The Immune System in the Pathogenesis and Prevention of Prion Diseases

Annissa Furr^1,2, Alan J Young^1,2* and Jurgen Richt³

¹Department of Veterinary and Biomedical Sciences, South Dakota State University

²Medgene Labs LLC, Brookings, SD

³Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS

*Corresponding Author:

Dr. Alan J. Young
Department of Veterinary and Biomedical Sciences
South Dakota State University
Brookings, SD. 57006, USA
Tel: 605-688-5982
Fax: 605-688-6003
Email: alan.young@sdstate.edu

Received Date: August 31, 2011; Accepted Date: January 27, 2012; Published Date: February 16, 2012

Citation: Furr A, Young AJ, Richt J (2012) The Immune System in the Pathogenesis and Prevention of Prion Diseases. J Bioterr Biodef S1:012. doi: 10.4172/2157-2526.S1-012

Copyright: © 2012 Furr A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Bovine spongiform encephalopathy (BSE) remains a significant threat to both human and animal health in the United States and in particular a threat to the agriculture industry. The diagnosis of BSE in a single cow within the United States in 2003 led to net losses of billions of dollars and additional studies have pointed to significant sociological consequences of BSE cases in Canada, particularly in terms of the dissolution of rural communities. Unlike other infectious diseases, BSE and other prion diseases are caused by a misfolded protein (PrPSc) that is highly resistant to normal nucleic acid-based disinfection and sterilization procedures. Disease progression is linked to the progressive conversion of a normal cellular molecule (PrPC) into the infectious form (PrPSc) mainly on cells of the immune and neurological systems, leading to formation of multimolecular fibrils associated with progressive neurodegeneration and dementia. Due to the unique nature of this infectious particle and the fact that the disease is caused by a misfolded form of a normal self-protein, development of vaccines and other therapeutics have been challenging. The molecular
mechanisms behind PrPSc targeting to lymph node germinal centers and the failure of immune recognition, remain unclear. Here, we review the current knowledge of prion disease biology, the role of the immune system in disease transmission and pathogenesis and efforts towards development of therapeutics and vaccines. While feed-based control of BSE has been successful in limiting transmission and spread of the disease, the recent description of apparently spontaneous and/or genetic forms of BSE underlies the importance of continued research into tools to counter this unique disease threat to the world economy as well as animal and human health.

Introduction

Bovine spongiform encephalopathy (BSE) is an ongoing threat to the cattle industry worldwide and is also of concern from a biodefense perspective because its causative agent, the infectious prion, is characterized both as a USDA-designated select agent and as a potential object for agroterrorism. The primary focus of prion diseases as a threat to the security of the United States should be the proven economic consequences of the identification of BSE-positive animals and animal products and its significant consequences for the US beef export market. It has been estimated that the 2003 case of BSE in the United States resulted in a net loss to the export market of between $3.2 and $4.7 billion, in addition to the ongoing sociological burden to the rural economy that continues to affect agricultural and associated industries [1,2]. The economic consequences of BSE are further exacerbated by the realization that the BSE agent, unlike other transmissible spongiform encephalopathy (TSE) agents, appears to transmit across species barriers and has resulted in a novel human prion disease, called variant Creutzfeldt-Jacob disease (vCJD), in at least 200 individuals to date [3]. TSEs of sheep (scrapie) had been recognized for more than 200 years when BSE was first recognized in the British cattle herd in 1986 [4]. The origin of the original case(s) of BSE remains an enigma, although several potential causes have been suggested. Hypotheses include (i) the inclusion of sheep- or goatderived scrapie agent-infected tissues in meat and bone meal fed to cattle [5], (ii) a previously undetected sporadic or genetic bovine TSE contaminating cattle feed [6], or (iii) origination from a human TSE through animal feed contaminated with human remains [7]. It is generally accepted that the practice of feeding meat and bone meal from affected animals back to the cattle amplified the disease in the European dairy and beef herds, and implementation of an outright ban on this process has significantly limited further occurrence of the disease. It was estimated that approximately 750,000 animals infected with the BSE agent were slaughtered and consumed by humans in Europe (mainly UK) between 1980 and 1996 [4]. Since that time, it has become largely accepted that BSE is caused by a promiscuous strain of prion pathogen that effectively crosses species barriers and affects both humans and multiple animal species and as a consequence over 200 cases of variant CJD (vCJD) and one case of goat BSE have been identified [3-8]. North America identified its first BSE case in May 2003 in a Canadian-born cow in Alberta, and in December 2003 the first American BSE case was diagnosed [9,10]. Although this animal was later identified as a Canadian import, the identification of two additional indigenous cases in 2005 and 2006 (82,84) clearly established the presence of BSE within the United States, albeit at significantly lower rates than those measured in most parts of Europe. Molecular characterization of these cases indicated three specific etiologies for BSE similar to human TSEs: infectious, genetic and sporadic [10,11]. Regardless of the cause, the severe economic consequences of BSE and its zoonotic potential were the most important aspects of implementing mitigation strategies, with an estimated cost to the US Cattle Markets of $3.2-4.7 billion, most of which was due to losses in the export market [1].

Overview of TSE History and Pathogenesis

Naturally occurring transmissible spongiform encephalopathies (TSEs) have been found in numerous mammalian species including deer and elk (chronic wasting disease, CWD), sheep and goats (scrapie), cattle (bovine spongiform encephalopathy, BSE), mink (transmissible mink encephalopathy, TME) and humans. TSEs of humans include sporadic, genetic and infectious forms of Creutzfeldt-Jakob disease, Kuru, Gerstmann-Sträussler-Scheinker syndrome and familial fatal insomnia. Of these diseases, only BSE agents appear capable of extensively crossing species barriers and are believed to be the cause of vCJD. As a group, these unique diseases pose a considerable health threat to humans and animals and impose a significant financial burden on the agricultural industry. Billions of dollars are lost each year due to trade restrictions, disease surveillance and herd destruction, which could be at least partially ameliorated by a sensitive antemortem test or effective vaccine strategies. Due to its unique nature, however, definitive ante mortem diagnostic tests and development of immune-based counter-therapies have been elusive.

The oldest of the TSEs or prion diseases is scrapie in sheep, which has been known since the middle of the 18th century [12]. It is believed that scrapie, like all TSEs, is caused by ingestion of material containing the altered form, PrP^Sc, of the normal cellular protein PrP^C. Clinical signs of TSEs include degenerative neurological problems, weight loss and marked changes in personality or behavior. In most prion diseases, incubation periods are measured in years, accounting for their early designation as “slow viral diseases” [12].

The normal cellular protein PrP^C is a glycosylphosphatidylinositolanchored plasma membrane protein that is highly conserved and encoded by a single copy host gene (Prnp) [13]. PrP^Cis found on nearly every cell in the body but is highly expressed by neuronal cells and most cells of the immune system. The molecular function of this ubiquitous cellular protein is not yet clear, but its importance in several cellular processes has been identified. As a result of these studies, PrP^C appears to be a pleiotropic protein involved in cellular trafficking, copper uptake, protection against oxidative stress, cell adhesion and differentiation, as well as cell signaling and protection against apoptotic activity [14-16]. Clearly, many of these functions are of importance to the immune system, although identification of specific immunological defects in Prnpknockout mice and cattle have not been reported [17,18]. In addition, PrP^C appears to have some basic physiological significance based on the fact that it is expressed and rather highly conserved in many species including humans, hamsters, rats, mice, cattle, sheep, goats and chickens [19]. However, it has been reported that mice engineered to lack the gene encoding PrP^C exhibit no developmental or behavioral problems except a higher susceptibility to seizures and no significant defects have been observed in prion-knockout cattle [17,20,21].

According to the “protein-only hypothesis”, prions are the causative agent of all TSEs including scrapie in sheep and CWD in deer and elk. Prions are infectious, structurally altered isoforms of the normal cellular proteins that are partially resistant to proteolytic degradation [14,22-24]. PrP^Sc and PrP^C are identical in their primary protein structure; therefore, changes in their secondary structure account for the structural and physiological differences between the two proteins. PrP^C is primarily alpha-helical in conformation with very few betasheets, while PrP^Sc is comprised of significantly fewer alpha-helices but is rich in beta-sheets, although this structure remains to be confirmed by x-ray crystallographic techniques. The mechanism by which PrP^C is converted to PrP^Sc remains elusive, but research indicates that the conversion occurs either on the cell surface, or intracellularly after PrP^C has been internalized for degradation [24]. PrP^C is therefore converted post-translationally to PrP^Sc. Once formed, the infectious PrP^Sc forms multimeric amyloid plaques as it accumulates, causing characteristic and identifiable accumulations within germinal centers of lymph nodes and affected central nervous system tissue. Within the CNS, this results in progressive cell death and the spongiform plaques identified in the brains of infected humans and animals [13,25]. The conversion of PrP^C into PrP^Sc is an autocatalytic process and newly formed PrP^Sc can then propagate the conversion of more PrP^C. In support of this, an in vitro amplification technique known as protein misfolding cyclic amplification (PMCA) has been used with great success in prion research and is the basis for numerous studies designed to identify small but infectious doses of PrP^Sc in test material. PMCA, which is analogous to PCR in principle, can cause the generation of PrP^Sc from PrP^C by using small amounts of PrP^Sc infectious protein as seed to catalyze the conversion of native or recombinant PrP^C in vitro [26]. Additional studies have confirmed that this newly-amplified PrP^Sc is in fact infectious in rodent models, although quantitative measurement of prions from other species has been more complex [27]. To date, therefore, mapping of disease pathogenesis has largely relied upon immunohistochemical detection of prion fibrils and biochemical detection of PrP^Sc.

Transmission of TSEs

Species specificity of TSEs

Although Prnp, the gene that encodes the normal cellular protein PrP^C, is highly conserved among species, TSE agents themselves are highly species-restricted. With the exception of the BSE agent, which appears highly capable of crossing species barriers, all other TSE agents tend to be restricted to closely-related species (Table 1). These natural species barriers may be overcome by experimental inoculation, but the mode of infection is generally not one that occurs in nature and the inoculating dose is usually substantially higher than would be expected in nature [28]. Following intraspecies experimental transmission, the incubation period of disease is normally significantly longer than in the natural host, although successive passages through the new species can to some extent ameliorate this [29]. Although not a natural host for prion diseases, murine models of TSEs have been developed either by adapting natural scrapie or TME agents to rodent models, or by engineering mice that express Prnp from sheep, deer, humans, and cattle [30]. These models have been extremely useful in examining the species restriction of prion diseases and development of bioassay systems for infected material from natural hosts. Furthermore, these experiments clearly indicate that transmission of disease depends upon matching the sequence of host PrP^C to the original species from which the PrP^Sc originated [31]. This relationship has been further confirmed using the in vitro PMCA assay [32].

Table 1: Known Transmissible Spongiform Encephalopathies and their Natural Hosts.

Genetic susceptibility to infectious TSEs: Scrapie and Chronic Wasting Disease

Genetic susceptibility to prion disease varies depending on target species. Isolation of specific polymorphisms affecting overall susceptibility of wild deer species has been difficult, however recent data may indicate that genotype can affect incubation times of experimental disease [33-36]. Other species, including sheep and elk, appear to have a possible genetic resistance or enhanced susceptibility to prion disease depending on very specific polymorphisms within the Prnp gene [37]. In sheep, amino acid substitutions at three specific codons appear to be most influential for determining susceptibility or resistance to disease progression. These three codons are: alanine (A) or valine (V) at codon 136, arginine (R) or histidine (H) at codon 154, and glutamine (Q) or arginine (R) at codon 171. Animals with the genotype of ARR are most resistant to infection with the scrapie agent, while animals with the genotype VRQ are most susceptible to disease [38]. In elk, one epidemiological study suggested that animals with a leucine at codon 132 may be resistant to infection with the CWD agent, whereas those with methionine are susceptible [29], although this could not be confirmed in experimentally infectedelk [39]. The codon 132 polymorphism is particularly intriguing, given that all patients suffering from vCJD to date are homozygous for methionine at the analogous codon 129 of the human Prnp gene [40,41]. The reason for this genetic resistance or enhanced susceptibility has yet to be explained, but may relate to either the conformation or expression level of native PrP^C in these individuals. Susceptible sheep have a higher percentage of cells expressing PrP^C on their surface than resistant animals. Furthermore, resistant sheep have a higher percentage of CD4+ and CD8+ cell populations following infection with the scrapie agent [42]. These differences may explain, at least in part, the genetic resistance to disease.

Lymphoid Pathogenesis of TSEss

Involvement of immune cell subsets in prion disease

Current research in prion immunology has suggested the possible involvement of multiple cell types of the lymphoreticular system (LRS) during disease progression. Interestingly, almost all cells of the LRS express high levels of PrP^Con their surface, which may be a reason why they are involved in the early stages of the disease [43,44]. Prion accumulation has also been found within granulomas of transgenic mice infected with prions [43], in lymphoid follicles of kidneys in white-tailed deer infected with the CWD agent [45], and has been identified at low levels within the muscle of mule deer infected with the CWD agent and in the muscle tissue of hamsters infected with the TME agent, potentially associated with local inflammation in those tissues [46,47]. This clearly implicates a role for the immune system as a potential target, if not responder, to the prion agent.

Mechanism of Prion Targeting to Lymph Nodes

General considerations

In many ways, targeting of PrP^Sc to germinal centers appears similar to antigen delivery and deposition within lymph nodes during normal immune responses. Although the current understanding of the function and mechanisms involved in antigen delivery and initiation of T and B cell responses has been reviewed elsewhere, a brief discussion of the normal process ofantigenic recognition and B cell stimulation is warranted in order to discuss similarities and differences associated with prion infection [48-50]. Following introduction into the nonlymphoid tissues of the body, antigen may be delivered to the regional lymph node in one of two mechanisms. Large, insoluble antigens appear to be engulfed by resident dendritic cells, which then actively enter the afferent lymphatics and travel to the draining lymph node. In contrast, small, soluble antigens appear to passively enter the lymph fluid and are then delivered to the outer boundary of the germinal center by virtue of normal lymph flow [51]. There is now evidence to suggest that B cells actively shuttle these antigens to the follicular dendritic cells (FDCs), non-hematopoietic cells found within the germinal center. These antigens are then believed to bind to the surface of FDCs by virtue of Fc or complement receptors, where they are passively “presented” to B cells for activation. Activated B cells can then be stimulated by these immobilized antigen complexes, and receive costimulation from follicular T cells or the FDCs themselves. FDCs possess several characteristics which further regulate B cell differentiation and activation, including cell-surface TLR4 and the chemokine CXCL13 (BLC) which recruits CXCR5 positive B cells to the germinal center [50- 52]. Following stimulation, B cells may differentiate into plasma cells, which actively secrete antibody, or memory B cells, in a mechanism which is not entirely understood. Associated with this differentiation, B cells downregulate expression of CXCR5, and upregulate CXCR4 which promotes their migration towards the dark zone and differentiation to plasma cells [50]. Although the majority of plasma cells migrate to the medulla of the lymph node to secrete antibody, memory B cells and so-called “lymphoblasts” leave the lymph node in the efferent lymph, ultimately migrating to other tissues of the body via the blood (Figure 1).

Figure 1: Schematized Model of PrPSc Interactions in Lymph Nodes:
1. PrPSc is delivered to the lymph node in the afferent lymph. Free PrPSc may directly activate complement to assist in delivery, or may be phagocytosed and delivered by migratory dendritic cells. 2. PrPSc is targeted to the Follicular Dendritic Cells, probably through the interaction of complement degradation products on the PrPSc and CD21/CD35 on the surface of the FDC. 3. PrPSc may be amplified by converting PrPcon FDCs to PrPSc, or by secretion in the form of exosomes which are then phagocytosed by Tingible Body Macrophages in the germinal center. 4. B cells interact with the “infected” FDCs, potentially in an abortive attempt to respond to the conformationally foreign PrPSc. 5. Mature B cells downregulate CXCR5 and upregulate CXCR4, migrate from the germinal center to become either antibody secreting plasma cells or memory B cells that may have been infected with PrPSc. 6. PrPSc-containing memory B cells leave the lymph node in the efferent lymph where they re-enter the blood and home to germinal centers of distant lymphoid tissues via CXCR5 interactions.

It is interesting to note that the early germinal center response to both T-dependent antigens and T-independent antigens is similar. However, while FDCs may provide important costimulatory signals to B cells in the absence of T cell help, T-independent responses tend to be far shorter than T-dependent responses, with germinal centers beginning to involute within 5 days of stimulation [49]. In the absence of antigen presentation to T cells and resulting T-helper cell responses, therefore, B cell differentiation and response is significantly abrogated, although still present and tends to result in production of low affinity IgM antibody. It is intriguing that the localization of the prion agent in lymphoid tissues following infection appears to parallel many of these mechanisms closely.

Mechanisms of PrP^Sc Delivery to Lymph Nodes

Antigen trafficking into the germinal center remains somewhat unclear, but recent studies using two-photon microscopy have allowed some insights into these events. Antigens that form immune complexes are retained on the surface of FDCs within germinal centers through specific receptors [48]. FDCs bind immune complexes via complement receptors CD21/CD35 that bind proteolytic components of C3 and C4. These captured immune complexes are then held in the germinal center to allow migrating immune cells to come in contact with and react to the antigens. Research using two-photon microscopy has shown that subcapsular macrophages and B cells are responsible for trafficking the immune complexes from the point of infection to the germinal center [53]. It is possible that prions, being larger than typical antigens, follow a pattern similar to immune complexes as they travel into the germinal center. Accumulation of prions is intensified throughout infection, suggesting that the LRS might be a continuous trafficking medium of prions throughout the course of infection. In addition, the LRS could also be a site of replication of the infectious agent. Upon examination of lymph nodes in varying stages of disease, it is obvious that in a susceptible, infected animal there is a steady increase in the amount of PrP^Sc as the disease progresses (unpublished observations). Assuming that the animal is not continually exposed to the infectious agent in the environment, it can be assumed that the increase in infectious material is achieved by conversion of host PrP^C to PrP^Sc during disease incubation.

Targeting of Soluble PrP^Sc to Germinal Centers

Complement, a collection of plasma proteins that make up part of the innate immune system, has the ability to bind to antigen and, through a catalytic amplification loop, activate cells of the immune system. Activation of each active component is accomplished by cleavage of an inactive precursor into an active form capable of activating the next component in the pathway and a cleavage product that may have additional bioactivity. Complement can be activated via the classical pathway, the alternative pathway, or the lectin pathway. Both the classical and alternative pathways have been associated with prion disease [54]. PrP^Sc has been demonstrated to directly activate complement component C1 in the absence of specific antibody, thereby initiating the classical complement pathway. Ultimately, this results in cleavage of the C3 component, presumably depositing C3b, C3d, and C4d complement components on the surface of the scrapie fibrils. FDCs trap and retain antigen through expressed complement receptors on their surface. Complement receptors CD21 and CD35 bind the complement proteolytic fragments C3b, iC3b, C3d and C4b [53,55]. Both CD21 and CD35 are critical in normal antigen targeting to FDCs and germinal centers [48]. Antigen complexes or PrP^Sc are then “presented” to B cells through the interaction of surface antibody on B cells and specific receptors on the surface of FDCs, supported by co-receptors CD21 and CD40. Together, surface adsorbed antigen and these co-receptors activate antigen-specific B cells in germinal centers and initiate both T-dependent and T-independent antibody responses to antigen [49]. Clearly, these processes are at least partially interrupted in prion-infected germinal centers, as there is no evidence for significant production of PrP^Sc-specific antibody, although recent reports suggest low-affinity PrP-specific antibody can be produced late in the disease process in mouse models [56]. Nonetheless, the targeting and accumulation of PrP^Sc on FDCs appears independent of antibody, and is likely at least partially dependent on complement activation [57]. Mice genetically deficient in antibody production can be infected with PrP^Sc with no observable differences in incubation time or production of PrP^Sc infectivity [57]. In contrast, mice that lack CD21/CD35 receptors demonstrate significantly prolonged incubation periods as compared to wild type susceptible mice [58]. Inactivation or elimination of C1q renders mice resistant to prion infection [57,59]. Cobra venom factor (CVF) non-specifically depletes the complement system of mice for up to 5 days. When mice are treated once with CVF and then immediately infected with the scrapie agent, these mice survive the CVF-untreated infected mice by at least 2 months [60]. This further indicates a significant involvement of the complement pathway in the early establishment of infection. In addition, these data imply that early trafficking to the LRS is vital for successful establishment of the disease: even though the alternative complement system is restored after 5 days, the mice retain substantial protection from infection [57].

Cell-Borne Delivery to Germinal Centers

It is generally accepted that initial prion entry in the intestine is mediated by direct uptake from the intestinal lumen by M-cells [61-65]. Following uptake, delivery and dissemination of the prion molecule is less clear, but there is significant evidence for a role for dendritic cell (DC) subsets in transport [66,67]. DCs have the ability to ingest antigen and retain the antigen for extended periods of time, and are believed to play a role in the normal transport of antigen between the tissues and lymph nodes [50,51]. Following infection of the gut, PrP^Sc-positive DCs can be detected in the intestinal lymph, presumably in transit to the regional lymph nodes [66]. In addition, tingible body macrophages in lymph nodes show infectivity and contain PrP^Sc at early and late stages of infection, and while generally not believed to be migratory they remain potential candidates for harboring or disseminating infection throughout the lymphoid system [68]. Macrophages carrying PrP^Sc have been identified in the lymph nodes of infected animals, and appear to act as the primary transport mechanism in animals that have been depleted of FDCs [55]. It therefore seems likely that the earliest stages of prion infection of the lymphoid system involve a normal antigeninduced process designed to clear foreign infections from the host, and that the conversion from antigen to pathogen occurs following delivery to the germinal center.

Amplification of Prions within Germinal Centers

Shortly after PrP^Sc delivery to the germinal center, the infectious protein accumulates within the light zone, similar to other antigens (Figure 1) [69]. Specifically, the PrP^Sc is targeted to follicular dendritic cells (FDCs) within the germinal centers that remain positive by immunohistochemistry throughout infection [70]. FDCs are longlived, non-dividing cells of epithelial origin that can retain antigen complexes on their surface for extended periods [71,72]. Although their precise origin remains unknown, it is clear that they are absolutely required for peripheral amplification of PrP^Sc during infection [18,73]. Murine models have further demonstrated a role for FDCs as the central cell type within lymph nodes for prion pathogenesis by treating mice with gamma-irradiation either before or after infection with PrP^Sc, a treatment that shows no effect on the establishment of prion disease [74]. Due to the fact that FDCs and neurons are mitotically inactive and therefore survive gamma-irradiation, while T cells, B cells and monocytes are susceptible, these studies demonstrated that FDCs and neuronal cells but not circulating lymphocytes are vital in the pathogenesis of prion disease [54]. Within the germinal center, FDCs constitutively express high levels of PrP^C, which presumably functions as a template for PrP^Sc replication, which results in the deposition of scrapie fibrils [73]. In cattle, there is no detectable amplification or deposition of prion protein in lymph node germinal centers following infection. It is interesting to note that cattle FDCs, unlike those of other species, express an antigenically unique isoform of PrP^C different from that observed in neuronal cells, potentially explaining the absence of peripheral replication of the prion agent in cattle [75]. Although pathological changes in lymphoid tissues infected with PrP^Sc have been difficult to define, one study observed abnormal FDC maturation cycles in the mesenteric lymph nodes of mice infected with the scrapie agent [76]. In general, these alterations limited the ability of FDCs to downregulate PrP^C and resulted in increased immune complex and PrP^Sc deposition. Together, these data suggest a central role for FDCs in prion amplification during disease incubation.

In addition to FDCs, tingible body macrophages within lymph nodes have also been identified as containing PrP^Sc [68]. These cells normally phagocytose dead and dying B cells within germinal centers, but may also play a central role in the clearance of PrP^Sc from affected tissues [77]. Macrophages are able to phagocytose small, PrP^Sc containing vesicles produced by infected FDCs, and elimination of macrophages from prion-infected mice results in significant enhancement in the level of PrP^Sc within tissues [78]. Interestingly, while resting macrophages can effectively phagocytose and clear PrP^Sc, activation of macrophages appears to decrease the ability of these cells to phagocytose and degrade the infectious material [79]. Furthermore, activated macrophages appear to increase PrP^C expression, potentially providing additional template PrP^C for agent amplification. Ultimately, this could lead to accelerated PrP^Sc replication during immune responses. Interestingly, the presence of bacterial colitis has recently been shown to increase susceptibility to oral prion infection [80]. It is important to note, however, that several studies appear to directly refute this conclusion. Specifically, activation of innate immunity through administration of Freund’s Complete adjuvant significantly prolongs prion disease in mouse models, and interfering with innate immunity through targeted activation of TLR-9 or knockout of TLR-4 accelerates the disease process [81,82]. Therefore, the effect of systemic immune activation in disease pathogenesis remains unclear.

Systemic Dissemination through the Lymphoreticular System

During the asymptomatic period of TSE prion pathogenesis there is significant peripheral spread of PrP^Sc throughout the lymphoid system. Although it is possible that free PrP^Sc may effectively enter the efferent lymph, and eventually the blood, causing prionemia, a more likely explanation is the infection and dissemination of recirculating lymphocytes. A number of studies have clearly demonstrated that the infectious agent could be transmitted between susceptible sheep or deer by blood transfusion [83-86]. In rodents, in vitro detection of PrP^Sc associated with buffy coat leukocytes has illustrated the importance of circulating leukocytes in agent dissemination [87,88]. More recently, several studies have directly implicated blood-borne B cells as the source of this infectivity [83,89]. The mechanism whereby B cells become infected and potentially transmit PrP^Sc to distant lymph nodes remains unclear; however, it seems likely that it is acquired directly from PrP^Scinfected FDCs in germinal centers. Whether this acquisition of PrP^Sc is part of a normal immunological process, or incidental as a result of PrP^C expression by B cells, remains to be investigated. It is interesting to note that CXCR4 expression is increased associated with normal B cell maturation within lymph nodes, and that CXCR4 ligation by CXCL13 on murine olfactory cells has been reported to increase PrP^C expression [50,90]. Regardless of the mechanism, it seems likely that migratory B cells may be a major factor in the dissemination of PrP^Sc throughout the lymphoid system of affected animals.

Immune Response to Prions

There does not appear to be a significant protective immune response to PrP^Sc infection, presumably due to the fact that the primary sequence of PrP^Sc is identical to PrP^C and hence subject to self-tolerance [91]. Since both T and B cells are selected during development to ignore self-antigens in order to develop self-tolerance, one would not expect it to react to an antigen with the same amino acid sequence asPrP^C, a normal cellular protein. This concept is supported by the fact that production of PrP-specific antibody and sensitized T cells can only be accomplished effectively in Prnp-knockout mice [92,93]. Recently, low-affinity IgM antibodies specific for PrP have been reported in experimental mice following clinical signs associated with scrapie, suggesting that long-term infection may result in a T-independent response to PrP^Sc [56]. Despite tolerance, there is substantial lymphoreticular system (LRS) involvement in prion disease. The LRS, primarily the lymph nodes, is a source of accumulation and possible propagation of PrP^Sc well before the agent is established in the brain, and therefore the circulating immune system remains a primary target for development of ante mortem diagnostic assays [69,94]. Prions accumulate quickly in the lymph nodes and remain there throughout the extended asymptomatic period, whereas they only accumulate in the brain in the later stages of the disease. Incubation periods vary greatly based on species. Incubation times for experimental sheep scrapie are normally 1-2 years, while humans can incubate prion disease in excess of 50 years before showing any clinical signs [31,95]. Therefore, it may eventually be possible to treat prion diseases after infection but before PrP^Sc accumulation has been established in the brain. As long as accumulation in the brain is inhibited, the host appears to suffer no ill effects from the disease [95].

Immune-Based Approaches to Prion Disease Therapy

Effective prevention and treatment of prion diseases is focused on two major areas: Preexposure prevention, and post-exposure prophylaxis. To date, attempts to prevent prion disease through vaccination of normal individuals has been limited, whereas progress has been made in distinct therapeutic approaches to limit prion fibril replication following infection.

There is no measurable immune response against prions during normal disease progression [96], however, a number of studies have focused on the development of appropriate immunization strategies to develop immunity to PrP^C, PrP^Sc, or both [96]. Attempts to develop vaccination strategies in murine models have been hindered by (i) selftolerance to the primary structure of the PrP molecule (ii) potential autoimmune responses to self- PrP^C induced by vaccination and (iii) inability to provide protection to cerebral tissues due to immunological preference of the brain, and impermeability of the blood-brain barrier [96]. Similar difficulties have been illustrated in studies designed to uncover effective vaccination strategies for Alzheimer’s disease. Nonetheless, studies have demonstrated the effectiveness of anti-PrP antibodies in blocking both infection and disease progression in vitro. Specifically, anti-PrP antibodies directed against distinct PrP epitopes have been shown to inhibit prion replication in affected cell lines [97- 99]. In later work, one of these antibodies was engineered as a singlechain antibody (scFv) fragment to allow greater tissue penetration, and was found to retain its ability to inhibit PrP^Sc conversion in vitro [100]. In an attempt to provide direct, local production of inhibitory antibodies at the site of replication, an adeno-associated virus type 2 was used deliver cDNA of several anti-PrP scFv antibodies in a mouse model of prion infection. While there was apparently little correlation between the affinity of individual scFv fragments for PrP^C and their ability to inhibit disease, several constructs did effectively lengthen survival of experimental mice [101].

Additional studies in animal models were also shown to have some success. Mice rendered transgenic for the heavy chain of the 6H4 anti-PrP monoclonal antibody were resistant to prion infection, and both direct immunization of wild-type mice with recombinant PrP^C or passive vaccination with PrP-specific antisera delayed disease onset [102-104]. Significantly higher doses of passive antibody were shown to block disease, even when provided to infected mice up to one month after exposure to PrP^Sc-containing material [105]. This may then represent a potential therapeutic strategy when the date of exposure is known, but is less likely to prove a useful therapy in most clinical or agricultural situations. Passive vaccination is ineffective following development of clinical signs, however.

In terms of direct immunization, a number of strategies have been attempted to bypass selftolerance mechanisms and stimulate active immunity to prevent infection. These include approaches using bacterially-expressed full-length PrP, dimeric PrP, synthetic PrP peptides and polypeptides with various adjuvants and various combinations of the above [106-109]. Unfortunately, anti-PrP titers in all cases tended to be low and more importantly all attempts were nonprotective against prion infection. In an attempt to exploit the speciesspecific differences in PrP^C, wild-type mice were immunized with a cDNA coding for human PrP^C linked to a T-cell immunostimulatory epitope. In these studies, tolerance was effectively broken in mice given three successive injections of the cDNA construct, as evidenced by generation of autoantibodies directed against the native prion protein [110]. More recently, research has focused on the initiation of T-cell based immunity in order to break the natural tolerance to PrP^C. In one such study, dendritic cells were pre-loaded with prion protein peptides and injected into wild-type mice at 15 day intervals [111]. Spleen cells and sera collected 10 days after the final challenge demonstrated that T cells could be induced to produce a good lymphokine response as defined by Interferon-gamma and IL-4 release. When administered prior to challenge with the scrapie agent, PrP-loaded DCs were capable of slowing the disease progression, suggesting that initiating an anti- PrP T cell response may be one means of providing some protection against disease. It has also been suggested that, analogous to cancer therapies, the inoculation of PrPspecific T cells may be used to slow development of prion disease in affected individuals [93,112].

In order to avoid potential autoimmune responses of prion vaccination, investigators have targeted regional immunity at the portal of entry (the mucosa) rather than systemic immunity which could lead to adverse effects. Goni et al. expressed PrP in attenuated Salmonella as a live oral vaccine, which resulted in a significant delay in onset of prion disease in mice [113]. Vaccines based on Salmonella vectors have been used previously in ruminants, and may be an inexpensive route to prevent the spread of prion disease in animals [114]. It will be crucial, however, to limit immunity to the gut, as systemic (and in particular neurological) immunity may lead to significant neuropathology. Following direct intracerebral injection of anti-PrP antibodies, both hippocampal and cerebellar neurons were targeted with bivalent but not monovalent IgG antibodies [115]. While this may be due to direct activation of complement and initiation of Antibody-Dependent Cellular Cytotoxicity, it is equally possible that direct crosslinking of the neuronal PrP resulted directly in neurotoxicity. These questions must be resolved prior to implementation of antibody-based prevention and therapy for prion diseases. An alternative approach would be to directly target the PrP^Sc molecule, while maintaining tolerance to native PrP^C. This approach has had some success in murine models, in which a Tyrosine-Tyrosine-Arginine (YYR) epitope of PrP^C was conjugated to a leukotoxin-carrier protein to induce PrP^Sc-specific immunity [116]. In a murine model system, this vaccine regimen produced a strong PrPSc-specific IgG response, suggesting potential for development of a PrPSc-specific vaccine in the absence of autoimmune complications. It is noteworthy that IgG1 titers were found also within the cerebral spinal fluid and mucosal secretions, suggesting the potential for systemic protection against prion infection. Clearly, more work is necessary, however the potential for active protection against TSEs by direct vaccination remains a possible, and highly desirable, resolution to the potential economic and health threat of prion agents.

Acknowledgment

This work was supported by the NIAID-NIH PO1 AI 77774-01 “Pathogenesis, Transmission and Detection of Zoonotic Prion Diseases”, and NIAID-NIH grant 1R15AI072757. The authors thank April Beyer for assistance in preparation of the manuscript.

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