to prevent infection by the scrapie agent (Miekka et al., 2003~.

186 ADVANCING PRION SCIENCE A new chromatography-based process for manufacturing immune globulin to be given intravenously (IGIV) was evaluated to determine whether the process would remove hamster scrapie prions added to the source plasma material. Western blot analysis measured a large reduction in PrPSc, and assays in hamsters showed no infectivity after i.c. inoculation of six hamsters followed for 250 days (Trejo et al., 20031. This result looks very promising for this isolated end product. However, blood fractionation studies (Brown et al., 1998) indicate that the potential burden of prions in the immunoglobulin fraction is much less than would be found in other plasma derivatives. Also these findings can not be generalized to other blood products manufactured using different processes. Another strategy used to eliminate prions is to filter them out of the product during the manufacturing process. NanofiItration has shown great promise in eliminating viruses and prions from plasma products (Burnouf and Radosevich, 20031. In a recent study, Planova~ filters of 15 nanometer (nary) pore size reduced mouse-adapted scrapie prions that had been added to human albumin to such a low level that infectivity was eliminated as determined by a mouse infectivity assay (Tateishi et al., 20011. It is possible, however, that test conditions may create aggregation of the prions that favors their removal. Burnouf and Radosevich (2003) reported that in one experiment by Tateishi's group, residual infectivity was found in the 10 nm filtrate when the detergent sarkosy! (1 percent) was added to the plasma, reducing aggregation of priors. However, in another experiment, when Tateishi's group added sarkosy! (0.5 percent) to plasma, their 15 and 10 nm filters appeared to remove the infectivity as demonstrated in mice inocu- lated with filtrates at dilutions of 10-i or less (Tateishi et al., 20011. Al- though this application of the technology is heartening, it does not fully address the needs. Advancing this method and others, however, would be an important step in the direction of having safer blood derived products. Recommendation 7.3: Fund research to develop novel methods for removing prions from or inactivating prions in blood products and tissues in vitro, using physical, chemical, or immune mechanisms alone or in combination. [Priority 21 Preventing Transmission of TSE Agents in Transplanted Human Tissues As previously noted, many iatrogenic CJD cases have been attributed to infected processed aura mater allografts and a small number to infected corneal transplants (Hogan et al., 19991. Thus, processed human aura mater, corneas, and other eye tissues (e.g., retinas) are thought to present the greatest risk of TSE transmission by implantation (FDA TSEAC, 2001~. In addition, substantial amounts of PrPSc accumulate in the lymphoid tis- sues of vC]D patients (Bruce et al., 2001~.

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 187 Several measures taken by FDA and other organizations have reduced the risk of iC}D due to tissue implants in the United States. Nearly 20 years ago, FDA ceased approving the U.S. sale of human cadaveric pituitary hor- mones, responsible for a large number of iC}D cases worldwide (personal communication, D. Asher, FDA, August 2003~. In 1990, FDA issued its first safeguards to minimize the possibility of TSE transmission through processed aura mater allografts (FDA CDRH, 1999~. There has been no confirmed U.S. case of iC}D due to this product since March 1997. In fact, NPDPSC has seen only 4 cases of iC}D out of nearly 1,100 suspect human TSE cases since 1997, when the center was established (Chapter 6, Table 6-1~. FDA does not require providers of human cells, tissues, and cellular prod- ucts (HCT/Ps) intended for human transplantation to register with FDA or to give the agency a list of their products, although the agency proposed these requirements 7 years ago (FDA CBER, 1997~. The World Health Organization (WHO) recommended in 1997 that processed human aura mater grafts no longer be used, especially in neuro- surgery, unless no alternative was available (FDA CDRH, 1999~. This rec- ommendation was endorsed by FDA's TSE Advisory Committee (TSEAC) with the caveat that "the final decision regarding the use of processed hu- man aura mater should be left to the discretion of the treating neurosur- geon" (FDA CDRH, 1999:3~. Since then, FDA's Center for Devices and Radiological Health (CDRH) has issued guidelines, not regulations, to mini- mize the risk of TSE transmission by processed human aura mater implan- tation (FDA CDRH, 1999~. Among other precautions, this guidance rec- ommends that manufacturers: · Obtain a medical history to select aura mater donors who are free of neurological disease at the time of death. · Use suitability criteria similar to those for donors of blood (see Box 5-1 in Chapter 5~. · Perform gross and histological examination of the brain to exclude donors with possible evidence of TSE-related changes. · Include a disinfection step in the manufacturing process. · Prevent cross-contamination of aura mater from different donors by blocking commingling during collection and processing. CDRC is developing updated guidance regarding processed aura mater al- lografts (FDA CDRH, 2002~. FDA's Center for Biologics Evaluation and Research (CBER), which regulates corneas and other human cells and tissues intended for transplant, requires these materials to be tested for various infectious diseases (FDA, 1997~. Since there is no antemortem test for TSEs in humans, however, providers of corneas for implantation generally reject prospective donors

188 ADVANCING PRION SCIENCE who would be deferred from donating blood because they resided in Eu- rope during the high-risk period for BSE transmission to humans, possibly exposing them to the infectious agent (see Chapter 9, Table 9-2) (personal communication, D. Asher, FDA, August, 2003~. Stricter deferral criteria could entail the exclusion of older donors, who are more likely to have sC}D, or the examination of potential donors' brain tissue for signs of TSE. However, TSEAC concluded in 2001 that imposing more stringent selec- tion criteria on potential donors of corneas would prevent the United States from meeting the demand for corneal transplants (FDA TSEAC, 2001~. To further minimize the risk of disease transmission, CBER prohibits the commingling of human tissues from different donors during processing (FDA, 1997~. In addition, the Eye Bank Association of America and the American Association of Tissue Banks have adopted voluntary standards to prevent the transplant of TSE-infected tissue (personal communication, D. Asher, FDA, August, 2003~. TSE infectivity has been detected in a variety of human organs besides brains and eyes, albeit less consistently and in much smaller amounts. Pri- mate bioassays of tissue homogenates from the organs of human TSE cases (mostly sC}D) revealed infectivity in 10 percent of tested human spleens, It percent of livers, 18 percent of kidneys, 20 percent of lymph nodes, and 50 percent of lungs (Brown et al., 1994~. The regulation of human solid organs intended for transplantation is conducted by the United Network for Organ Sharing under contract with the Health Resources and Services Administra- tion, U.S. Department of Health and Human Services. INACTIVATION OF PRIONS ON SURFACES AND IN THE ENVIRONMENT Avoidance of exposure to TSE agents is a principal preventive strategy. However, there are sites in which the presence of prions may be unavoid- able. These sites include hospitals and veterinary clinics; research and refer- ence laboratories; and operating rooms having contact with TSE-infected people, animals, or tissues. In such situations, it is appropriate to use inac- tivation procedures to eliminate prions so they will not contaminate equip- ment or infect people. Inactivation becomes an especially critical issue when a prior-exposed surgical instrument is being used on a patient. Today, hospitals lack both an antemortem test for human TSEs (except a brain biopsy) and satisfactory methods to disinfect medical equipment potentially contaminated with a TSE agent. The few disinfection methods believed to have the power to eliminate TSE infectivity on medical equip- ment (Department of Health, 2003; Weber and Rutala, 2002; WHO, 2000) are caustic and potentially harmful to both hospital personnel and the equip- ment itself. New disinfection methods are sorely needed. It would be ideal

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 189 to have a safe, inexpensive disinfection procedure against prions or the TSE agent for routine use in all hospital disinfection protocols. Since human TSEs incubate for years, it is not unusual for a patient diagnosed with suspected TSE to have undergone an invasive medical pro- cedure during the years when the suspected infection was incubating. Under such circumstances, hospital staff must trace back all patients who may have been exposed to the instruments used on the suspect TSE case, deter- mine the patients' level of risk, and decide what to tell them (COD Incidents Panel, 2001; Weber and Rutala, 2002~. Anecdotal evidence suggests that many surgeons are reluctant to perform invasive procedures on a suspected TSE patient, leaving hospitals to undertake draconian infection-control measures for what may or may not be a case. Finally, as mentioned in Chapter 6, most pathologists in the United States will not perform an au- topsy on suspect TSE cases mainly for fear of contaminating their equip- ment (Belay, 2003~. Clearly, the lack of a safe, routine, affordable proce- dure for inactivating the TSE agent on surfaces has significant consequences. Prions are unusually difficult to inactivate a unique characteristic that differentiates them from infectious living organisms such as viruses, bacte- ria, and fungi. Excellent reviews regarding prion inactivation have been published (Taylor, 1999, 2000~. Table 7-3 summarizes the types of agents used to inactivate prions and their effectiveness. Dry and steam heat, or- ganic and nonorganic chemicals, and various types of ionizing and nonion- izing radiation have been employed. In addition to the agent used, certain factors associated with the source material can influence inactivation. Fac- tors that would increase resistance to inactivation include a larger amount or a higher ID50 level of infectivity of the material being treated, macerated material versus intact tissue, dried material, contact with glass or steel sur- faces, or protein fixation by a chemical or heating (Taylor, 19991. These factors promote thermos/ability and thus greater resistance to inactivation. Also, different prion strains respond differently to the same inactivation procedure (Taylor et al., 2002~. Studies in the 1980s (Brown et al., 1986; Kimberlin et al., 1983) em- ployed mouse assays and mouse-adapted prion strains to develop recom- mendations on the effectiveness of inactivation procedures. Since some prions from certain mouse strains were more resistant to inactivation than others, the recommendations were based on the conditions that would inac- tivate the hardiest strain tested. These recommendations included providing for an additional margin of safety by increasing the concentration of the chemical agent and lengthening the time of heat treatment. Recommendations from these earlier studies were later shown to be inadequate for other prion strains or in other test conditions. For instance, testing showed that autoclaving inactivated priors, but that a subpopula- tion of the protein in test material could become "fixed" and be more resis-

190 TABLE 7-3 Agents Used to Deactivate Prions ADVANCING PRION SCIENCE Evidence for Complete Inactivation Class of Agent Agent/Conditions Not Successful Successful Radiation Microwave X Ultraviolet X Ionizing X Dry Heat 600°C/5-15 mina X 360°C/24 hr 220°C/20 min X 160°C/24 hr X Moist Heat (autoclave) Gravity displacement 121°C/90 min X 132°C/90 min Xb 132°C/4.5 hr xc Porous loading 134-138°C/18 min X (debated) Chemicals Acid/bases 2 M NaOH/2 hr X Alkylating agents Formalin X Glutaraldehyde X Acetyl ethylenimine X ~ propyl lactone X Ethylene oxide X Detergents Sodium dodecyl sulfate X Halogens 2% iodine/4 hr X Sodium hypochlorite X~ (20,000 ppm/lhr) Dichloroisocyanurate X Organic solvents Chloroform X Ethanol X Phenol X Hexane X Heptane X Perchlorethylene X Petroleum X

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs TABLE 7-3 Continued 191 Evidence for Complete Inactivation Class of Agent Chemicals (continued) Oxidizing agents Chlorinedioxide Hydroperoxide Peracetic acid Agent/Conditions Not Successful Successful Salts Chaotropes Proteolytic Enzymes Combinations Sodium metaperiodate Potassium permanginate Guanidine thiocyanate >4 M Guanidine hydrochloride Urea Trypsin Pronase Quiagen Proteinase K 1 M NaOH/121 °C/30-60 min GD 1 M NaOH/121°C/90 min GD 2 M NaOH/121°C/30 min GD 1 M NaOH/boiling/lmin X X X X X X X X X X X X (debated) xe xf Xg X~ aBrown et al. (2000b). bErnst and Race (1993). CPrusiner et al. (1984). ~Kimberlin et al. (1983). eErnst and Race (1993). fPrusiner et al. (1984). Taylor et al. (1997). hTaylor (2000). NOTE: GD = gravity displacement autoclave; M = molar; NaOH = sodium hydroxide. SOURCE: Adapted from Taylor (2000).

92 ADVANCING PRION SCIENCE tent to temperature inactivation. That phenomenon was demonstrated in a study in which 60 percent of the animals injected with infectious material previously treated at 134°C became infected, compared with 72 percent that were injected by material treated at 138°C (Taylor, 19991. This study revealed that autoclaving alone is not a sufficient method for inactivating priors. One study that appeared to show the ability of sodium hydroxide to inactivate prions was subsequently found to have significant design pitfalls. In a later study, when the caustic chemical was neutralized before injection into the assay animal, the material was found to contain residual infectivity (Taylor et al., 19941. Earlier studies had diluted the sodium hydroxide so it could be injected into the assay animals without acute toxicity. Unfortu- nately this procedure also reduced the prion levels so that infectivity was not detected. Failure to identify fully satisfactory methods for inactivating prions with a single type of deactivation treatment led investigators to believe that com- bination methods might be a more effective approach. Several studies have since shown that a combination of heat and sodium hydroxide is a reliable way to inactivate priors. However the latter chemical is caustic to some equipment and must be handled carefully because of safety concerns. Most of the methods shown in Table 7-3 have little if any effect in inactivating priors. Some can reduce infectivity many-fold but only a very few are reliable. Because of this concern, it is recommended that surgical instruments having been in contact with known CTD-positive patients or patients known to be at a much elevated risk of acquiring CTD be discarded rather than disinfected (Taylor, 20001. The rarity of CTD and the high stakes associated with infection make this an attractive option. The Department of Health in the United Kingdom recommends that, at a minimum, for brain biopsies involving nonfocal lesions, the surgical equipment should be quar- antined until CTD can be ruled out. This policy was established in response to an unfortunate incident in which 24 patients were exposed to the same surgical equipment used on a CTD patient whose diagnosis was initially unknown (Mayor, 20031. Policies recommending disposal or quarantining of equipment are problematic, however, when dealing with fiberoptic equip- ment, which is both expensive to replace and difficult to disinfect, especially when contaminated with prions (Dombrovski et al., 20031. The concern about transfer of infectivity through instrumentation is not merely theoretical. The same surgical stereotactic encephalographic elec- trode implanted into the brain of a patient with CJD infected two subse- quent patients, aged 17 and 23, despite the use of typical sterilization proce- dures (Bernoulli et al., 19771. Proof that this electrode was the source of the prion infection was convincingly obtained in a subsequent experimental animal study. Two years had passed since the silver electrode had been used in the last human patient. The electrode had been cleaned three times and

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 193 had been disinfected repeatedly with both ethanol and formaldehyde vapor. Yet when that same electrode was implanted in the brain of a chimpanzee, the agent was transmitted to the animal (Gibbs et al., 19941. The evidence for iatrogenic transmission of prions has been summa- rized (Brown et al., 2000a) and is mentioned in Chapter 5, but the special concern about transferring the prion agent on surgical instruments merits additional discussion. The type of surface appears to be an important fea- ture. Steel and glass are extremely common surfaces for surgical equipment and in surgical environments generally. Steel and glass have both been shown to thermostabilize prions from inactivation (Asher et al., 1986, 19871. A series of intriguing studies in mice has revealed just how intractable prions are once they have become fixed onto a steel surface (Flechsig et al., 2001; Weissmann et al., 20021. The mode! for those studies involved the insertion of prior-contaminated stainless steel wires into the brains of uninfected mice. The initial experiment revealed that it took as little as 5 minutes of contact to contaminate the wire and transfer the agent to a recipient mouse with the same degree of infectivity as occurred by direct injection of 30 Al of a 1 percent brain homogenate into the brain. In a second experiment, recipient mice were exposed to contaminated wire for different time periods. It took only 30 minutes of exposure to transfer the agent to four of four mice in that exposed group although the incubation period was extended, indicating a lesser degree of infectivity from the brief contact. The investigators indicated that they had demonstrated, in unpub- lished studies, similar infectivity using gold wire, revealing that prions coated on plastic surfaces transmitted infection in cell culture assays (Weissmann et al., 20021. This mode! may be useful in screening new meth- ods for inactivating priors, but the investigators caution that larger surface- contact with brain material, use of the vCTD prion strain, and testing in nonhuman primates would ultimately need to be performed to ensure the efficacy of any new sterilization technique for surgical instruments (Weissmann et al., 20021. Such research is worthy and could prevent the unnecessary quarantining or discarding of valuable surgical equipment. It also might offer a more environmentally friendly method of decontaminat- . . . 1ng surglca" . operatorles. Recommendation 7.4: Fund research to develop standard assays for the detection of PrPSc or TSE infectivity on the surfaces of reus- able medical instruments and materials, as well as research to de- velop better methods to disinfect such instruments and materials. [Priority 21 The inactivation of prions contaminating a relatively small contained space, such as a laboratory work surface, an operating room table, or a

194 ADVANCING PRION SCIENCE surgical instrument, poses challenges, but the inactivation of prions in a large contained space, such as an abattoir or a large open space, such as a pasture, poses far greater ones. Knowledge of the natural degradation of prions in the environment is extremely limited. Studies from Iceland showed that scrapie-free sheep brought into a pasture where scrapie had been preva- lent 3 years previously, but where no animals had grazed in the interim, came down with scrapie, suggesting that pasture land could remain infected for many years (Palsson, 19791. More recently, Brown and Gaj~usek (1991) showed that scrapie agent from a hamster brain homogenate mixed with soil could survive burial in a garden for 3 years. Standard methods for evaluating the presence and decay of TSEs in the natural environment are not available, and this has hampered our understanding of the threat posed by long-term environmental contamination by priors. Recommendation 7.5: Fund research to develop standard test methods for detecting prion contamination in environmental samples. [Priority 31 The longevity of TSE infectivity, specifically in soil, has tremendous implications for the proper disposal of animals infected with a TSE agent, as well as the offal and rendered material from these animals. For years, burial of dead farm animals has been a common and acceptable practice. However, large-scale burial of animal material potentially contaminated by TSE agents poses a significant risk (Scientific Steering Committee, 2003b). New methods are being evaluated to avoid the need for burial of TSE- contaminated material. One method that has received much attention is treatment at 150°C for 3 hours in concert with high-pressure alkaline hy- drolysis. The European Commission's Scientific Steering Committee (2003a) could not certify this method as effective; however, that committee cited laboratory studies demonstrating that under similar conditions, large re- ductions in infectivity occurred but the method did not eliminate all infec- tivity. The committee urged further study suggesting that if such studies showed evidence for continued infectivity, residual waste products would need to be incinerated and placed into a control landfill. One cannot overrstate the importance of identifying safe methods for disposing of animals and animal tissues infected with a TSE agent. Disposal would immediately become an issue if the United States were to experience an outbreak of BSE; the United Kingdom and Europe already face the prob- lem. Recognizing this, USDA APHIS (2003b) proactively published a notice of proposed rulemaking in January 2003 in which it requested public com- ment on safe, reliable ways to dispose of animals and tissues infected with a TSE agent. Present research on this subject has not progressed far enough to provide a sufficient scientific foundation for policymaking. Therefore, the United States must intensify research into disposal methods for infected

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 195 animals and animal tissues. Given that the United Kingdom and Europe need such methods urgently, this area of research appears ripe for interna- tional collaboration. Recommendation 7.6: Fund research to identify safe, cost-effec- tive disposal mechanisms for animals and rendered waste infected with agents of transmissible spongiform encephalopathies. This research would best be conducted with a multidisciplinary ap- proach involving experts in such fields as prion biology, biochem- istry, environmental engineering, and commercial disposal tech- nology. [Priority 21 VACCINATION AS A PREVENTIVE STRATEGY For more than 200 years, people have successfully combated infectious diseases by using vaccines to mobilize the immune system to protect against invading pathogens. The approach has worked so well that immunization is among the 10 most successful achievements in public health in the twenti- eth century (CDC, 19991. Yet vaccination has a dubious role in the preven- tion of prion diseases. Most experts believe the body is immunologically tolerant to prions because the aberrantly folded protein has the same pri- mary protein structure as normal body protein. The basic substrate prion protein appears to be poorly immunogenic (Heppner et al., 20011. The im- mune response to bacteria or viruses which are much larger and contain foreign proteins is considerably more robust and much better understood. The host recognizes antigens on bacteria and viruses and elaborates protec- tive antibodies. Attenuating or manufacturing these identical antigens so that they are harmless to the host yet create the protective antibody re- sponse is the essence of vaccination. This classic immune response does not . . Occur Wit" ~ pr1ons. Investigators appear to be undaunted by the special challenges intro- duced by the atypical immune response seen in prion infections. One group has immunized mice with a recombinant PrP protein, which resulted in antibody production that slightly prolonged the incubation period (Sigurdsson et al., 20021. The mice were challenged with a mouse scrapie strain by intraperitoneal (i.p.) inoculation. The animals produced measur- able antibody, and those with the higher antibody titers experienced longer delays in their incubation period before their uniform demise. This result suggested a dose-response relationship between the antibody and the longer survival. Additionally, the survival time was slightly prolonged when mice were vaccinated before their exposure as compared with 24 hours follow- ing exposure (Sigurdsson et al., 20021. Another team of investigators, using a mouse model, demonstrated that

196 ADVANCING PRION SCIENCE injecting mice i.c. with a relatively low dose of SY, a mild strain of mouse- adapted CAD, followed by challenge with FU, a more virulent COD strain, delayed death and suppressed the expression of clinical disease in these animals by many months, up to approximately 2 years following inocula- tion of the SY agent (Manuelidis and Lu, 20031. These results support ear- lier work by Dickinson and colleagues (Dickinson et al., 1968, 19721. Manuelidis and Lu speculated that the innate immunity possibly involving myeloid microglial cells may have been involved in the host response they observed. The exploration of ways to elicit active immunity by vaccination as a strategy for TSE prevention, while intriguing, will probably not progress much further, even in animals, without a better understanding of the basic immunobiology and molecular biology of prpC and PrPSc. Even if animal experiments clearly showed that vaccination prevented prion disease, the transfer of that information to human trials and to a human vaccine would be many years away. It is clear that prions behave differently from classic pathogens. But even if prions or prion fragments do not make good immunogens, there is a growing body of evidence that the host does have an immune response to them. B-lymphocytes and follicular dendritic cells of the immune system appear to play a significant role in the extraneural pathway of prion dis- eases (Mabbott and Bruce, 20011. Thus it is conceivable that modulation of this immune activity, in a manner to be determined, might be effective in . . . . preventing or treating pr1on c 1seases. The most promising near-term strategy employing the principles of im- munity involves the development of specific antibodies. If antibodies could be tailored to bind to certain specific locations on prion protein or prefer- ably prions themselves, such that further conversion of prpC to PrPSc were disrupted or a critical pathogenic molecular pathway were inhibited, this might serve as an excellent prophylactic or therapeutic approach. Signifi- cant advances are being made in this area, as will be discussed in the next section. PROGRESS IN THERAPY FOR TSEs The evidence for success in treating TSEs is meager and incomplete. The reasons for this limited success are multiple and include uncertainty regarding the underlying pathophysiology of prion diseases, the difficulty of identifying disease at an early preclinical stage without sensitive detec- tion tests, the problem of getting any treatment agent past the blood-brain barrier, the toxicity associated with therapeutic agents, and the enormous challenge of translating gains from cell culture or animal studies to human

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 197 use. Adding to these important obstacles is the reality that prion diseases, which are devastating to victims and their loved ones, are quite rare; thus the allocation of research funds for work on TSEs has historically been limited as well. However, research directed at this protein-folding disorder would likely have crossover value in advancing understanding of other, more common neurodegenerative disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, and Parkinson's dis- ease. And fortuitously for sufferers of prion diseases, the reverse is also true. This section briefly reviews progress made in prion disease therapy. It ad- dresses not only the use of drugs, but also gene therapy and treatment with engineered antibodies. A recent review of drug therapy for TSEs (Brown, 2002) notes the large number of animal studies seeking to discover candidate therapeutic agents. The large majority of those studies led to dead ends, but a few showed some promise. A broad array of studies has been conducted with many different classes of therapeutic drugs. Most have shown little efficacy. Brown (2002) lists of more than 60 drugs that have been studied, highlighting those few agents that showed some efficacy in modifying the disease course. These older experiments in animals were aimed at a putative unknown infectious agent. More recent experiments involving cell-free conversion systems, cell cultures, and animals, undertaken after prions were discovered, have been directed at preventing prions from entering the brain or modifying them once in brain cells within the central nervous system (Brown, 20021. The timing of the therapeutic dosing can limit interpretation of drug efficacy studies. Drugs being studied have often been given shortly before or shortly after challenge with infectious priors. The efficacy of a drug used in that fashion may be quite different from that of the same drug given later, at the time of disease expression. The longer is the delay in giving the thera- peutic agent after the infectious challenge, the less favorable are the results (Brown, 20021. Many of the newer experiments have been conducted in cell cultures systems as well as animals. Cell culture systems serve as excellent models for screening new drug agents, but they are limited utility because the kinet- ics of drug absorption, distribution, inactivation, degradation, and excre- tion cannot be studied (Brown, 20021. Thus any positive effects must be further demonstrated using in viva systems. The mechanisms of action of the drugs used to suppress or reverse prion progression are variable and in many cases not fully characterized. The general concept is that there are multiple targets of opportunity to attack prions in the body. A listing of those targets appears in Box 7-2. Many drugs appear to have a direct or indirect effect on the protein folding capacity and convertibility of prion protein to priors. Some operate

198 ADVANCING PRION SCIENCE within the periphery, while others have their effects in the central nervous system. A list of drug classes and drug agents showing some level of effec- tiveness appears in Table 7-4. The translation of a scientific discovery in the laboratory to animals and then on to human studies prior to commercial use of a new drug is a laborious, lengthy, and highly structured process that takes many years. One shortcut strategy has been to administer drugs already FDA-approved for some other medical conditions to patients having a TSE. Although this strategy is sound, the results have been highly disappointing. These attempts have involved very few patients with a small number of drugs (Brown, 2002~. Also, because of the rarity of human TSEs, the ability to test a candi- date drug in hundreds of individuals, as is done in clinical trials, is limited. The dilemma of not having an approved drug to treat human TSEs, yet having drugs in the formulary that have shown some positive effect against prions in cell or animal testing, has led to some controversial decisions on the appropriateness of administering such drugs to patients dying from CJD. An example was reported recently in the United Kingdom (Dyer, 2003~. A teenage boy, symptomatic from a vCJD infection, was given pentosan polysulfate, a drug used in North America to treat interstitial bladder infec- tions. Studies conducted a decade ago in mice showed that this drug, when given prior to prion exposure, protected animals challenged with 100 LD50 of prions (Diringer and Ehiers, 1991; Doh-ura et al., 2002~. Since the drug is not thought to cross the blood-brain barrier, it had to be administered directly into the brain by a catheter, a procedure that carries a risk of cere-

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 199 TABLE 7-4 Drug Classes and Agents Used Experimentally to Treat TSEs Drugs Whose Action Occurs in Peripheral Stages of Disease Drugs Whose Effect Is in Both the Peripheral Stages of Disease and the Central Nervous System Polyanions Dextran sulfate HPA-23 Pentosan polysulfate Sulfonated dyes Congo red Tetrapyrroles Porphyrins and phthalocyanines Anthracyclines Iodotoxorubicin Lymphotoxin p-receptor IgG fusion protein Antibacterials Dapsone Tetracyclineb Polyene antibiotics MS-8029 Branched polyamines Cysteine protease inhibitors Acridine derivatives Quinacrinea Ph en othi azine s A. . . nt~paras~t~cs r ouramln p-breaker peptides Synthetic peptides Anti-PrP antibodies aKorth et al. (2001) bForloni et al. (2002) SOURCE: Adapted from Brown (2002), Tables 1 and 2 and supplementary table. bral hemorrhage and infection. This case was widely debated and legally challenged, eventually leading to a court decision that the drug could be . . . g~ven to t" ,~s pat~ent. Despite the controversy over using existing formulary drugs for human prion diseases, research continues to offer new possibilities. A recent study using chronically scrapie-infected mouse neuroblastoma cells (ScN2a) showed that tricyclic derivatives of acridine and phenothiazine may have some potential for treatment of patients with CTD (Korth et al., 2001~. The investigators screened several candidate drugs, two of which had already been approved for human use: quinacrine used as an antimalarial, and chlo- rpromazine, used as an antipsychotic. Both drugs inhibited PrPSc formation and cleared the prior-infected N2a cells. The quinacrine was 10-fold more potent than the chlorpromazine. The fact that both drugs cross the blood- brain barrier is an additional reason for their use in treating patients with

200 ADVANCING PRION SCIENCE C]D. At least two patients with C]D apparently have been treated with quinacrine, and a more extensive study is planned (Love, 20011. Some caution, however, is appropriate given the recent case report of serious liver toxicity in a patient receiving quinacrine for sC]D (Scoazec et al., 20031. Very recently, this acridine class of drugs was manipulated to create a dimeric motif, bis-acridine. This new class and its derivatives, when studied in cell culture systems, appeared to show even greater potency than its relative, quinacrine (May et al., 20031. Another team, building on results seen in cell culture studies, evaluated quinacrine in a murine mode! (Collins et al., 20021. The study results were disappointing, failing to show prolonged survival of mice when treated with quinacrine orally. All mice survived the same length of time whether they were treated at 5 days, 65 days, or not at all following i.c. challenge with a mouse-adapted C]D prion agent. The authors concede that using an alter- native route of exposure, such as parenterally, might have resulted in a different outcome. They caution that different prion strains can respond differently to therapeutic agents tested in the same animal species (Collins et al., 20021. Barret and colleagues (2003) also evaluated quinacrine. They studied the drug's effect in both ScN2a cells and a murine model. Like previous researchers, they show that quinacrine inhibited the formation of PrPSc in cells but did not appear to be effective in the animal model. They note that this result supports observations reported by others in both ani- mal and human studies, and they question its use for C]D monotherapy. A commonly used antibiotic, tetracycline, has shown the ability to hinder the formation of amyloid fibrils and reverse protease resistance of the PrPSc in human strains of sC]D in cultured cells (Tagliavini et al., 20001. A later study using vC]D and BSE prion strains in hamsters showed that when tetracycline was incubated with the infectious prions prior to intrac- erebral inoculation, it partly suppressed the agent and prolonged survival of the hamsters (Forioni et al., 20021. The survival outcomes were correlated with to the amount of infectivity and the concentration of the drug. When the drug was incubated with dilute inoculum and then injected into the hamsters, one-third did not develop disease. The researchers suggest that in situations in which an exposure was recognized, such as an iatrogenic expo- sure, the drug could be useful since the prions would initially be at low levels. They comment on the structural or chemical similarities of tetracy- cline to other drugs known to suppress priors, such as iododoxorubicin, Congo red, tetrapyrroles, and acridine derivatives. Finally, they indicate that minocycline and doxycycline forms of the drug are available and cross the blood-brain barrier (Forloni et al., 20021. There are no published re- ports at present that show the effectiveness of tetracycline in human pa- tients with TSEs, however. Another therapeutic strategy is to attack structural targets along the

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 201 prion/prion protein interfaces with the hopes of inhibiting conversion. Syn- thetic peptides with the same amino acid sequences as those from the cen- tral region of the hamster's prion protein inhibited conversion of prpC to PrPSc in both mice and hamsters (Chabry et al., l 999) in cell-free conversion systems, and in ScN2a cell culture studies. A similar study of synthetic pep- tides in cell-free systems revealed that the inhibition was due to binding of the peptide on the prpC that blocked conversion to PrPSc (Horiuchi et al., 2001). Other synthetic peptides have been used to disrupt the prominent p- sheet structure of the priors. A synthetic 13-residue peptide based on amino acid residues from the conserved central region (spanning residues 115-122 and additional proline residues) was incubated with purified mice and hu- man strains of PrPSc and then introduced to Chinese hamster ovary cells and to mice (Soto et al., 20001. When the PrPSc was incubated with the peptide, resistance to proteinase K resistance was decreased in the cell stud- ies, and longer incubation periods were observed in the mouse infectivity assay. The investigators estimated that there had been a 90 to 95 percent reduction in infectivity. They also claimed a reduction of the p-sheet struc- ture of the PrPSC and an increased proportion of or helix structure (Soto et al., 20001. Gene Therapy for TSEs The application of gene therapy to treat prion disease in humans ap- pears feasible yet quite far in the future. Gene therapy, even when it in- volves well characterized vectors, genes, and disease conditions is in its in- fancy. A recent incident involving a retroviral vector in blood stem cells may have led unexpectedly to the development of a leukemia-type condi- tion in two children. That incident has resulted in even greater caution and oversight regarding this new mode of therapy (FDA, 20031. Nevertheless, some innovative experimental work in the laboratory holds promise for this method of therapy. A mutant Prep gene was engi- neered that resulted in the deletion of eight amino acids between residues 114 and 121 of the prpC prion protein. This deletion spanned most of the central amyloidogenic region of PrPSc. Using a mammalian viral vector to carry the mutant and normal Prnp genes, investigators transfected scrapie- infected mouse neuroblastoma cells. Expression of prpC protein having this deletion resulted in a dominant negative inhibitory effect on the PrPSc. The altered protein would not serve as a substrate as would normal PrPC, and it reduced the existing levels of PrPSc in the infected cells (Holscher et al., 19981. Another group investigated this dominant negative inhibition in vivo. They inserted Prnp genes having amino acid substitutions into transgenic

202 ADVANCING PRION SCIENCE mice. The inserted substitutions were known to be the polymorphic alleles Q171R and Q218K, which confer resistance to prion disease in sheep and humans, respectively. The prion protein produced by these mutant genes successfully inhibited the PrPSc inoculated into the brains of transgenic knockout mice, as well as mice that had both null and wild-type Prep genes (Perrier et al., 20021. Another highly innovative experiment was aimed at disrupting the PrPC-PrPsc interface. The investigators used a soluble, dimeric form of PrP created in transgenic mice. Vectored genes inserted in these mice resulted in their prpC being expressed as two full-length prpC molecules fused to the heavy chain of a human immunoglobulin. This Fcy (PrP-Fc2) dimer was reported to interact with PrPSc that was delivered by i.c. and i.p. routes to these experimental mice. The interaction significantly delayed onset of dis- ease in the mice, although all succumbed eventually (Meter et al., 20031. These encouraging cell and animal experiments suggest that modifica- tion of gene expression can deter PrPSc conversion. However, all the mo- lecular events that result from gene modifications need to be understood. A single point mutation can lead to familial COD. Unfortunately, the altered mechanisms introduced as a result of that point mutation are still not clear. Until those mechanisms are clarified, the use of replacement genes as a therapeutic modality is unacceptably risky. Agents That Modulate or Augment Immunity In the near term, a more promising treatment strategy is based on the use of agents that affect immune mechanisms. Such strategies might involve a broad effect on the host's innate immune system or highly specific synthe- sized antibodies designed to attack a precise epitope on prion protein or its . , 1sororm. Regarding the more generic approach, such immune modulators as oligodeoxynucleotides containing CpG dinucleotides (CpG DNA) have been used to activate the innate immune system (Agrawal and Kandimalla, 20021. These dinucleotides are contained in bacteria, but only rarely in the eukary- otic cells of animals. Because these agents activate the host immune re- sponse they have been used to boost the effects of vaccines, antibodies, allergens, and antigens (Agrawal and Kandimalla, 20021. Several different kinds of cytokines are activated by CpGs, as shown in Plate 7-1. Multiple pathways are at work simultaneously. The application of CpG oligonucle- otides in prion therapy was demonstrated by Sethi and colleagues (20021. After infecting mice with the scrapie agent i.p., they administered CpG by i.p. injection either at the time of scrapie inoculation or 7 hours later. CpG was given daily for 4 days except in one group which was treated for 20 days. In all treatment groups, but not controls, survival was extended; in

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 203 addition, the group receiving the 20 days of treatment survived without illness beyond 330 days. The investigators were unable to determine the mechanism for these effects, but speculated that they were due to the stimu- lation of toll-like receptor (TLR) 9-expressing cells, such as macrophages, monocytes, and dendritic cells (Seth) et al., 20021. A more direct approach is to modulate a specific protein of interest, such as prion protein, with a specific agent, namely antibodies. As previ- ously noted, there appears to be no natural antibody response by a host to priors. However, antibodies can be produced exogenously by novel meth- ods and administered prophylactically to the host organism. There are en- couraging studies showing that antibodies directed at PrP apparently block conversion to PrPSc. For example, researchers used a monoclonal antibody, 6H4, previously shown to bind PrP in the region spanning amino acid resi- dues 144 to 152 (Korth et al., 1997) to block infection of mouse neuroblas- toma (N2a) cells with mouse scrapie agent (Enari et al., 20011. When these cells were preincubated with 6H4 antibodies at the time of exposure to scrapie PrPSc, infection of the cells was not observed. When the antibody was added after the cells had been infected and were in a static state, the antibodies caused a reduction in the amount of PrPSc. This result suggested that equilibrium of PrPSc production and degradation existed and that it could be altered (Enari et al., 20011. Another study, also using scrapie-infected cells (ScN2a), screened sev- eral possible recombinant antibody fragments, known as Fabs, for their ability to clear PrPSc. The investigators noted significant activity associated with Fab 18, and also observed that the decrement of PrPSc was dose re- lated. They caution that future in vivo studies must recognize that Fabs have a short half-life in the body and do not cross the blood-brain barrier (Peretz et al., 20011. In vivo studies have proceeded using antibodies. One group examined the administration of several different monoclonal antibodies in mice. Mice were inoculated with antibodies i.p. at the same time they were inoculated i.p. with a mouse scrapie agent (Sigurdsson et al., 20031. The antibodies were readministered weekly until sacrifice. The antibodies prolonged sur- vival of the mice compared with controls given no antibody or standard IgG. The result of one antibody in particular, 8B4, was notable in that 10 percent of the animals receiving a diluted level of prions did not develop disease, and no toxicity was observed during the study (Sigurdsson et al., 20031. Another encouraging report was recently published by a group study- ing monoclonal antibodies in mice. In this study, using two different test antibodies, the investigators showed that even when the i.p. administration of the antibodies was delayed to 7 or 30 days after i.p. inoculation of the scrapie agent, all the mice survived and remained healthy for more than 500

204 ADVANCING PRION SCIENCE days which was 300 days longer than control mice (White et al., 20031. While very hopeful, the researchers offer caveats that the antibodies did not work when given after the onset of symptoms, suggesting that the blood- brain barrier may limit their use to prophylaxis during the incubation pe- riod. The auhors note further that although they saw no evidence for au- toimmunity, it is a possibility to be considered (White et al., 20031. At least one research team combined concepts of gene therapy and an- tibody therapy by creating transgenic mice that could produce antiprion antibody endogenously. Using some clever genetic engineering, they trans- ferred genes into knockout mice. The transgene was derived from a hybri- doma that expressed monoclonal antibody to murine PrPC. Once in the mouse, the transgene expressed a single-chain variable antibody fragment (scFv) that had anti-PrP binding attributes (Heppner et al., 20011. Follow- ing i.p. inoculation of these transgenic mice with scrapie priors, no infectiv- ity was seen in either the knockout mice (Prnp°/° ~ or mice that had one null allele and one Prnp allele (Pnrp°/+~. Nor could PrPSc be detected in the spleen (Heppner et al., 20011. The study team observed no overt symptoms of autoimmune disease but were cognizant of that potential. The investigators are hesitant to recommend such complex gene-altering strategies as those they described but are optimistic about the potential for active and passive immunization strategies used in prophylaxis or therapy for prion diseases (Heppner et al., 20011. Recommendation 7.7: Fund research to develop new therapeutic agents, including antibodies, that either block the conversion of prpc to PrPSc or disrupt the molecular mechanisms of pathogenesis of transmissible spongiform encephalopathies after this conversion has taken place. The most promising approach appears to be ratio- nal drug design, which begins with knowledge of the tertiary struc- ture of the protein or molecule that the therapeutic agent will tar- get. [Priority 11 Summary of Outlook for TSE Therapy The work in progress to develop therapeutic agents for TSEs is reveal- ing that, in cell culture and animal models, experimental agents can affect the accumulation of prions and prolong the survival of animals. At present, drug treatment in humans is limited to drugs that have been used for other medical conditions and been shown to be relatively safe. To date, no drugs or other agents have demonstrated consistent or prolonged success in treat- ing human TSEs. This failure relates in part to the use of candidate thera- pies very late in the course of disease. Also, it is unknown whether the efficacious outcome of one therapeutic agent for a particular TSE can be extrapolated to other TSEs.

ASSESSMENT OF STRATEGIES TO PREVENT AND TREAT TSEs 205 Significant acceleration in identifying effective therapeutic agents for TSEs will require scientific breakthroughs. The main obstacle to rapid progress is the same as that which is constraining rapid development of diagnostics (see Chapter 41: fundamental knowledge gaps with regards to the molecular mechanisms, immunobiology, and pathogenesis of prion dis- ease. The heartening news, however, is that breakthroughs in TSE diagnos- tics will likely translate quickly into progress in the development of thera- peutic agents because both diagnostics and therapeutics will target the structural peculiarities of priors. Diagnostics and therapies are inextricably linked for another vital reason: Therapies will likely be more successful if administered early in the preclinical stage of infection, when prions exist in the host at very low titers. Thus, having a diagnostic test sensitive enough to detect prions very early in the incubation period, long before the onset of symptoms, will likely lead to the best outcomes for persons or animals be- ing treated for prion diseases. REFERENCES Agrawal S. Kandimalla ER. 2002. Medicinal chemistry and therapeutic potential of CpG DNA. Trends in Molecular Medicine 8(3):114-121. Agriculture and Agri-Food Canada. 2003. Fact SI7eet: Trade. [Online]. Available: http:// www.agr.gc.ca/cb/trade/factsheet_e.phtml [accessed June 8, 2003]. Anderson RM, Donnelly CA, Ferguson NM, Woolhouse ME, Watt CJ, Udy HJ, MaWhinney S. Dunstan SP, Southwood TR, Wilesmith JW, Ryan JB, Hoinville LJ, Hillerton JE, Aus- tin AR, Wells GA. 1996. Transmission dynamics and epidemiology of BSE in British cattle. Nature 382(6594):779-788. APHIS and FSIS (Animal and Plant Health Inspection Service and Food Safety and Inspection Service). 2002. Bovine Spongiform Encepl7alopatI7y (B. SK) Response Plan Summary. Washington, DC: USDA APHIS. Asher DM, Pomeroy KL, Murphy L, Gibbs CJ Jr, Gajdusek DC. 1987. Abstract: Attempts to Disenfect Surfaces Contaminated witI7 Etiologic Agents of tI7e Spongiform Encepl7alopa- tI7ies. Presentation at the VIIth International Congress of Virology, Alberta, Canada. Asher DM, Pomeroy KL, Murphy L, Rohwer RG, Gibbs CJ Jr, Gajdusek DC. 1986. Abstract: Practical Inactivation of Scrapie Agent on Surfaces. Presentation at the IX International Congress of Infectious and Parasitic Diseases, Munich. Barret A, Tagliavini F. Forloni G. Bate C, Salmona M, Colombo L, De Luigi A, Limido L, Suardi S. Rossi G. Auvre F. Adjou KT, Sales N. Williams A, Lasmezas C, Deslys JP. 2003. Evaluation of quinacrine treatment for prion diseases. Journal of Virology 77(15):8462- 8469. Belay E. 2003. CJD Surveillance in tI7e United States. Presentation to the IOM Committee on Transmissible Spongiform Encephalopathies: Assessment of Relevant Science, Meeting 4. Washington, DC: National Academy Press. Bernoulli C, Siegiried J. Baumgartner G. Regli F. Rabinowicz T. Gajdusek DC, Gibbs CJ Jr. 1977. Danger of accidental person-to-person transmission of Creutzieldt-Jakob disease by surgery. Lancet 1(8009):478-479. Brown P. 2002. Drug therapy in human and experimental transmissible spongiform encepha- lopathy. Neurology 58(12):1720-1725.

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