© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PrPSc copies. The exact mechanism governing this conversion is still under dispute, but it is widely accepted that it does not involve modifications of the primary structure of the protein.

The physiological and pathological PrP isoforms display distinct conformations. The N-terminal region of PrPC is highly unstructured, as opposed to the globular C-terminal region, which contains predominantly a-helices and only a minor region encompassing twostranded β sheet [2]. Infrared spectroscopy and circular dichroism data indicate clear differences in the secondary structure of PrPC and PrPSc, in which equal amounts of a-helices and β sheets can be found [3]. This conformational difference is believed to be at the basis of the biochemical differences observed between the two isoforms, namely, the partial proteinase K resistance, the reduced solubility, and the fibril-producing potential displayed by PrPSc [4]. To date, the only known difference at the chemical level is associated with the oxidation level of the methionine residues, which was found to be elevated in PrPSc compared to PrPC [5].

The physiological role of the prion protein remains obscure. Its high level of conservation among species would indicate that PrPC is of crucial importance to the organism; however, PrP−/− mice are viable, developmentally and behaviorally normal, and do not display a prominent phenotype except for the complete protection against prion diseases [6, 7]. PrPC has since been implicated in a variety of cellular functions, including cell proliferation, differentiation and survival, protection against oxidative stress, and synaptic function (reviewed in [8, 9]). Further evidence suggests it may play a role in the immune system. In line with this, it has been recently reported that PrP−/− mice display lower numbers of CD4 T cells and lymphoid tissue inducer (LTi) cells as well as impaired splenic T zone structures [10]. Moreover, immune responses have been reported during prion diseases progression, suggesting the involvement of the immune system in disease pathogenesis, and immune-based approaches have yielded some of the most promising results toward protection and/or treatment of spongiform encephalopathies. In this chapter these exciting aspects of prion biology will be discussed.

## **2. PrP and the immune system**

#### **2.1. PrPC expression patterns in cells of the immune system**

Even though PrPC is predominantly expressed in the central and peripheral nervous system [11, 12], elevated protein expression levels have also been reported in many cells of the immune system. In long-term hematopoietic stem cells (HSCs), PrPC expression levels are raised and PrPC has been suggested as a marker for these cells [13]. PrPC expression is retained throughout maturation either toward the myeloid [14] or the lymphoid lineage [15, 16]. Interestingly, along the granulocyte maturation lineage, PrPC expression is downregulated [17].

Among cells of the lymphoid lineage, T cells, monocytes, and natural killer (NK) cells express higher PrPC levels compared to B lymphocytes [18]. PrPC expression levels are regulated and can vary greatly across different T-cell subtypes: CD8+ cells display higher expression levels than CD4+ cells, and between CD4+ cells, CD25+ expresses 4.5-fold higher *Prnp* levels than CD25− cells [19], while CD45RO+ memory T lymphocytes express higher PrPC levels compared to naïve CD45RA+ T lymphocytes [16]. It would thus appear that PrPC expression levels in cells of the immune system are dynamic, indicating that PrPC may play a role in the immune system.

#### **2.2. PrPC function in the immune system**

PrPSc copies. The exact mechanism governing this conversion is still under dispute, but it is widely accepted that it does not involve modifications of the primary structure of the protein. The physiological and pathological PrP isoforms display distinct conformations. The N-terminal region of PrPC is highly unstructured, as opposed to the globular C-terminal region, which contains predominantly a-helices and only a minor region encompassing twostranded β sheet [2]. Infrared spectroscopy and circular dichroism data indicate clear differences in the secondary structure of PrPC and PrPSc, in which equal amounts of a-helices and β sheets can be found [3]. This conformational difference is believed to be at the basis of the biochemical differences observed between the two isoforms, namely, the partial proteinase K resistance, the reduced solubility, and the fibril-producing potential displayed by PrPSc [4]. To date, the only known difference at the chemical level is associated with the oxidation level of the methionine residues, which was found to be elevated in PrPSc compared to PrPC [5].

The physiological role of the prion protein remains obscure. Its high level of conservation among species would indicate that PrPC is of crucial importance to the organism; however, PrP−/− mice are viable, developmentally and behaviorally normal, and do not display a prominent phenotype except for the complete protection against prion diseases [6, 7]. PrPC has since been implicated in a variety of cellular functions, including cell proliferation, differentiation and survival, protection against oxidative stress, and synaptic function (reviewed in [8, 9]). Further evidence suggests it may play a role in the immune system. In line with this, it has been recently reported that PrP−/− mice display lower numbers of CD4 T cells and lymphoid tissue inducer (LTi) cells as well as impaired splenic T zone structures [10]. Moreover, immune responses have been reported during prion diseases progression, suggesting the involvement of the immune system in disease pathogenesis, and immune-based approaches have yielded some of the most promising results toward protection and/or treatment of spongiform encephalopathies. In this chapter these exciting aspects of prion biology

Even though PrPC is predominantly expressed in the central and peripheral nervous system [11, 12], elevated protein expression levels have also been reported in many cells of the immune system. In long-term hematopoietic stem cells (HSCs), PrPC expression levels are raised and PrPC has been suggested as a marker for these cells [13]. PrPC expression is retained throughout maturation either toward the myeloid [14] or the lymphoid lineage [15, 16]. Interestingly,

Among cells of the lymphoid lineage, T cells, monocytes, and natural killer (NK) cells express higher PrPC levels compared to B lymphocytes [18]. PrPC expression levels are regulated

cells, CD25+

cells display higher expression

expresses 4.5-fold higher *Prnp* lev-

memory T lymphocytes express higher PrPC levels

along the granulocyte maturation lineage, PrPC expression is downregulated [17].

will be discussed.

134 Prion - An Overview

levels than CD4+

els than CD25−

**2. PrP and the immune system**

**2.1. PrPC expression patterns in cells of the immune system**

and can vary greatly across different T-cell subtypes: CD8+

cells, and between CD4+

cells [19], while CD45RO+

Despite evidence that PrPC may be associated with the function of the immune system, its role remains unclear. PrP−/− mice do not display gross overt effects, at least under normal conditions. However, evidence indicates that when PrP−/− mice are subject to immunological stress their phenotype may deviate from normal. To test whether PrPC may act as a regulator of cellular immunity, the effect PrPC deficiency may have on the course of experimental autoimmune encephalomyelitis (EAE) was assessed [20]. EAE is an inflammatory demyelinating disease of the central nervous system (CNS), triggered by the injection of brain extracts, proteins of the CNS such as the myelin basic protein and the myelin oligodendrocyte glycoprotein (MOG) or peptides from these proteins to experimental animals, usually mice and rats. EAE is widely used as an animal model for multiple sclerosis and acute disseminated encephalomyelitis but is also considered the prototype for T-cell–mediated autoimmune disease in general [21]. It was found that PrP−/− mice displayed a more aggressive disease onset and no clinical improvement during the chronic phase of the disease. These clinical findings were in agreement with the increased cytokine gene expression in MOG-primed PrP−/− cells and indicate that PrPC could be involved in the attenuation of T-cell-dependent neuroinflammation.

Similar results were obtained when *Prnp* expression was silenced via treatment with small interfering ribonucleic acid (siRNA) targeting *Prnp*. In this case, siRNA administration led to effective *Prnp* silencing in the lymphoid tissue, but not the central nervous system. In agreement to the results obtained with PrP−/− mice, siRNA-mediated *Prnp* silencing led to marked worsening of EAE [22]. In a series of elegant experiments, it was shown that the central nervous system autoimmune disease was modulated at all stages of the disease and that PrPC regulates activation of T lymphocytes mediated by the T-cell receptor (TCR), differentiation, and survival, thus identifying PrPC as a regulator of cellular immunological homeostasis. The proposed immunomodulatory properties when considered in conjunction with (i) the protein's expression patterns, which overlap with immune-privileged organs and (ii) the observation that only minor phenotypes can be associated with PrP−/− mice under physiological conditions, but rather striking ones under stress and particularly under inflammation in immune-privileged organs, has led to the hypothesis that PrPC may be involved in immune quiescence, protecting immune-privileged organs, such as the brain [23].

## **3. Prion disease pathogenesis and the immune system**

The central event in the pathogenesis of all forms of transmissible spongiform encephalopathies is the conversion of PrPC to the more thermodynamically stable PrPSc by PrPSc via a mechanism which remains at large obscure [24]. Regrettably, the actual conversion mechanism is not the only missing piece of the prion disease pathogenesis puzzle, and not much is known on how the infectious agent enters the host or how it is transported from the periphery to the central nervous system. A series of experiments using animal models of TSEs have provided interesting data on pathogenesis.

Parenteral—usually intracranial or intraperitoneal—administration of the pathogen to hamsters or mice is among the most widely used animal TSE models. Such models are particularly useful, since most of the naturally acquired TSE cases both in humans and animals are contracted via peripheral—through the alimentary tract—exposure to the pathogen [25]. While these models provide a wealth of information regarding pathogenesis, it later became evident that different mechanisms are involved in the pathogenesis of prion infection following the intraperitoneal or the oral route of infection [26], and other factors such as the pathogen strain and the host species and/or strain can also have a major impact on the mechanisms involved [27]. For example, in a recent study in sheep with different *Prnp* polymorphisms, which confer different levels of resistance to prion infection, it was observed that following intracranial administration of the pathogen, sheep with a "resistant" genetic polymorphism did not accumulate the pathogen in lymphoid tissues [28]. Even more strikingly, it has recently been reported that the role of the immune system might be limited in case of genetic prion disease. In a murine model of late onset genetic Creutzfeldt-Jakob disease, PrPSc has not been detected in the lymph nodes or the spleens of the transgenic mice at all ages and stages of disease, indicating that in this case conversion of PrPC to PrPSc occurs predominantly or even exclusively within the CNS [29].

Prion pathogenesis can be divided into phases, some of which may take place in parallel: (i) peripheral prion exposure and uptake, (ii) peripheral pathogen replication, (iii) migration through the peripheral nervous system to the CNS, and (iv) centrifugal spread from the CNS back to the periphery [25, 27]. Despite PrPSc can be detected in various sites following peripheral exposure, especially in the lymphatic system, signs of pathology, including neurodegeneration, spongiosis, and gliosis are only found within the CNS. It is important to stress that as the means available evolve, our understanding of the phenomena taking place also improves. For instance, detection of PrPSc in the brains of some peripherally challenged hamsters as early as 4 and 9 days following challenge was recently reported [30].

M cells, which are epithelial cells specialized for transepithelial transport found in the follicleassociated epithelia of the small and large intestines, tonsils and adenoids [31], were shown capable of transcytosing the TSE infectious agent *in vivo* [32]. In addition to M cells, other epithelial cells may be involved in the uptake of the pathogen in a ferritin-mediated mechanism [33]. The pathogen is first detected in gut-associated lymphoid tissue (GALT), including Peyer's patches and mesenteric lymph nodes [26]. Evidence from in vitro studies indicates that the GALT in the small rather than the large intestine plays a major role in PrPSc accumulation and eventually neuroinvasion [34].

It is not yet clear how the pathogen is transported from the entry site to the lymphoid tissue. It has been hypothesized that following pathogen uptake by M cells, the infectious agent can be transported to the M cells' intraepithelial pocket, where it can be processed by macrophages, B- and T- lymphocytes residing within this pocket or the dendritic cells, macrophages, and lymphocytes situated immediately beneath the intraepithelial pocket [35]. Of these cells, macrophages and dendritic cells appear the most plausible candidates for effective transport of the pathogen. In line with this assumption, PrP accumulations were detected in various types of macrophages following TSE infection [36–38]. However, the role macrophages undertake remains obscure, as *in vivo* experiments have shown that macrophages may also be involved in clearing the pathogen [39, 40]. It seems that the role of the macrophages following prion infection depends on the infectious dose and the agent strain [41]. Macrophages may also be important for the delivery of the infectious agent to the neural cells, and in this case, cell death may play an important role. In recent *in vitro* studies, it was determined that coculture of killed, PrPSc-infected macrophages with N2a-3 neuroblastoma cells accelerated PrPSc transmission to the neuronal cells [42]. Dendritic cells on the other hand can be ideally located to transport the pathogen following uptake by the M cells, and some of them have already been shown to be able to transport PrPSc without degrading it [43, 44].

known on how the infectious agent enters the host or how it is transported from the periphery to the central nervous system. A series of experiments using animal models of TSEs have pro-

Parenteral—usually intracranial or intraperitoneal—administration of the pathogen to hamsters or mice is among the most widely used animal TSE models. Such models are particularly useful, since most of the naturally acquired TSE cases both in humans and animals are contracted via peripheral—through the alimentary tract—exposure to the pathogen [25]. While these models provide a wealth of information regarding pathogenesis, it later became evident that different mechanisms are involved in the pathogenesis of prion infection following the intraperitoneal or the oral route of infection [26], and other factors such as the pathogen strain and the host species and/or strain can also have a major impact on the mechanisms involved [27]. For example, in a recent study in sheep with different *Prnp* polymorphisms, which confer different levels of resistance to prion infection, it was observed that following intracranial administration of the pathogen, sheep with a "resistant" genetic polymorphism did not accumulate the pathogen in lymphoid tissues [28]. Even more strikingly, it has recently been reported that the role of the immune system might be limited in case of genetic prion disease. In a murine model of late onset genetic Creutzfeldt-Jakob disease, PrPSc has not been detected in the lymph nodes or the spleens of the transgenic mice at all ages and stages of disease, indicating that in this case con-

version of PrPC to PrPSc occurs predominantly or even exclusively within the CNS [29].

early as 4 and 9 days following challenge was recently reported [30].

tion and eventually neuroinvasion [34].

Prion pathogenesis can be divided into phases, some of which may take place in parallel: (i) peripheral prion exposure and uptake, (ii) peripheral pathogen replication, (iii) migration through the peripheral nervous system to the CNS, and (iv) centrifugal spread from the CNS back to the periphery [25, 27]. Despite PrPSc can be detected in various sites following peripheral exposure, especially in the lymphatic system, signs of pathology, including neurodegeneration, spongiosis, and gliosis are only found within the CNS. It is important to stress that as the means available evolve, our understanding of the phenomena taking place also improves. For instance, detection of PrPSc in the brains of some peripherally challenged hamsters as

M cells, which are epithelial cells specialized for transepithelial transport found in the follicleassociated epithelia of the small and large intestines, tonsils and adenoids [31], were shown capable of transcytosing the TSE infectious agent *in vivo* [32]. In addition to M cells, other epithelial cells may be involved in the uptake of the pathogen in a ferritin-mediated mechanism [33]. The pathogen is first detected in gut-associated lymphoid tissue (GALT), including Peyer's patches and mesenteric lymph nodes [26]. Evidence from in vitro studies indicates that the GALT in the small rather than the large intestine plays a major role in PrPSc accumula-

It is not yet clear how the pathogen is transported from the entry site to the lymphoid tissue. It has been hypothesized that following pathogen uptake by M cells, the infectious agent can be transported to the M cells' intraepithelial pocket, where it can be processed by macrophages, B- and T- lymphocytes residing within this pocket or the dendritic cells, macrophages, and lymphocytes situated immediately beneath the intraepithelial pocket [35]. Of these cells, macrophages and dendritic cells appear the most plausible candidates for effective transport of

vided interesting data on pathogenesis.

136 Prion - An Overview

B lymphocytes were initially identified as the cells involved in replication of the TSE infectious agent [45], but this hypothesis was later revised, and the role of B lymphocytes in prion pathogenesis was associated with the regulated maturation of follicular dendritic cells (FDCs) [46]. Initial experiments with splenectomized or thymectomized mice indicated the dispensable role of T lymphocytes in the replication of the agent [47], whereas fractionation [48] and irradiation [49] experiments indicated that replication of the pathogen depends on radioresistant cells, localized within the stromal compartment of the spleen. FDCs fulfill all these criteria, and their crucial role for replication of the pathogen was confirmed in a series of experiments, in which depletion of mature FDCs led to prolongation of the incubation period of the disease [46, 50–53]. FDCs are of stromal origin, reside in the primary B lymphocytes follicles and germinal centers of lymphoid tissues, and are non-phagocytic and non-migratory. As a result of their large surface area and longevity, FDCs are capable of trapping and retaining antigen in its native state for months to years. FDCs retain antigens in the form of immune complexes, consisting of antigen-complement components and/or antibody and trap these complexes either through complement receptors CR1 and CR2 or through FcRIIb and FcεRII antibody receptors [35]. In agreement with the role FDCs undertake in prion pathogenesis and the involvement of complement components and receptors in antigen trapping by FDCs, it was found that the absence of complement components (C1q, C2, C3, and factor B) and cellular complement receptor can have an adverse effect on the accumulation of PrPSc in the spleen [54, 55]. However, the inability to completely inhibit disease progression via depletion of mature FDCs [46, 51], in addition to observations confirming that propagation of prion diseases is possible even in the absence of mature FDCs [41, 56–58], indicates that possibly a different cell type—most probably MOMA-1-positive macrophages [41]—is responsible for replication of the pathogen. These differences in the cell types required for pathogenesis were attributed to the dose and agent strain [41].

Peripheral replication of the pathogen precedes neuroinvasion, during which the pathogen is transported within the CNS. Both the enteric and autonomic nervous systems are believed to participate in the transport of the infectious agent [36, 59, 60]. The exact mechanism governing transport of the pathogen to the CNS remains unidentified, and has been reported to be both PrPC-dependent [61, 62] and independent [63]. Interestingly, it was reported that the transfer speed of intraperitoneally administered prions relies to the distance between FDCs and splenic nerve endings [64, 65].

The immune system is greatly implicated in the peripheral pathogenesis of prion diseases but fails to provide protection. Until recently, no response against the prion pathogen has been described, and this was linked to tolerance effects due to widespread expression of the physiological isoform of the prion protein throughout the body, which prevents the host from mounting a humoral or cellular immune response against PrPSc following infection [66]. On the contrary, PrP−/− mice mount a robust immune response against PrP, indicating the immunogenicity of the protein. Lately it was shown that TSE infection can have adverse effects on the maturation cycle of FDCs, causing an abnormality in immune function [67]. Given the crucial role the immune system plays in the peripheral pathogenesis of prion diseases, it could be argued that it promotes rather than protects against prion pathogenesis. In agreement with this, increased susceptibility to intraperitoneal challenge with TSE agents following repetitive immunization was recently reported [68].

## **4. Harnessing the immune system against prion diseases**

Since the immune system plays an ambivalent role in prion disease pathogenesis, the question emerged whether suppressing the immune system would be the most appropriate approach [69]. Targeting the FDCs was already proven a viable approach, providing partial protection in an animal model of prion diseases and minimizing the infectivity of the peripheral tissue of the afflicted animals [46, 50–52]. Disruption of the FDCs also appears to be the protective mechanism against TSEs following repetitive CpG administration [70]. CpG had previously been administered as a stimulator of innate immunity and was shown effective at providing partial protection in an animal model of TSEs [71]. In this case CpG was administered to stimulate the macrophages and enhance phagocytosis of the pathogen. Indeed, repetitive administration provided partial protection against TSEs [71], but as it was later shown, this protection was due to disruption of the lymphoid follicles rather than stimulation of the macrophages [70]. Interestingly, disruption of the FDCs has also been observed following immunization of wild-type mice with recombinant murine PrP aggregates and is at least in part responsible for the observed partial protection when the immunized mice were challenged with a murine strain of TSEs [72].

The first indications that the immune system might prove effective against prion diseases stemmed from *in vitro* experiments, where it was shown that treatment of TSE-infected cell cultures with monoclonal anti-PrP antibodies could effectively inhibit PrPSc replication and on some occasions clear infectivity [73–75]. Proof of principle that immunization against prion diseases can be effective against prion diseases was provided later using transgenic mice, capable of producing anti-PrP antibodies. These mice, in contrast to wild-type controls, failed to succumb to disease following challenge with a mouse-adapted scrapie strain [76]. Similarly, passive immunization of wild-type mice, by administration of anti-PrP antibodies was found to provide protection against prion diseases [77].

From this initial series of experiments, valuable conclusions emerged, most importantly, that immunization is an efficient means of therapy rather than protection, against prion diseases. Moreover, the safety of these procedures was confirmed, since immunization against a selfantigen could always give rise to autoimmunity. Given the identical primary structure PrPC and PrPSc share, adverse reactions stemming from the reaction of the anti-PrP antibodies with PrPC could be expected. Autoimmunity was not induced by these immunization approaches, and furthermore the "dispensable" role of the prion protein for the appearance of a physiological phenotype was already known from studies on PrP-/- animals [6], as well as from transgenic animals with conditional depletion of the prion protein [78] and provided an extra layer of security. However, other findings raised some concerns over the safety of administration of anti-PrP antibodies, since it was found that intracerebral administration of anti-PrP monoclonal antibodies can give rise to cross-linking of PrP molecules on adjacent neurons and eventually cell death, triggered possibly by the initiation of death signaling [79]. These effects are clearly not associated with autoimmunity, but rather with impaired cell signaling.

#### **4.1. Passive immunization approaches**

The immune system is greatly implicated in the peripheral pathogenesis of prion diseases but fails to provide protection. Until recently, no response against the prion pathogen has been described, and this was linked to tolerance effects due to widespread expression of the physiological isoform of the prion protein throughout the body, which prevents the host from mounting a humoral or cellular immune response against PrPSc following infection [66]. On the contrary, PrP−/− mice mount a robust immune response against PrP, indicating the immunogenicity of the protein. Lately it was shown that TSE infection can have adverse effects on the maturation cycle of FDCs, causing an abnormality in immune function [67]. Given the crucial role the immune system plays in the peripheral pathogenesis of prion diseases, it could be argued that it promotes rather than protects against prion pathogenesis. In agreement with this, increased susceptibility to intraperitoneal challenge with TSE agents following repetitive

Since the immune system plays an ambivalent role in prion disease pathogenesis, the question emerged whether suppressing the immune system would be the most appropriate approach [69]. Targeting the FDCs was already proven a viable approach, providing partial protection in an animal model of prion diseases and minimizing the infectivity of the peripheral tissue of the afflicted animals [46, 50–52]. Disruption of the FDCs also appears to be the protective mechanism against TSEs following repetitive CpG administration [70]. CpG had previously been administered as a stimulator of innate immunity and was shown effective at providing partial protection in an animal model of TSEs [71]. In this case CpG was administered to stimulate the macrophages and enhance phagocytosis of the pathogen. Indeed, repetitive administration provided partial protection against TSEs [71], but as it was later shown, this protection was due to disruption of the lymphoid follicles rather than stimulation of the macrophages [70]. Interestingly, disruption of the FDCs has also been observed following immunization of wild-type mice with recombinant murine PrP aggregates and is at least in part responsible for the observed partial protection when the immunized mice were challenged

The first indications that the immune system might prove effective against prion diseases stemmed from *in vitro* experiments, where it was shown that treatment of TSE-infected cell cultures with monoclonal anti-PrP antibodies could effectively inhibit PrPSc replication and on some occasions clear infectivity [73–75]. Proof of principle that immunization against prion diseases can be effective against prion diseases was provided later using transgenic mice, capable of producing anti-PrP antibodies. These mice, in contrast to wild-type controls, failed to succumb to disease following challenge with a mouse-adapted scrapie strain [76]. Similarly, passive immunization of wild-type mice, by administration of anti-PrP antibodies

From this initial series of experiments, valuable conclusions emerged, most importantly, that immunization is an efficient means of therapy rather than protection, against prion diseases. Moreover, the safety of these procedures was confirmed, since immunization against a self-

immunization was recently reported [68].

138 Prion - An Overview

with a murine strain of TSEs [72].

was found to provide protection against prion diseases [77].

**4. Harnessing the immune system against prion diseases**

The first indications that passive immunization could prove useful at protecting against prion diseases emerged from studies in which mice genetically modified to produce an anti-PrP monoclonal antibody (6H4μ) were fully protected against prion diseases [76]. In a more classical approach, monoclonal anti-PrP antibodies (ICSM18 and ICSM35) were administered intraperitoneally to wild-type mice briefly after intraperitoneal inoculation with the pathogen or when the first clinical signs appeared. When the antibodies were administered after the inoculation, animals receiving the antibodies survived approximately 300 days more than control mice, and the accumulation of infectivity in the peripheral tissue was markedly reduced [77]. Intraperitoneal administration of a different antibody (6D11) immediately after intraperitoneal administration of the pathogen also proved its protective efficacy, since mice receiving the antibody survived longer by approximately 36.9% compared to control mice. In a recent study, a pharmacokinetic and pharmacodynamic analysis following intraperitoneal administration of various anti-PrP antibodies was carried out. The ability of an antibody to form long-lasting complexes with PrPC was found to positively correlate with its efficacy in delaying peripheral accumulation of PrPSc and, in agreement with this, intraperitoneal administration of the monoclonal antibody BAR216 led to a statistically significant prolongation of survival of the mice [80].

The therapeutic efficacy of intracerebral administration of anti-PrP monoclonal antibodies was evaluated in two recent studies. In the first one, monoclonal antibody 4H11 (F(ab′)2 and IgG) was intraventricularly administered to transgenic mice overexpressing PrP using osmotic pumps from d85 to d100 following intraperitoneal challenge with a mouse-adapted bovine spongiform encephalopathy (BSE) strain. The mice were not protected by this regimen, and they succumbed to disease concomitantly with the control mice. Furthermore, mice treated with the antibodies developed neuronal cell death, associated with administration of the antibodies. In addition to previously reported results [79], linking cell death to PrP crosslinking events, in this study, emerged that PrP cross-linking is not the only mechanism mediating cell death; "coating" the whole cell surface PrP with antibodies or antibodies fragments could induce other toxic signals [81]. In the second study, intraventricular administration of antibodies 106, 110, 31C6, and 44B1 to wild-type mice was not linked with neuronal cell death; however, only a minor prolongation of survival and in one of the two tested animal models was achieved following administration of the monoclonal antibodies [82]. Differences in the epitopes recognized by the antibodies used in these two studies as well as the use of PrP overexpressing versus wild-type mice could account for the different results obtained regarding neuronal cell death. Of note, neuronal cell death has been challenged in another, more recent study, and it would be safe to assume that toxic effects are associated with the epitope and the dosage of the antibodies used [83].

A completely different passive immunization approach was used in two other studies; based on the discovery of the non-integrin 37/67 kDa lamin receptor (LRP/LR) as an interaction partner for both isoforms of PrP [84–86], polyclonal anti-LRP/LR [87] or single-chain Fv anti-LRP/LR antibodies [88] were intraperitoneally administered to wild-type mice as protective means in a mouse model of prion diseases. On both occasions, peripheral PrPSc accumulation was reduced; however, partial protection was only achieved with the polyclonal antibodies. This difference in the efficacy was attributed to differences in the pharmacokinetics and dosage regimen; polyclonal antibodies have a half-life of approximately 14 days in the blood, whereas the single-chain antibodies have a half-life of only 12 h. Moreover, the polyclonal antibodies were administered for 12 weeks, starting 1 week before administration of the pathogen, whereas the single-chain antibodies for 8 weeks. Passive immunization approaches are summarized in **Table 1**.

#### **4.2. Active immunization approaches**

Although passive immunization does protect against prion diseases, it provides a narrow window for intervention, i.e., antibodies must be administered shortly after exposure to the pathogen. In this regard, active immunization against the prion protein, which provides protection against the diseases similarly to a conventional vaccine, could prove a much more useful approach. Nevertheless, the prion protein-associated tolerance effects which prevent the immune system from mounting an immune response against the prion protein hinder development of such approaches [66].

Despite the tolerance effects, initiation of a humoral immune response against the prion protein was achieved, albeit with mediocre results in terms of protection against the disease. In the first reports, wild-type mice were immunized with recombinant murine prion protein mixed with complete Freund's adjuvant (CFA) and challenged with a mouse-adapted scrapie strain either concomitantly with the immunization (rescue treatment) or following its completion (prophylactic treatment). Although the mice developed antibodies against the prion protein, only mice of the prophylactic treatment group were partially protected against the pathogen; mice of this group succumbed to disease with a delay of approximately 16d compared to control mice [89].

#### *4.2.1. Peptide-based active immunization*

Numerous strategies were implemented to overcome the tolerance effects and promote generation of anti-prion antibodies. The most obvious approach was to use prion peptides properly modified to enhance the antigenicity of the protein (summarized in **Table 2**). Following this rationale, wild-type animals were immunized with prion protein peptides [90–93], PrP

was achieved following administration of the monoclonal antibodies [82]. Differences in the epitopes recognized by the antibodies used in these two studies as well as the use of PrP overexpressing versus wild-type mice could account for the different results obtained regarding neuronal cell death. Of note, neuronal cell death has been challenged in another, more recent study, and it would be safe to assume that toxic effects are associated with the epitope and the

A completely different passive immunization approach was used in two other studies; based on the discovery of the non-integrin 37/67 kDa lamin receptor (LRP/LR) as an interaction partner for both isoforms of PrP [84–86], polyclonal anti-LRP/LR [87] or single-chain Fv anti-LRP/LR antibodies [88] were intraperitoneally administered to wild-type mice as protective means in a mouse model of prion diseases. On both occasions, peripheral PrPSc accumulation was reduced; however, partial protection was only achieved with the polyclonal antibodies. This difference in the efficacy was attributed to differences in the pharmacokinetics and dosage regimen; polyclonal antibodies have a half-life of approximately 14 days in the blood, whereas the single-chain antibodies have a half-life of only 12 h. Moreover, the polyclonal antibodies were administered for 12 weeks, starting 1 week before administration of the pathogen, whereas the single-chain antibodies for 8 weeks. Passive immunization approaches

Although passive immunization does protect against prion diseases, it provides a narrow window for intervention, i.e., antibodies must be administered shortly after exposure to the pathogen. In this regard, active immunization against the prion protein, which provides protection against the diseases similarly to a conventional vaccine, could prove a much more useful approach. Nevertheless, the prion protein-associated tolerance effects which prevent the immune system from mounting an immune response against the prion protein hinder

Despite the tolerance effects, initiation of a humoral immune response against the prion protein was achieved, albeit with mediocre results in terms of protection against the disease. In the first reports, wild-type mice were immunized with recombinant murine prion protein mixed with complete Freund's adjuvant (CFA) and challenged with a mouse-adapted scrapie strain either concomitantly with the immunization (rescue treatment) or following its completion (prophylactic treatment). Although the mice developed antibodies against the prion protein, only mice of the prophylactic treatment group were partially protected against the pathogen; mice of this group succumbed to disease with a delay of approximately 16d

Numerous strategies were implemented to overcome the tolerance effects and promote generation of anti-prion antibodies. The most obvious approach was to use prion peptides properly modified to enhance the antigenicity of the protein (summarized in **Table 2**). Following this rationale, wild-type animals were immunized with prion protein peptides [90–93], PrP

dosage of the antibodies used [83].

140 Prion - An Overview

are summarized in **Table 1**.

**4.2. Active immunization approaches**

development of such approaches [66].

compared to control mice [89].

*4.2.1. Peptide-based active immunization*

**Table 1.** Summary of studies based on passive immunization against prion diseases.

dimers [94–96], or PrP aggregates [72]. In addition to homologous prion protein immunization [89], which provided proof of principle that active immunization can have a protective role against prion diseases, immunization with heterologous prion peptides also provided rather encouraging results [97]. In an attempt to enhance the immunogenicity of the prion peptides, various adjuvants, including Freund's adjuvant, Montanide IMS-1313, TiterMax, CpG, anti-OX40 antibodies—antibodies against the signaling molecule CD134, which recently has been shown to break T cell tolerance—and keyhole limpet hemocyanin, were used [95, 98], as well as different vaccine formulations, including encapsulation of the CpG-antigen complex in polylactide-coglycolide microspheres [96]. Interestingly, an early report indicates that immunization with complete Freund's adjuvant alone can provide partial protection in a mouse model of prion diseases through an unidentified mechanism [99]. Based on the extremely strong adjuvant effect exerted by heat-shock proteins, PrP molecules chemically cross-linked [100] or fused [72] to recombinant bacterial heat-shock proteins were also used to immunize wild-type mice and lead to the production of antibodies that recognized recombinant PrP.

Despite the widely accepted notion that PrPSc is not immunogenic and that the immune system does not provide protection against PrPSc in wild-type animals, when highly purified proteinase K-resistant PrPSc, originating from murine brains afflicted with an animal model of prion diseases was coadministered with CpG [101] or administered immobilized on Dynabeads coated with antibodies against PrP [102] a humoral immune response, which providing partial protection in animal model of prion diseases was elicited.

Although the protective role of the aforementioned, peptide-based approaches was not investigated on all occasions, it became evident that using various approaches the self-tolerance effects can be overcome and immune reactions against the prion protein can be obtained. However, it appears that protection against TSEs is restricted to antibodies capable of recognizing the native cell-surface PrPC [95]. This requirement was met by antibodies known to provide protection against TSEs, e.g., ICSM18 [77] and 6H4 [76], whereas other antibodies capable of recognizing recombinant PrP but unable to provide protection against TSEs also failed to recognize native PrPC [72, 95].

#### *4.2.2. DNA vaccines*

dimers [94–96], or PrP aggregates [72]. In addition to homologous prion protein immunization [89], which provided proof of principle that active immunization can have a protective role against prion diseases, immunization with heterologous prion peptides also provided rather encouraging results [97]. In an attempt to enhance the immunogenicity of the prion peptides, various adjuvants, including Freund's adjuvant, Montanide IMS-1313, TiterMax, CpG, anti-OX40 antibodies—antibodies against the signaling molecule CD134, which recently has been shown to break T cell tolerance—and keyhole limpet hemocyanin, were used [95, 98], as well as different vaccine formulations, including encapsulation of the CpG-antigen complex in polylactide-coglycolide microspheres [96]. Interestingly, an early report indicates that immunization with complete Freund's adjuvant alone can provide partial protection in a mouse model of prion diseases through an unidentified mechanism [99]. Based on the extremely strong adjuvant effect exerted by heat-shock proteins, PrP molecules chemically cross-linked [100] or fused [72] to recombinant bacterial heat-shock proteins were also used to immunize wild-type mice and lead to the production of antibodies that recog-

NP Clearance of

**Epitope Administration protocol** *In vitro* **assay** *In vivo* **assay Reference**

Clearance of PrPSc in ScN2a cells

PrPSc in ScGT2 cells

Minor delay of incubation time in immunized versus control mice

NP [117]

[116]

Despite the widely accepted notion that PrPSc is not immunogenic and that the immune system does not provide protection against PrPSc in wild-type animals, when highly purified proteinase K-resistant PrPSc, originating from murine brains afflicted with an animal model of prion diseases was coadministered with CpG [101] or administered immobilized on Dynabeads coated with antibodies against PrP [102] a humoral immune response, which

Although the protective role of the aforementioned, peptide-based approaches was not investigated on all occasions, it became evident that using various approaches the self-tolerance effects can be overcome and immune reactions against the prion protein can be obtained. However, it appears that protection against TSEs is restricted to antibodies capable of recognizing the native cell-surface PrPC [95]. This requirement was met by antibodies known to provide protection against TSEs, e.g., ICSM18 [77] and 6H4 [76], whereas other antibodies

providing partial protection in animal model of prion diseases was elicited.

nized recombinant PrP.

**Antibody name**

142 Prion - An Overview

EB8, DC2, DE10, EF2

**Antibody type and target**

Monoclonal EB8: 26–34aa;

DC2: 35–46aa; DE10: 44–52aa and EF2: 47–52aa

Undefined Intraperitoneal

**Table 1.** Summary of studies based on passive immunization against prion diseases.

administration twice weekly starting 2 or 28 days after administration of the pathogen or at onset of the clinical symptoms

W226 Monoclonal, scFV

> In addition to peptide-based vaccines, DNA vaccines were also used to promote immune responses against the prion protein. In this case, nucleic acid encoding for the prion protein is administered to animals, wherein the nucleic acid is translated to the corresponding protein and an immune response is initiated. The first attempt at raising anti-PrP antibodies using DNA vaccines was only successful in PrP−/− mice, whereas the same approach failed to give rise to anti-PrP antibodies in wild-type mice [103]. Induction of anti-PrP antibodies using DNA vaccines in wild-type mice was triggered when the mice were immunized with a DNA construct coding for the murine prion protein fused to the lysosomal targeting signal from lysosomal integral membrane protein type II (LIMPII). Immunization with this construct leads to a remarkable delay on the onset of disease symptoms, which was not followed by a similar prolongation of survival interval. This discrepancy in the obtained results was attributed to immunopathology mediated by PrP-specific antibodies induced by the DNA vaccine used and constitutes the first report of adverse effects following active prion immunization [104].

> In a different approach, DNA vaccines were used to prime wild-type mice, followed by peptide immunizations to further boost immune responses. Although this approach was successful when PrP-/- mice were immunized, very low antibody titers and only marginal protection were achieved when tested on wild-type mice [105]. In a recent report, wild-type mice were immunized with cDNA coding for human PrPC fused to a T-cell stimulatory peptide. These mice developed a strong humoral immune response against the native protein, and although a bioassay was not carried out, the produced antibodies were capable of recognizing the native conformation of murine PrPC, which—as already mentioned—constitutes a strong indicator of protective efficiency against prion diseases [106]. Studies based on DNA vaccines are summarized in **Table 3**.

#### *4.2.3. Immunization with PrP-displaying viral constructs*

A different approach to overcome the tolerance effects and stimulate the production of anti-PrP antibodies in wild-type mice is the expression of the prion protein on the surface of viral particles (summarized in **Table 4**). Virus-like particles (VLPs) are much better B lymphocytes immunogens than monovalent proteins and would be expected to trigger a stronger humoral immune response by passing tolerance.

In a first attempt, retroviral particles displaying the C-terminal portion of murine PrP were used to immunize wild-type mice. These mice developed anti-PrP antibodies, capable of recognizing the native form of PrPC, thus displaying strong therapeutic potential [107]. A similar approach was used to insert the 9-amino-acid-, prion-pathogenesis associated-peptide pertaining to the murine/rat prion protein into the L1 major capsid protein of bovine papillomavirus type 1. These VLPs were used to immunize both wild-type rats and rabbits. The anti-sera

**Antigen Animals**

Various murine PrP peptides

144 Prion - An Overview

Recombinant murine PrP chemically cross-linked to bacterial heatshock proteins

Recombinant murine PrP

Recombinant murine PrP dimer

Recombinant murine prion peptide 105–125 linked to keyhole limpet hemocyanin and recombinant murine prion 90–230

Mouse prion peptides 31–50 and 211–230

Various murine prion peptides and adjuvants

Murine prion peptides 39–67, 98–127, 143–172, and 158–187 with CFA or CpG

**immunized**

Wild-type mice, rabbits

**Humoral response T-cell**

**responses**

Wild-type mice + NP NP Prolongation of

+ NP Polyclonal

Wild-type mice + NP NP Prolongation of

Wild-type mice NP NP NP Prolongation of

Wild-type mice + ND FACS to detect

Wild-type mice + NP NP Reduction of proteinase

Wild-type mice + NP NP NP [100]

sera produced reduced PrPSc synthesis in prion-infected cell lines

binding of the produced antibodies on native PrP

Wild-type mice + + NP NP [98]

*In vitro* **assays** *In vivo* **assays Reference**

K-resistant prion protein in a scrapieinfected tumor transplant

survival interval in a mouse model of prion disease

survival interval in a mouse model of prion disease

survival interval in a mouse model of prion disease, even when only the adjuvant Complete Freund's Adjuvant (CFA) is administered

Statistically insignificant prolongation of survival time in a mouse model of prion disease

NP [94]

[93]

[89]

[92]

[99]

[95]

**Table 2.** Summary of studies on peptide-based active immunization against prion diseases.

**Table 3.** Summary of studies on DNA vaccination against prion diseases.

**Table 4.** Studies on active immunization approaches, using PrP-displaying virus constructs.

collected from both immunized species recognized native PrPSc, and importantly immune serum from the immunized rabbit prevented synthesis of PrPSc in scrapie-infected cell lines [108]. In a more recent approach, dendritic cells transduced with adenoviruses encoding the human prion protein were used to immunize wild-type mice. These mice developed antibodies against the murine prion protein as well, which provided partial protection against TSEs, as shown by the reduction in splenic PrPSc accumulation and prolongation of survival interval in a murine model of TSEs [109].

#### *4.2.4. Mucosal immunization*

**Vaccine Immunized**

DNA vaccine encoding either murine PrP or murine PrP fused to ubiquitin or to a lysosomal targeting signal

146 Prion - An Overview

DNA vaccine encoding murine PrP linked to helper T-cell epitopes Combination of DNA and peptide immunization

DNA vaccine encoding human PrP fused or not to a tetanus toxin stimulatory T-cell epitope and PrP protein boost

DNA vaccine encoding human PrP fused to ubiquitin, lysosomal integral membrane protein type II lysosometargeting signal or an ER-targeting signal in conjunction with PrP vaccination

**animals**

PrP−/− and wildtype mice

**Humoral response**

Achieved in PrP−/− mice, very low titer in wild-type mice

**Table 3.** Summary of studies on DNA vaccination against prion diseases.

Wild-type mice + + NP Prolongation of

Detected in PrP−/− mice but not wild-type mice

Wild-type mice + NP FACS to detect

**T-cell responses** *In vitro* **assays** *In vivo* **assays Reference**

asymptomatic period and accumulation of disease associated PrP, but not of survival interval. Death of the immunized mice was attributed to neurodegeneration associated with production of anti-PrP

Not effective [105]

NP [106]

[104]

antbodies

FACS to detect binding of the produced antibodies on native PrP positive with PrP−/− mice sera, negative with wild-type mice sera. PrP−/− mice sera reduced PrPSc levels in prion-infected cell lines

binding of the produced antibodies on native PrP

Wild-type mice + + NP NP [120]

To date, the only active immunization strategy providing complete protection against prion diseases is mucosal immunization. To trigger mucosal immunization, either transgenic, liveattenuated *Salmonella typhimurium* or cholera toxin is used. Both the attenuated *S*. *typhimurium* and the cholera toxin induce the production of IgA, which is the main immunoglobulin found in mucous secretions and is particularly abundant in the secretions of the gastrointestinal tract.

To induce mucosal immunization, a live-attenuated *S*. *typhimurium* vaccine strain engineered to express one [110] or two copies [110–112] of mouse [110, 112] or deer [111] PrP was administered orally to mice and deer, respectively. In a different approach, a murine PrP fragment was coadministered with cholera toxin either orally or intranasally [113]. The immunized animals were then orally challenged with a murine model of TSEs or chronic wasting disease (CWD)—infected brain homogenate to evaluate the protective potential of the immunization. Although both approaches promoted the generation of anti-PrP IgA, protection afforded by the

**Table 5.** Mucosal vaccination approaches.

immunized animals varied greatly. Animals immunized with the attenuated PrP-expressing *Salmonella* survived significantly longer than control animals, and most importantly some of them were completely protected, remaining disease-free [110–112]. On the other hand, the cholera toxin was used to induce production of IgA, mice were only partially protected against oral exposure to the infectious agent, and modest prolongation of the survival interval was observed, without any mice remaining symptoms-free [113]. Studies dealing with mucosal immunization are summarized in **Table 5**.

Although mucosal immunization is only effective following oral exposure, it is important to remember that the gut is the major route of entry for prion diseases such as CWD in white-tailed deer, BSE in cattle, and variant Creutzfeldt-Jakob disease and kuru in humans. Furthermore, mucosal vaccination can be properly designed to induce a primarily humoral immune response and is unlikely to produce a significant immune response within the brain, thus minimizing the risk of appearance of adverse reactions [112].

#### **5. Future perspectives**

Despite fervent research and some very encouraging results, many facets of the involvement of the immune system in prion pathogenesis remain obscure, and a powerful immunoprotective tool has yet to emerge. Passive immunization with anti-prion antibodies and mucosal immunization were the only two approaches to provide satisfactory results but have a series of limitations associated with the narrow window of intervention and the route of infection. However, immune-based therapeutics both in their more classical immunization-based form or more modern, immunomodulatory form [114] hold great promise for prion diseases and other protein-misfolding diseases.

## **Author details**

Konstantinos Xanthopoulos1 , Dimitra Dafou2 , Eirini Kanata1 and Theodoros Sklaviadis1 \*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Pharmaceutical Sciences, School of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece

2 Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece

## **References**

immunized animals varied greatly. Animals immunized with the attenuated PrP-expressing *Salmonella* survived significantly longer than control animals, and most importantly some of them were completely protected, remaining disease-free [110–112]. On the other hand, the cholera toxin was used to induce production of IgA, mice were only partially protected against oral exposure to the infectious agent, and modest prolongation of the survival interval was observed, without any mice remaining symptoms-free [113]. Studies dealing with mucosal

Although mucosal immunization is only effective following oral exposure, it is important to remember that the gut is the major route of entry for prion diseases such as CWD in white-tailed deer, BSE in cattle, and variant Creutzfeldt-Jakob disease and kuru in humans. Furthermore, mucosal vaccination can be properly designed to induce a primarily humoral immune response and is unlikely to produce a significant immune response within the brain,

Despite fervent research and some very encouraging results, many facets of the involvement of the immune system in prion pathogenesis remain obscure, and a powerful immunoprotective tool has yet to emerge. Passive immunization with anti-prion antibodies and mucosal immunization were the only two approaches to provide satisfactory results but have a series of limitations associated with the narrow window of intervention and the route of infection. However, immune-based therapeutics both in their more classical immunization-based form or more modern, immunomodulatory form [114] hold great promise for prion diseases and

immunization are summarized in **Table 5**.

**Table 5.** Mucosal vaccination approaches.

**Vaccine Immunized**

Orally administered *S. typhimurium* LVR01 expressing one or two copies of mouse PrP

Orally administered *S. typhimurium* LVR01 expressing two copies

Intranasally, intragastrically, or intraperitoneally administered murine PrP90–231 and cholera

148 Prion - An Overview

toxin

of elk PrP

**animals**

White-tailed deers

**Humoral response**

**T-cell responses** *In vitro* **assay**

+ NP NP Significant prolongation

Wild-type mice + NP NP Significant prolongation

Wild-type mice + NP NP Marginal prolongation

*In vivo* **assay Reference**

[110, 112]

[113]

[111]

of survival interval in a mouse model of prion

of survival interval in a mouse model of prion disease following intranasal administration

of survival interval in an elk model of prion disease. One immunized animal remained asymptomatic

disease

**5. Future perspectives**

other protein-misfolding diseases.

thus minimizing the risk of appearance of adverse reactions [112].

[31] Kraehenbuhl, J.P. and Neutra, M.R., *Epithelial M cells*: *differentiation and function*. Annu Rev Cell Dev Biol, 2000. **16**: pp. 301–332.

[14] Burthem, J., et al., *The normal cellular prion protein is strongly expressed by myeloid dendritic*

[15] Cashman, N.R., et al., *Cellular isoform of the scrapie agent protein participates in lymphocyte*

[16] Li, R., et al., *The expression and potential function of cellular prion protein in human lympho-*

[17] Dodelet, V.C. and Cashman, N.R., *Prion protein expression in human leukocyte differentia-*

[18] Durig, J., et al., *Differential constitutive and activation*-*dependent expression of prion protein in*

[19] Isaacs, J.D., Jackson, G.S., and Altmann, D.M., *The role of the cellular prion protein in the*

[20] Tsutsui, S., et al., *Absence of the cellular prion protein exacerbates and prolongs neuroinflammation in experimental autoimmune encephalomyelitis*. Am J Pathol, 2008. **173**(4): pp. 1029–1041.

[21] Miller, S.D. and Karpus, W.J., *Experimental autoimmune encephalomyelitis in the mouse*.

[22] Hu, W., et al., *Pharmacological prion protein silencing accelerates central nervous system autoimmune disease via T cell receptor signalling*. Brain, 2010. **133**(Pt 2): pp. 375–388.

[23] Bakkebo, M.K., et al., *The cellular prion protein*: *a player in immunological quiescence*. Front

[24] Riesner, D., *Biochemistry and structure of PrP*(*C*) *and PrP*(*Sc*). Br Med Bull, 2003. **66**: pp.

[25] Beekes, M. and McBride, P.A., *The spread of prions through the body in naturally acquired*

[26] Prinz, M., et al., *Oral prion infection requires normal numbers of Peyer*'*s patches but not of*

[27] Aguzzi, A. and M. Heikenwalder, *Pathogenesis of prion diseases*: *current status and future*

[28] Greenlee, J.J., et al., *Lack of prion accumulation in lymphoid tissues of PRNP ARQ*/*ARR sheep intracranially inoculated with the agent of scrapie*. PLoS One, 2014. **9**(9): p. e108029.

[29] Friedman-Levi, Y., et al., *Genetic prion disease*: *no role for the immune system in disease patho-*

[30] Chen, B., Soto, C., and Morales, R., *Peripherally administrated prions reach the brain at subinfectious quantities in experimental hamsters*. FEBS Lett, 2014. **588**(5): pp. 795–800.

*transmissible spongiform encephalopathies*. FEBS J, 2007. **274**(3): pp. 588–605.

*enteric lymphocytes*. Am J Pathol, 2003. **162**(4): pp. 1103–1111.

*outlook*. Nat Rev Microbiol, 2006. **4**(10): pp. 765–775.

*genesis*?. Hum Mol Genet, 2014. **23**(15): pp. 4134–4141.

*human peripheral blood leucocytes*. Br J Haematol, 2000. **108**(3): pp. 488–495.

*immune system*. Clin Exp Immunol, 2006. **146**(1): pp. 1–8.

Curr Protoc Immunol, 2007. **Chapter 15**: p. Unit 15 1.

*cells*. Blood, 2001. **98**(13): pp. 3733–3738.

*activation*. Cell, 1990. **61**(1): pp. 185–192.

*tion*. Blood, 1998. **91**(5): pp. 1556–1561.

Immunol, 2015. **6**: p. 450.

21–33.

150 Prion - An Overview

*cytes*. Cell Immunol, 2001. **207**(1): pp. 49–58.

[62] Glatzel, M. and Aguzzi, A., *PrP*(*C*) *expression in the peripheral nervous system is a determinant of prion neuroinvasion*. J Gen Virol, 2000. **81**(Pt 11): pp. 2813–2821.

[47] Fraser, H. and Dickinson, A.G., *Studies of the lymphoreticular system in the pathogenesis of*

[48] Clarke, M.C. and Kimberlin, R.H., *Pathogenesis of mouse scrapie*: *distribution of agent in the*

[49] Fraser, H. and Farquhar, C.F., *Ionising radiation has no influence on scrapie incubation period*

[50] Mabbott, N.A., et al., *Temporary inactivation of follicular dendritic cells delays neuroinvasion*

[51] Mabbott, N.A., et al., *Follicular dendritic cell dedifferentiation by treatment with an inhibitor of the lymphotoxin pathway dramatically reduces scrapie susceptibility*. J Virol, 2003. **77**(12):

[52] Mohan, J., Bruce, M.E., and Mabbott, N.A., *Follicular dendritic cell dedifferentiation reduces scrapie susceptibility following inoculation via the skin*. Immunology, 2005. **114**(2): pp.

[53] Mabbott, N.A., et al., *Temporary blockade of the tumor necrosis factor receptor signaling pathway impedes the spread of scrapie to the brain*. J Virol, 2002. **76**(10): pp. 5131–5139.

[54] Klein, M.A., et al., *Complement facilitates early prion pathogenesis*. Nat Med, 2001. **7**(4): pp.

[55] Mabbott, N.A., et al., *Temporary depletion of complement component C3 or genetic deficiency*

[56] Oldstone, M.B., et al., *Lymphotoxin*-*alpha*- *and lymphotoxin*-*beta*-*deficient mice differ in susceptibility to scrapie*: *evidence against dendritic cell involvement in neuroinvasion*. J Virol, 2002.

[57] Shlomchik, M.J., et al., *Neuroinvasion by a Creutzfeldt*-*Jakob disease agent in the absence of B cells and follicular dendritic cells*. Proc Natl Acad Sci U S A, 2001. **98**(16): pp. 9289–9294.

[58] Manuelidis, L., et al., *Follicular dendritic cells and dissemination of Creutzfeldt*-*Jakob disease*. J

[59] Baldauf, E., Beekes, M., and Diringer, H., *Evidence for an alternative direct route of access for the scrapie agent to the brain bypassing the spinal cord*. J Gen Virol, 1997. **78** (Pt 5): pp.

[60] McBride, P.A., et al., *Early spread of scrapie from the gastrointestinal tract to the central nervous system involves autonomic fibers of the splanchnic and vagus nerves*. J Virol, 2001. **75**(19):

[61] Race, R., Oldstone, M., and Chesebro, B., *Entry versus blockade of brain infection following oral or intraperitoneal scrapie administration*: *role of prion protein expression in peripheral*

*of C1q significantly delays onset of scrapie*. Nat Med, 2001. **7**(4): pp. 485–487.

*scrapie*: *the role of spleen and thymus*. J Comp Pathol, 1978. **88**(4): pp. 563–573.

*pulp and stroma of infected spleens*. Vet Microbiol, 1984. **9**(3): pp. 215–225.

*in mice*. Vet Microbiol, 1987. **13**(3): pp. 211–223.

*of scrapie*. Nat Med, 2000. **6**(7): pp. 719–720.

pp. 6845–6854.

225–234.

152 Prion - An Overview

488–492.

1187–1197.

pp. 9320–9327.

**76**(9): pp. 4357–4363.

Virol, 2000. **74**(18): pp. 8614–8622.

*nerves and spleen*. J Virol, 2000. **74**(2): pp. 828–833.

[96] Kaiser-Schulz, G., et al., *Polylactide*-*coglycolide microspheres co*-*encapsulating recombinant tandem prion protein with CpG*-*oligonucleotide break self*-*tolerance to prion protein in wildtype mice and induce CD4 and CD8 T cell responses*. J Immunol, 2007. **179**(5): pp. 2797–2807.

[79] Solforosi, L., et al., *Cross*-*linking cellular prion protein triggers neuronal apoptosis in vivo*.

[80] Feraudet-Tarisse, C., et al., *Immunotherapeutic effect of anti-PrP monoclonal antibodies in TSE mouse models: pharmacokinetic and pharmacodynamic analysis*. J Gen Virol, 2010. **91**(Pt

[81] Lefebvre-Roque, M., et al., *Toxic effects of intracerebral PrP antibody administration during*

[82] Song, C.H., et al., *Effect of intraventricular infusion of anti*-*prion protein monoclonal antibodies on disease progression in prion*-*infected mice*. J Gen Virol, 2008. **89**(Pt 6): pp. 1533–1544.

[83] Klohn, P.C., et al., *PrP antibodies do not trigger mouse hippocampal neuron apoptosis*. Science,

[84] Gauczynski, S., et al., *The 37*-*kDa*/*67*-*kDa laminin receptor acts as the cell*-*surface receptor for*

[85] Morel, E., et al., *Bovine prion is endocytosed by human enterocytes via the 37 kDa*/*67 kDa lam-*

[86] Gauczynski, S., et al., *The 37*-*kDa*/*67*-*kDa laminin receptor acts as a receptor for infectious prions and is inhibited by polysulfated glycanes*. J Infect Dis, 2006. **194**(5): pp. 702–709. [87] Zuber, C., et al., *Anti*-*LRP*/*LR antibody W3 hampers peripheral PrPSc propagation in scrapie*

[88] Zuber, C., et al., *Single chain Fv antibodies directed against the 37 kDa*/*67 kDa laminin receptor*

[89] Sigurdsson, E.M., et al., *Immunization delays the onset of prion disease in mice*. Am J Pathol,

[90] Arbel, M., Lavie, V., and Solomon, B., *Generation of antibodies against prion protein in wildtype mice via helix 1 peptide immunization*. J Neuroimmunol, 2003. **144**(1–2): pp. 38–45. [91] Magri, G., et al., *Decrease in pathology and progression of scrapie after immunisation with syn-*

[92] Schwarz, A., et al., *Immunisation with a synthetic prion protein*-*derived peptide prolongs survival times of mice orally exposed to the scrapie agent*. Neurosci Lett, 2003. **350**(3): pp. 187–189.

[93] Souan, L., et al., *Modulation of proteinase*-*K resistant prion protein by prion peptide immuniza-*

[94] Gilch, S., et al., *Polyclonal anti*-*PrP auto*-*antibodies induced with dimeric PrP interfere efficiently with PrPSc propagation in prion*-*infected cells*. J Biol Chem, 2003. **278**(20): pp.

[95] Polymenidou, M., et al., *Humoral immune response to native eukaryotic prion protein correlates with anti*-*prion protection*. Proc Natl Acad Sci U S A, 2004. **101** Suppl 2: pp. 14670–14676.

*as therapeutic tools in prion diseases*. Mol Immunol, 2008. **45**(1): pp. 144–151.

*thetic prion protein peptides in hamsters*. Vaccine, 2005. **23**(22): pp. 2862–2868.

*the course of BSE infection in mice*. Prion, 2007. **1**(3): pp. 198–206.

*the cellular prion protein*. EMBO J, 2001. **20**(21): pp. 5863–5875.

*inin receptor*. Am J Pathol, 2005. **167**(4): pp. 1033–1042.

*infected mice*. Prion, 2007. **1**(3): pp. 207–212.

*tion*. Eur J Immunol, 2001. **31**(8): pp. 2338–2346.

Science, 2004. **303**(5663): pp. 1514–1516.

6): pp. 1635–1645

154 Prion - An Overview

2012. **335**(6064): p. 52.

2002. **161**(1): pp. 13–17.

18524–18531.

**Provisional chapter**

## **Genetic Resistance to Prion Diseases Genetic Resistance to Prion Diseases**

## Yalçın Yaman and Cemal Ün Yalçın Yaman and Cemal Ün

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66916

#### **Abstract**

[110] Goni, F., et al., *Mucosal vaccination delays or prevents prion infection via an oral route*.

[111] Goni, F., et al., *Mucosal immunization with an attenuated Salmonella vaccine partially protects white*-*tailed deer from chronic wasting disease*. Vaccine, 2015. **33**(5): pp. 726–733. [112] Goni, F., et al., *High titers of mucosal and systemic anti*-*PrP antibodies abrogate oral prion infection in mucosal*-*vaccinated mice*. Neuroscience, 2008. **153**(3): pp. 679–686.

[113] Bade, S., et al., *Intranasal immunization of Balb*/*c mice against prion protein attenuates orally acquired transmissible spongiform encephalopathy*. Vaccine, 2006. **24**(9): pp. 1242–1253.

[114] Sacquin, A., et al., *Prolongation of prion disease*-*associated symptomatic phase relates to CD3*+ *T cell recruitment into the CNS in murine scrapie*-*infected mice*. Brain Behav Immun,

[115] Sadowski, M.J., et al., *Anti*-*PrP Mab 6D11 suppresses PrP*(*Sc*) *replication in prion infected myeloid precursor line FDC*-*P1*/*22L and in the lymphoreticular system in vivo*. Neurobiol

[116] Petsch, B., et al., *Biological effects and use of PrPSc*- *and PrP*-*specific antibodies generated by immunization with purified full*-*length native mouse prions*. J Virol, 2011. **85**(9): pp.

[117] Didonna, A., et al., *Characterization of four new monoclonal antibodies against the distal*

[118] Pilon, J.L., et al., *Immunization with a synthetic peptide vaccine fails to protect mule deer* (*Odocoileus hemionus*) *from chronic wasting disease*. J Wildl Dis, 2013. **49**(3): pp. 694–698.

[119] David, M.A., Jones, D.R., and Tayebi, M., *Potential candidate camelid antibodies for the treatment of protein*-*misfolding diseases*. J Neuroimmunol, 2014. **272**(1–2): pp. 76–85. [120] Han, Y., et al., *Immune responses in wild*-*type mice against prion proteins induced using a DNA prime*-*protein boost strategy*. Biomed Environ Sci, 2011. **24**(5): pp. 523–529.

Neuroscience, 2005. **133**(2): pp. 413–421.

2012. **26**(6): pp. 919–930.

4538–4546.

156 Prion - An Overview

Dis, 2009. **34**(2): pp. 267–278.

*N*-*terminal region of PrP*(*c*). PeerJ, 2015. **3**: p. e811.

Prions are abnormal isoforms of the host‐encoded cellular prion proteins which are mis‐ folding in its three‐dimensional structure acquire pathogenicity. Prions cause transmis‐ sible spongiform encephalopathy (TSEs) in humans and some animal species including sheep, goats, cattle, cat, deer and elk. TSEs, also called "prion diseases," cause irreversible neurodegeneration in the central nervous system and are always fatal. Cellular prion proteins are encoded by prion protein gene (*PRNP*) in mammals; moreover, it is known that the variations in the *PRNP* gene have influence on the resistance and/or incubation period of the TSEs. It is well‐documented that after exposure to the pathogenic prions, development of some TSEs depend on the host *PRNP* genotype, for example, scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, Creutzfeldt‐Jakob disease (CJD) and kuru in humans, as well. In this chapter, genetic resistance to prion diseases will be reviewed.

**Keywords:** TSE, prion disease, PRNP, genetic resistance

## **1. Introduction**

It is known that conformational changes in prion protein cause Creutzfeldt‐Jakob disease (CJD) in humans, scrapie disease in sheep and goats [1, 2], bovine spongiform encephalopa‐ thy (BSE) in cattle, feline spongiform encephalopathy in cat, and wasting disease in deer and elk.

Polymorphisms inside the prion protein‐coding gene (*PRNP*) in humans and also in some mammalian species have been appeared to impact disease susceptibility and pathologies [3]. In human population, kuru and CJD are profoundly related with polymorphism in codon 129. All CDJ affected individuals are known to be homozygous for methionine amino acid in codon 129 while at the same codon heterozygote individuals seem most resistant to kuru [4, 5]. Also, it is known that there is a high correlation between the polymorphisms in codons

136, 154, and 171 of the PRNP gene and the level of susceptibility to scrapie in sheep [3, 6, 7]. In cattle, numerous studies were carried out for discovering a relationship amongst BSE and polymorphisms in cattle genome [8–12]. The studies about BSE‐affected animals in Germany and USA represented the influence of *PRNP* promoter polymorphisms on BSE susceptibility in cattle [13, 14]. The impacts of insertion‐deletion (indel) polymorphisms within a location 1.6 kbp upstream of exon 1 and inside intron 1 (23‐bp and 12‐bp, respectively) on BSE suscep‐ tibility are determined by further analyses in cattle [15–17]. Despite the fact that cattle with the ‐/‐23 bp promoter genotype and the –/12 bp intron 1 genotype have both been significantly connected with BSE, it could not be reached any consensus on which genotype is most identi‐ fied with BSE [13, 15, 16, 18]. In addition, indel polymorphisms that affect the sensitivity of classical BSE appear not to be pertinent to other transmissible spongiform encephalopathies in cattle [19]. Until now, the incidence of *PRNP* gene promoter polymorphisms has been iden‐ tified in some cattle in Asia [20, 21], Europe [13, 16, 18, 22] and America [14, 23].

## **2. Resistance in humans**

There exist various types of human prion disease such as Creutzfeldt‐Jakob disease (CJD), fatal familial insomnia (FFI), and Gerstmann Sträussler‐Scheinker syndrome (GSS). Related to the cause of the illness they exist in three main forms: Genetic, sporadic and acquired. Genetic form of the disease is caused by a mutation in prion protein‐coding gene (*PRNP*), whereas acquired form occurs by the transmission of disease from an animal or another human dis‐ ease. The cause of sporadic form is not clear up to now [24–26].

The human prion‐coding gene consists of two exons and the second one contains the whole open reading frame. It is known that a valine amino acid at position 129 of the human prion protein provide resistancy to the Creutzfeldt‐Jakob disease. Both Valin129Valin and Methionine129Methionine genotypes are resistant to the disease, whereas Methionine129Methionine genotypes are susceptible [27, 28]. Another polymorphism at codon 219 was reported to be related with development of Creutzfeldt‐Jakob disease in Japanese population [29].

## **3. Resistance in small ruminants**

Scrapie is a neurodegenerative disease of sheep and goats. As with other transmissible spon‐ giform encephalopathies (TSE) which affect humans and animal species, scrapie is always fatal and characterized by long incubation periods ranging from months to years, vacuola‐ tion, neuronal loss and astrocytosis in the central nervous system (CNS) and has no inflam‐ matory or immune responses [30]. The earliest reports of the scrapie based on middle of 1700s in Britain. Various terms such as "scrapie," "scratchie," "rubbers," "rickets" and "goggles" were used to indicate the disease [31].

It is thought that scrapie first occurred in the United Kingdom in the eighteenth century and following decades, particularly after World War II, the disease spread by importation of the infected animals. Scrapie has reported nearly all over the world, for example, Iceland (1878), Canada (1938), USA (1947), Australia (1952), Norway (1958), India (1961), Republic of South Africa (1966), Kenya (1970), Germany (1973), Brazil (1978), Yemen (1979), Sweden (1988), Cyprus (1989) and Japan (1990), reviewed in reference [30].

Scrapie has been known for over 250 years; therefore, it is regarded to be prototype of the TSEs [30]. Earlier, researchers thought that it was a hereditary disease, but later, according to the results of the experimental transmission studies, they were considered that "Scrapie was a natural infection and gained from ground". After seven years of working with several thousand breeding ewes within several hundred ewes were affected classical scrapie, H. B. Parry postulated some hypothesis that scrapie had a hereditary feature in a simple Mendelian autosomal recessive manner, development of the disease determined by genotype of the indi‐ viduals, and it was not a natural infection. They observed that in high‐incidence flocks, many scrapie diseased individuals had affected parent or progeny [32, 33]. Later studies revealed the evidences that scrapie is a transmissible infection [34] which is caused by a kind of pro‐ teins called "prion" [35], and development and/or incubation period of the disease under genetic control [36–40].

#### **3.1. Resistance in sheep**

136, 154, and 171 of the PRNP gene and the level of susceptibility to scrapie in sheep [3, 6, 7]. In cattle, numerous studies were carried out for discovering a relationship amongst BSE and polymorphisms in cattle genome [8–12]. The studies about BSE‐affected animals in Germany and USA represented the influence of *PRNP* promoter polymorphisms on BSE susceptibility in cattle [13, 14]. The impacts of insertion‐deletion (indel) polymorphisms within a location 1.6 kbp upstream of exon 1 and inside intron 1 (23‐bp and 12‐bp, respectively) on BSE suscep‐ tibility are determined by further analyses in cattle [15–17]. Despite the fact that cattle with the ‐/‐23 bp promoter genotype and the –/12 bp intron 1 genotype have both been significantly connected with BSE, it could not be reached any consensus on which genotype is most identi‐ fied with BSE [13, 15, 16, 18]. In addition, indel polymorphisms that affect the sensitivity of classical BSE appear not to be pertinent to other transmissible spongiform encephalopathies in cattle [19]. Until now, the incidence of *PRNP* gene promoter polymorphisms has been iden‐

tified in some cattle in Asia [20, 21], Europe [13, 16, 18, 22] and America [14, 23].

ease. The cause of sporadic form is not clear up to now [24–26].

There exist various types of human prion disease such as Creutzfeldt‐Jakob disease (CJD), fatal familial insomnia (FFI), and Gerstmann Sträussler‐Scheinker syndrome (GSS). Related to the cause of the illness they exist in three main forms: Genetic, sporadic and acquired. Genetic form of the disease is caused by a mutation in prion protein‐coding gene (*PRNP*), whereas acquired form occurs by the transmission of disease from an animal or another human dis‐

The human prion‐coding gene consists of two exons and the second one contains the whole open reading frame. It is known that a valine amino acid at position 129 of the human prion protein provide resistancy to the Creutzfeldt‐Jakob disease. Both Valin129Valin and Methionine129Methionine genotypes are resistant to the disease, whereas Methionine129Methionine genotypes are susceptible [27, 28]. Another polymorphism at codon 219 was reported to be related with development of Creutzfeldt‐Jakob disease in

Scrapie is a neurodegenerative disease of sheep and goats. As with other transmissible spon‐ giform encephalopathies (TSE) which affect humans and animal species, scrapie is always fatal and characterized by long incubation periods ranging from months to years, vacuola‐ tion, neuronal loss and astrocytosis in the central nervous system (CNS) and has no inflam‐ matory or immune responses [30]. The earliest reports of the scrapie based on middle of 1700s in Britain. Various terms such as "scrapie," "scratchie," "rubbers," "rickets" and "goggles"

It is thought that scrapie first occurred in the United Kingdom in the eighteenth century and following decades, particularly after World War II, the disease spread by importation of the

**2. Resistance in humans**

158 Prion - An Overview

Japanese population [29].

**3. Resistance in small ruminants**

were used to indicate the disease [31].

Sheep and goat prion protein‐coding gene (*PRNP*) which encodes the cellular prion protein located on chromosome 13 [41]. The gene structure of the sheep *PRNP* was determined by [40], they demonstrated that sheep *PRNP* encoded 256 amino acids and highly homologous with the *PRNP* gene of the other species. Furthermore, the authors suggest that arginine/ glutamine substitution in the 171th position of the sheep *PRNP* might have affected the scra‐ pie incubation period. According to the results of many subsequent study polymorphisms of 136th, 154th and 171th codons of ovine *PRNP* had a strong influence on susceptibility or resistance to the scrapie [8, 42–45].

Commonly encoded amino acids at three codons are as follows: alanine (A) or valine (V) at codon 136, arginine (R) or histidine (H) at codon 154 and glutamine (G), histidine (H) or arginine (R) at codon 171 and out of possible other combinations, common *PRNP* alleles are A136R154R171, A136R154Q171, A136R154H171, A136H154Q171 and V136R154Q171, (respectively, ARR, ARQ, ARH, AHQ and VRQ for short) [45, 46]. While ARR alleles related to resistance, VRQ is regarded as the most susceptible alleles. Until now, only three scrapie cases were reported in ARR homozygous sheep which are one case from Japan [47] and two cases from France and Germany [48]. Some studies on PrP genotype and their relevance to scrapie in scrapie diseased sheep are presented in **Table 1**.

There is no report about direct transmission from sheep to human in natural condition, nev‐ ertheless, scrapie can be transmitted interspecies by experimentally [59–61], furthermore, the cattle prion disease, Bovine spongiform encephalopathy (BSE) which is transmitted to human and causes a variant of Creutzfeldt‐Jakob disease (vCJD) [62], originated from the usage of scra‐ pie contaminated material in cattle nutrition [63]. Even, in a more recent study, natural scrapie isolate was successfully transmitted to a primate (cynomolgus macaque) suggesting that scrapie has zoonotic potential to primates including human [64]. Epidemiological connection with scra‐

**Table 1.** PrP genotype frequencies of the scrapie‐infected sheep in various countries.

pie, BSE and vCJD emerged public health concerns and lead to establishing scrapie eradication programs, including increasing the genetic resistance to scrapie in scrapie epidemic countries.

In 2001, Great Britain has established the "National Scrapie Plan" (NSP) intending to increase the frequencies of resistance alleles by selective breeding and eventually eradicate scrapie from British sheep herds. According to disease‐associated alleles, five risk groups were desig‐ nated from R1 to R5 where is R1 referring at the lowest risk and R5 at highest risk [65]. NSP scrapie risk groups can be seen in **Table 2**.

Reported case per year and estimated of the case number per million sheep according to risk groups in the United Kingdom (UK) are given in **Table 3**.

European Union (EU) Commission has issued a regulation in 2003 that required the establish of a selective breeding program for resistance to TSE in each sheep breed of member states [66]; therefore, European member states have been implementing breeding programs based on elimination of the most susceptible alleles while increasing resistant allele frequencies. For example, as a result of intensive genetic selection programs, particularly in high genetic merit flocks, ARR allele frequencies increased from 50 to 69% in the UK, 49 to 85% in France, 38 to 70% in the Netherlands and 47 to 70% in Italy [67].

**Table 2.** PrP genotypes and allocation of them into scrapie risk groups (adapted from reference [65]).

**Risk**

**PrP**

**Norway** *n*

**England** *n*

**England** *n*

**France** *n*

**France** *n*

**Ireland** *n*

**Italy** *n*

**The**

**Iceland** *n*

**Greece** *n*

**Japan** *n*

**Canada** *n*

**= 32 [49]**

**= 21 [50]**

**= 59 [51]**

**= 437 [52]**

**= 245 [53]**

**= 154 [54]**

**= 34 [55]**

**Netherlands**

**= 101 [56]**

**= 216 [57]**

**= 15 [47]**

**= 249 [58]**

160 Prion - An Overview

*n* **= 34 [45]**

**groups**

1 2

ARR/AHQ ARR/ARH ARR/ARQ

> 3

ARQ/ARH ARQ/AHQ AHQ/AHQ

ARH/ARH AHQ/ARH ARQ/ARQ

> 4

5

AHQ/VRQ ARH/VRQ ARQ/VRQ VRQ/VRQ

0.688

 0.000 **Table 1.**  Estimates of the number of reported cases of scrapie per million sheep of each risk groups in the UK (adapted from reference [46]).

Given the importance of the disease, a lot of genotyping studies on sheep *PRNP* have carried out in the almost all over the world such as; in New Zealand and Australia [68], Brazil [69], Israel, Palestine, and Jordan [70], Turkey [71], Egypt and Saudi Arabia [72] and East Asia [73], whether scrapie have reported or never been reported.

#### **3.2. Resistance in goats**

First natural scrapie case in goats was defined in 1942 [74]. Although goat scrapie has rare incidence compared with sheep, a surveillance program between 2002 and 2009 was per‐ formed according to the EU commission direction and over 3000 scrapie cases were reported in goats [75]. Scrapie cases occurring in natural condition in goats have been reported, partic‐ ularly throughout Europe [76–78]. Transmission of the scrapie from naturally affected sheep to goats which rearing together has often been observed [77, 79–81], in addition, transmission from goat to goat has been known [76].

In contrast to sheep, limited data are available related to scrapie resistance and *PRNP* alleles. Genotyping studies on goats *PRNP* have given various results in terms of disease suscepti‐ bility or resistance. Assessment of *PRNP* alleles in scrapie infected and non‐infected goats presented in **Table 4**.

As provided in **Table 4**, some relationships between caprine *PRNP* polymorphisms and scra‐ pie resistance were defined. Encoding of serine instead of glycine at codon 127 has decreased the probability of clinical manifestation of the disease [86]. Isoleucine‐methionine dimor‐ phism at codon 142 has found to be associated both experimental [88] and natural infection [86, 89]; furthermore, it is reported that [89] the presence of methionine‐isoleucine as het‐ erozygous at codon 142 has been provided resistance only in proline‐proline homozygous animal at codon 240. Encoding of arginine at codon 143 has provided limited protection to natural scrapie [80]. While the presence of asparagine instead of Serine or Aspartic acid at codon 146 has been found to be related to susceptibility to natural infection [78], it also has reported that the presence of Serine as heterozygous at the same codon has associated with the extended incubation period in oral challenging [90]. According to the results of various studies, arginine‐histidine dimorphism at codon 154 has provided limited resistance [78, 80, 83, 89]. The presence of glutamine/arginine as heterozygous at codon 211 has been found to

Given the importance of the disease, a lot of genotyping studies on sheep *PRNP* have carried out in the almost all over the world such as; in New Zealand and Australia [68], Brazil [69], Israel, Palestine, and Jordan [70], Turkey [71], Egypt and Saudi Arabia [72] and East Asia [73],

**Table 3.** Estimates of the number of reported cases of scrapie per million sheep of each risk groups in the UK (adapted

**Risk groups Case per year (***n***) Percentage of sheep Case per year per million (***n***)**

R1 0 21.3 0 R2 2.3 35.7 0.7 R3 104.9 23.9 57.8 R4 12 9.6 6.3 R5 381.8 9.6 1175.6

First natural scrapie case in goats was defined in 1942 [74]. Although goat scrapie has rare incidence compared with sheep, a surveillance program between 2002 and 2009 was per‐ formed according to the EU commission direction and over 3000 scrapie cases were reported in goats [75]. Scrapie cases occurring in natural condition in goats have been reported, partic‐ ularly throughout Europe [76–78]. Transmission of the scrapie from naturally affected sheep to goats which rearing together has often been observed [77, 79–81], in addition, transmission

In contrast to sheep, limited data are available related to scrapie resistance and *PRNP* alleles. Genotyping studies on goats *PRNP* have given various results in terms of disease suscepti‐ bility or resistance. Assessment of *PRNP* alleles in scrapie infected and non‐infected goats

As provided in **Table 4**, some relationships between caprine *PRNP* polymorphisms and scra‐ pie resistance were defined. Encoding of serine instead of glycine at codon 127 has decreased the probability of clinical manifestation of the disease [86]. Isoleucine‐methionine dimor‐ phism at codon 142 has found to be associated both experimental [88] and natural infection [86, 89]; furthermore, it is reported that [89] the presence of methionine‐isoleucine as het‐ erozygous at codon 142 has been provided resistance only in proline‐proline homozygous animal at codon 240. Encoding of arginine at codon 143 has provided limited protection to natural scrapie [80]. While the presence of asparagine instead of Serine or Aspartic acid at codon 146 has been found to be related to susceptibility to natural infection [78], it also has reported that the presence of Serine as heterozygous at the same codon has associated with the extended incubation period in oral challenging [90]. According to the results of various studies, arginine‐histidine dimorphism at codon 154 has provided limited resistance [78, 80, 83, 89]. The presence of glutamine/arginine as heterozygous at codon 211 has been found to

whether scrapie have reported or never been reported.

**3.2. Resistance in goats**

from reference [46]).

162 Prion - An Overview

presented in **Table 4**.

from goat to goat has been known [76].

Abbreviations of the amino acids: A, alanine; D, aspartic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan.

**Table 4.** The *PRNP* polymorphisms of scrapie‐infected/noninfected goats and association of polymorphisms with scrapie resistance.

be related to lower susceptibility [89], and the presence of lysine at codon 222 has been associ‐ ated with resistance to both natural [83, 89, 93] and oral [90] or intracerebral challenging [94].

Apart from these polymorphisms, an allele of caprine *PRNP*, which encodes shorter cellu‐ lar prion protein, has been reported. An experimental transmission to a goat carrying this allele as heterozygote has died after an unusually long incubation period [95]. In addition, a novel 28 bp insertion in the promoter region of caprine *PRNP* was found by [96] in healthy Chinese native goat breeds. Although there is no information with respect to disease resis‐ tance, some associations between this insertion/deletion polymorphism and production trait were reported.

Influences of the remaining codons over scrapie resistance or susceptibility in goats are not known yet. Currently available data on genetic resistance to scrapie are considered insuffi‐ cient to establish selective breeding programs in goats.

#### **3.3. Atypical scrapie in sheep and goats**

Norwegian researchers have recognized a novel type of scrapie case in 1998 which has unusual histopathological features comparing with classical scrapie. The geographical distri‐ bution of the disease indicated that it might be spontaneous scrapie, not a contagious disease. This atypical form of scrapie designated as Nor98 by the authors [97]. Later studies conducted on archived tissue specimens revealed that atypical scrapie is not a new disease and has been existed at least from late 1980s in the UK herds [98, 99]. In the following years, many atypical scrapie cases were reported in sheep and/or goats from [100–103], North America [104] and New Zealand [105], as well.

Atypical cases have appeared to relate with the *PRNP* genotypes considered relatively resis‐ tant to classical scrapie. Sheep which are carrier of AHQ allele have found to be more sus‐ ceptible to atypical scrapie; moreover, unlike classical scrapie, it was demonstrated that the presence of phenylalanine at codon 141 strongly associated with atypical cases [51, 53, 100, 106–109]. Interestingly, according to results of case control studies, while VRQ allele which is the most classical scrapie have found to be related to low incidence in atypical scrapie [51, 53, 108], the most resistant ARR allele associated with higher incidence [53, 107, 109]. Distribution of *PRNP* genotypes and roles of codon 141 on atypical scrapie resistance dem‐ onstrated in **Table 5**.

Although there is very limited data about relationship atypical scrapie and *PRNP* genotypes in goats, it has been reported that the presence of histidine at codon 154 may associated with atypical cases in goats, as well [103, 109].

European selective breeding programs against to classical scrapie in sheep already elimi‐ nating the AHQ and AFRQ alleles which have demonstrated to relate with atypical scrapie susceptibility; however, the major problem about ARR (resistant to classical scrapie but sus‐ ceptible to atypical scrapie) and VRQ (susceptible to classical scrapie but resistant to atypical scrapie) alleles remains to be solved.

**Table 5.** *PRNP* genotypes according to codons 136, 154 and 171 (and codon 141 if the presence of phenylalanine residue) and association with atypical scrapie.

## **4. Resistance in cattle**

be related to lower susceptibility [89], and the presence of lysine at codon 222 has been associ‐ ated with resistance to both natural [83, 89, 93] and oral [90] or intracerebral challenging [94].

Apart from these polymorphisms, an allele of caprine *PRNP*, which encodes shorter cellu‐ lar prion protein, has been reported. An experimental transmission to a goat carrying this allele as heterozygote has died after an unusually long incubation period [95]. In addition, a novel 28 bp insertion in the promoter region of caprine *PRNP* was found by [96] in healthy Chinese native goat breeds. Although there is no information with respect to disease resis‐ tance, some associations between this insertion/deletion polymorphism and production trait

Influences of the remaining codons over scrapie resistance or susceptibility in goats are not known yet. Currently available data on genetic resistance to scrapie are considered insuffi‐

Norwegian researchers have recognized a novel type of scrapie case in 1998 which has unusual histopathological features comparing with classical scrapie. The geographical distri‐ bution of the disease indicated that it might be spontaneous scrapie, not a contagious disease. This atypical form of scrapie designated as Nor98 by the authors [97]. Later studies conducted on archived tissue specimens revealed that atypical scrapie is not a new disease and has been existed at least from late 1980s in the UK herds [98, 99]. In the following years, many atypical scrapie cases were reported in sheep and/or goats from [100–103], North America [104] and

Atypical cases have appeared to relate with the *PRNP* genotypes considered relatively resis‐ tant to classical scrapie. Sheep which are carrier of AHQ allele have found to be more sus‐ ceptible to atypical scrapie; moreover, unlike classical scrapie, it was demonstrated that the presence of phenylalanine at codon 141 strongly associated with atypical cases [51, 53, 100, 106–109]. Interestingly, according to results of case control studies, while VRQ allele which is the most classical scrapie have found to be related to low incidence in atypical scrapie [51, 53, 108], the most resistant ARR allele associated with higher incidence [53, 107, 109]. Distribution of *PRNP* genotypes and roles of codon 141 on atypical scrapie resistance dem‐

Although there is very limited data about relationship atypical scrapie and *PRNP* genotypes in goats, it has been reported that the presence of histidine at codon 154 may associated with

European selective breeding programs against to classical scrapie in sheep already elimi‐ nating the AHQ and AFRQ alleles which have demonstrated to relate with atypical scrapie susceptibility; however, the major problem about ARR (resistant to classical scrapie but sus‐ ceptible to atypical scrapie) and VRQ (susceptible to classical scrapie but resistant to atypical

cient to establish selective breeding programs in goats.

**3.3. Atypical scrapie in sheep and goats**

New Zealand [105], as well.

onstrated in **Table 5**.

atypical cases in goats, as well [103, 109].

scrapie) alleles remains to be solved.

were reported.

164 Prion - An Overview

Bovine spongiform encephalopathy (BSE), the cattle prion disease, belongs to animal TSE's which has been characterized histopathological changes in the CNS as with scrapie. It is newly diagnosed prion disease, which has been never known until 1986 [110]. BSE became epidemic during the 1980s in the UK as a result of the changing rendering process and allow‐ ing to enter the prion contaminated product to cattle nutrition, and it is estimated that the exposure began in the early 1980s [110]. Having transmitted to human and causing a new variant of Creutzfeldt‐Jakob disease (CJD) [62] which is a human prion disease acquired from consumption of the meat products of the BSE diseased cattle [111], BSE has been regarded by the World Health Organization [112] as zoonotic. Unlike CJD, vCJD has diagnosed in younger people in the UK [113], latter in France [114]. Up to 2003, 135 vCJD cases have reported from the UK and 6 cases from France (reviewed in reference [115]).

BSE could transmit to sheep and goats by experimental routes [116] and development of the disease seemed to be affected by the *PRNP* genotype of the individual [88, 117]; furthermore, it was reported that BSE in goats can be occur in natural conditions [118, 119].

Because of the zoonotic potential and the ability to spread between species of the BSE, it has raised the public health concerns and enforced to governments to take control and preventive measures; moreover, researchers have intensified to reveal the genetic background of the disease.

Early studies on association between *PRNP* genotype of cattle and development of the BSE have focused on two known polymorphisms; the HindII restriction site and an octapeptide repeated sequence in the coding region of the cattle *PRNP*, but no relationship between these genotypes and BSE infection has found [120, 121]; however, although lack of detailed genetic information, some clues were obtained suggesting that BSE might be in linkage with host *PRNP* genotype [9].

In the following years, hundreds of nucleotide changes and insertions/deletions (indel) were identified in bovine *PRNP* [13, 122, 123, 124], including a 12 base pair (bp) indel within the intron 1 and a 23 bp indel within the promoter region [13, 122]. Case control studies showed that distribution of these two indel polymorphisms were different between healthy and BSE affected cattle and insertion alleles presumably connected with disease resistance [13]; more‐ over, it has demonstrated that insertion alleles related to the lower prion protein level com‐ pared with deletion alleles and may differentiate of the BSE incubation period [15]. Further studies have supported the relationship between BSE resistance and 23 bp/12 bp indel geno‐ types that are given in **Table 6**.

Although the clear association has been shown between *PRNP* indel genotypes and BSE inci‐ dence, there are some paradoxical situations at breed level, for example, it was reported that although Brown breeds have higher allelic frequency of insertion alleles, at the same time, these breeds have higher prevalence of BSE [17]. However, beside of the primary measures for prevention from circulation of BSE agents and exposure to both animal and human, selective breeding can offer a secondary strategy to eliminate the BSE.

Apart from classical BSE, two more types of the disease have been diagnosed by histopatho‐ logical examinations; H‐type and L‐type, both of two types classified as atypical BSE and have been observing sporadically. While H‐type BSE characterized with higher molecular mass [126], L‐type BSE which is also named as bovine amyloidotic spongiform encephalopathy (BASE), characterized with lower molecular mass and has diverse glycopattern of pathogenic prion proteins [127].

It is reported that *PRNP* 23 and 12 bp indel polymorphism do not provide the genetic resis‐ tance, neither to naturally occurring atypical BSE nor to experimentally inoculated other TSEs [16]. Although very limited data, several atypical cases with extremely rare [128] glutamate to lysine mutation in codon 211 (E211K), which is homologous with human E200K mutation in

people in the UK [113], latter in France [114]. Up to 2003, 135 vCJD cases have reported from

BSE could transmit to sheep and goats by experimental routes [116] and development of the disease seemed to be affected by the *PRNP* genotype of the individual [88, 117]; furthermore,

Because of the zoonotic potential and the ability to spread between species of the BSE, it has raised the public health concerns and enforced to governments to take control and preventive measures; moreover, researchers have intensified to reveal the genetic background of the disease.

Early studies on association between *PRNP* genotype of cattle and development of the BSE have focused on two known polymorphisms; the HindII restriction site and an octapeptide repeated sequence in the coding region of the cattle *PRNP*, but no relationship between these genotypes and BSE infection has found [120, 121]; however, although lack of detailed genetic information, some clues were obtained suggesting that BSE might be in linkage with host

In the following years, hundreds of nucleotide changes and insertions/deletions (indel) were identified in bovine *PRNP* [13, 122, 123, 124], including a 12 base pair (bp) indel within the intron 1 and a 23 bp indel within the promoter region [13, 122]. Case control studies showed that distribution of these two indel polymorphisms were different between healthy and BSE affected cattle and insertion alleles presumably connected with disease resistance [13]; more‐ over, it has demonstrated that insertion alleles related to the lower prion protein level com‐ pared with deletion alleles and may differentiate of the BSE incubation period [15]. Further studies have supported the relationship between BSE resistance and 23 bp/12 bp indel geno‐

Although the clear association has been shown between *PRNP* indel genotypes and BSE inci‐ dence, there are some paradoxical situations at breed level, for example, it was reported that although Brown breeds have higher allelic frequency of insertion alleles, at the same time, these breeds have higher prevalence of BSE [17]. However, beside of the primary measures for prevention from circulation of BSE agents and exposure to both animal and human, selective

Apart from classical BSE, two more types of the disease have been diagnosed by histopatho‐ logical examinations; H‐type and L‐type, both of two types classified as atypical BSE and have been observing sporadically. While H‐type BSE characterized with higher molecular mass [126], L‐type BSE which is also named as bovine amyloidotic spongiform encephalopathy (BASE), characterized with lower molecular mass and has diverse glycopattern of pathogenic

It is reported that *PRNP* 23 and 12 bp indel polymorphism do not provide the genetic resis‐ tance, neither to naturally occurring atypical BSE nor to experimentally inoculated other TSEs [16]. Although very limited data, several atypical cases with extremely rare [128] glutamate to lysine mutation in codon 211 (E211K), which is homologous with human E200K mutation in

it was reported that BSE in goats can be occur in natural conditions [118, 119].

the UK and 6 cases from France (reviewed in reference [115]).

*PRNP* genotype [9].

166 Prion - An Overview

types that are given in **Table 6**.

prion proteins [127].

breeding can offer a secondary strategy to eliminate the BSE.

**Table 6.** The distribution of the *PRNP* 23 bp indel and 12 bp indel genotypes according to breeds, in both healthy and BSE‐affected cattle.

the *PRNP* gene, has determined, suggesting that association to atypical BSE resistance may be exist [129, 130], but could not confirm by following studies [131, 132]. Transmissibility of the H‐type atypical BSE to cattle which is carrying the E211K mutation was demonstrated [133], on the other hand, some evidences have obtained that the E211K is a germ line mutation, thus, may cause inherited BSE that can be transmitted genetically [130].

## **5. Resistance in water buffaloes**

During the BSE epidemic in 1980s, it can be assumed that BSE and/or scrapie contaminated by‐products most likely have entered in to water buffalo (Bubalus bubalis) nutrition systems, as well. EU member states have approximately 409 thousand of buffaloes, where 90% of those have been reared in Italy [134]. Between 2001 and 2005, 128 BSE cases in cattle have been reported from Italy [135]. Along with cattle, bison, sheep, goats and some exotic ruminants, water buffaloes have been considered as TSE‐related risk factors [136]; nevertheless, no BSE or any other TSE has ever been reported in water buffaloes [137] neither in Italy nor the rest of the world.

Only few studies on indel polymorphisms of the water buffalo *PRNP* gene have conducted to compare with cattle *PRNP*. According to the results, 12 and 23 bp indel polymorphisms have been existed in water buffalo, as well. Furthermore, insertion alleles which are relate to BSE resistance have observed more frequent than those in cattle [138–141] that is given in **Table 7**.

As seen in **Table 7**, almost all buffalo breeds, except Thai river buffalo, are carrying mostly insertion alleles either at 23 or 12 bp indel loci. This may be an explanation for why buffaloes putatively resistant to BSE.

**Table 7.** 23 and 12 bp allele frequencies of healthy water buffaloes reared in Asian and European states.

The SPRN gene, which belongs to the prion protein gene family, encodes the shadow pro‐ tein. Shadow protein shares characteristic features with cellular prion protein, suggesting the existence of a functional relation with prion proteins [143]. A comparative study revealed that the SPRN gene has species‐specific indel polymorphisms in cattle and buffaloes and causes different promoter activity and expression levels [144]. Furthermore, according to the results of more recent study, molecular structure of buffalo cellular prion protein is different from cattle, but similar to those of rabbits, dog and horse which are considered low susceptible to TSEs [145]. These molecular and structural differences may be another explanation with regard to TSEs resistance in buffaloes.

## **Author details**

**5. Resistance in water buffaloes**

of the world.

168 Prion - An Overview

**Table 7**.

putatively resistant to BSE.

Buffalo

Azikheli Buffalo

Kundhi Buffalo

Nili Ravi Buffalo

Turkey Anatolian

Turkey Anatolian

Buffalo

Murrah Buffalo

During the BSE epidemic in 1980s, it can be assumed that BSE and/or scrapie contaminated by‐products most likely have entered in to water buffalo (Bubalus bubalis) nutrition systems, as well. EU member states have approximately 409 thousand of buffaloes, where 90% of those have been reared in Italy [134]. Between 2001 and 2005, 128 BSE cases in cattle have been reported from Italy [135]. Along with cattle, bison, sheep, goats and some exotic ruminants, water buffaloes have been considered as TSE‐related risk factors [136]; nevertheless, no BSE or any other TSE has ever been reported in water buffaloes [137] neither in Italy nor the rest

Only few studies on indel polymorphisms of the water buffalo *PRNP* gene have conducted to compare with cattle *PRNP*. According to the results, 12 and 23 bp indel polymorphisms have been existed in water buffalo, as well. Furthermore, insertion alleles which are relate to BSE resistance have observed more frequent than those in cattle [138–141] that is given in

As seen in **Table 7**, almost all buffalo breeds, except Thai river buffalo, are carrying mostly insertion alleles either at 23 or 12 bp indel loci. This may be an explanation for why buffaloes

**Country Breed** *n* **In % Del % In % Del %**

Pakistan Nili Buffalo 66 94 6 86 14 [139] Ravi Buffalo 39 97 3 83 17

Indonesia River Buffalo 14 100 0 100 0 [142]

Germany River Buffalo 11 100 0 100 0 [140]

**Table 7.** 23 and 12 bp allele frequencies of healthy water buffaloes reared in Asian and European states.

Thai River Buffalo 45 53 47 84 16

Poland River Buffalo 29 100 0 100 0

20 100 0 95 5

34 97 3 88 12

122 94 6 87 13

20 100 0 100 0

**23 bp indel alleles 12 bp indel alleles References**

106 92 8 86 14 [138]

89 100 0 100 0 [141]

Yalçın Yaman1 and Cemal Ün2 \*

## **References**

[19] Brunelle BW, Hamir AN, Baron T, Biacabe AG, Richt JA, Kunkle RA, Cutlip RC, Miller JM, Nicholson EM. Polymorphisms of the prion gene promoter region that influ‐ ence classical bovine spongiform encephalopathy susceptibility are not applicable to other transmissible spongiform encephalopathies in cattle. Journal of Animal Science. 2007;**85**(12):3142–47. doi:10.2527/jas.2007‐0208

[7] Tongue SC, Wilesmith JW, Cook CJ. Frequencies of prion protein (PrP) genotypes and distribution of ages in 15 scrapie‐affected flocks in Great Britain. Veterinary Record.

[8] Goldmann W, Hunter N, Benson G, Foster JD, Hope J. Different scrapie‐associated fibril proteins (PrP) are encoded by lines of sheep selected for different alleles of the sip gene. Journal of General Virology. 1991a;**72**:2411–2417. doi:10.1099/0022‐1317‐72‐10‐2411 [9] Neibergs HL, Ryan AM, Womack JE, Spooner RL, Williams JL. Polymorphism analysis of the prion gene in BSE‐affected and unaffected cattle. Animal Genetics. 1994;**25**(5):313–17.

[10] Hunter N, Foster JD, Goldmann W, Stear MJ, Hope J, Bostock C. Natural scrapie in a closed flock of Cheviot sheep occurs only in specific PrP genotypes. Archives of Virology

[11] Walawski K, Czarnik U. Prion protein octapeptide‐repeat polymorphism in Polish Black

[12] Heaton MP, Leymaster KA, Freking BA, Hawk DA, Smith TP, Keele JW, Snelling WM, Fox JM, Chitko‐McKown CG, Laegreid WW. Prion gene sequence variation within diverse groups of U.S. sheep, beef cattle, and deer. Mammalian Genome. 2003;**14**:765–

[13] Sander P, Hamann H, Pfeiffer I, Wemheuer W, Brenig B, Groschup MH, Ziegler U, Distl O, Leeb T. Analysis of sequence variability of the bovine prion protein gene (*PRNP*) in German cattle breeds. Neurogenetics. 2004;**5**(1):19–25. doi:10.1007/s10048‐003‐0171‐y [14] Seabury CM, Womack JE, Pedrahita J, Derr JN. Comparative PRNP genotyping of U.S. cattle sires for potential association with BSE. Mammalian Genome. 2004;**15**(10):828–833.

[15] Sander P, Hamann H, Drögemüller C, Kashkevich K, Schiebel K, Leeb T. Bovine prion protein gene (*PRNP*) promoter polymorphisms modulate *PRNP* expression and may be responsible for differences in bovine spongiform encephalopathy susceptibility. Journal

[16] Juling K, Schwarzenbacher H, Williams JL, Fries R. A major genetic component of BSE

[17] Haase B, Doherr MG, Seuberlich T, Drögemüller C, Dolf G, Nicken P, Schiebel K, Ziegler U, Groschup MH, Zurbriggen A, Leeb T. *PRNP* promoter polymorphisms are associ‐ ated with BSE susceptibility in Swiss and German cattle. BMC Genetics. 2007;**8**:15.

[18] Kashkevich K, Humeny A, Ziegler U, Groschup MH, Nicken P, Leeb T, Fischer C, Becker CM, Schiebel K. Functional relevance of DNA polymorphisms within the promoter region of the prion protein gene and their association to BSE infection. FASEB Journal.

of Biological Chemistry 2005;**280**(45):37408–14. doi:10.1074/jbc. M506361200

susceptibility. BMC Biology. 2006;**4**:33. doi:10.1186/1741‐7007‐4‐33

and White cattle. Journal of Applied Genetics. 2003;**44:**191–195.

2004;**154**: 9–16.

170 Prion - An Overview

1996;**141**:809–824. doi:10.1007/BF01718157

777. doi:10.1007/s00335‐003‐2283‐y

doi:10.1007/s00335‐004‐2400‐6

doi:10.1186/1471‐2156‐8‐15

2007;**21**:1547–1555.

[45] Belt PB, Muileman IH, Schreuder BE, Bos‐de Ruijter J, Gielkens AL, Smits MA. Identification of five allelic variants of the sheep PrP gene and their association with natu‐ ral scrapie. Journal of General Virology 1995; **76**: 509–17. doi:10.1099/0022‐1317‐76‐3‐509

[31] M'Gowan JP. Investigation into the disease of sheep called "Scrapie" (Traberkrankheit [or] La Tremblante): with reference to its association with sarcosporidiosis. William

[32] Parry HB. Scrapie: a transmissible hereditary disease of sheep. Nature. 1960;**185**:441–43.

[33] Parry HB. Scrapie: a transmissible and hereditary disease of sheep. Heredity. 1962;

[34] Dickinson AG, Stamp JT, Renwick CC. Maternal and lateral transmission of scrapie in sheep. Journal of Comparative Pathology. 1974;**84**(1):19–25. doi:10.1016/0021‐9975(74)90023‐1

[35] Prusiner SB. Novel proteinaceous infectious particles cause scrapie. Science.

[36] Dickinson AG, Meikle VM, Fraser H. Identification of a gene which controls the incuba‐ tion period of some strains of scrapie agent in mice. Journal of Comparative Pathology.

[37] Foster J, Dickinson A. The unusual properties of CH1641, a sheep‐passaged isolate of

[38] Hope J, Hunter N. Scrapie‐associated fibrils, PrP protein and the sinc gene. Ciba

[39] Hunter N, Foster JD, Dickinson AG, Hope J. Linkage of the gene for the scrapie‐ associated fibril protein (PrP) to the sip gene in Cheviot Sheep. Veterinary Record.

[40] Goldmann W, Hunter N, Foster JD, Salbaum JM, Beyreuther K, Hope J. Two alleles of a neural protein gene linked to scrapie in sheep. Proceedings of the National Academy of

[41] Iannuzzi L, Palomba R, Di Meo GP, Perucatti A, Ferrara L. Comparative FISH‐Mapping of the prion protein gene (*PRNP*) on cattle, river buffalo, sheep and goat chromosomes.

[42] Clouscard C, Beaudry P, Elsen JM, Milan D, Dussaucy M, Bounneau C, Schelcher F, Chatelain J, Launay JM, Laplanche JL. Different allelic effects of the codons 136 and 171 of the prion protein gene in sheep with natural scrapie. Journal of General Virology.

[43] Laplanche JL, Chatelain J, Westaway D, Thomas S, Dussaucy M, Brugere‐Picoux J, Launay M. PrP polymorphisms associated with natural scrapie discovered by denaturing gradi‐

ent gel electrophoresis. Genomics. 1993;**15**(1):30–37. doi:10.1006/geno. 1993.1006

[44] Hunter N, Goldmann W, Benson G, Foster JD, Hope J. Swaledale sheep affected by natu‐ ral scrapie differ significantly in PrP genotype frequencies from healthy sheep and those selected for reduced incidence of scrapie. Journal of General Virology 1993;**74**:1025–31.

Blackwood & Sons; 1914. 116P.

1982;**216**(4542):136–44. doi:10.1126/science.6801762

1968;**78**(3):293–99. doi:10.1016/0021‐9975(68)90005‐4

Foundation Symposium. 1988;**135**:146–63.

1989;**124**(14):364–66.

1995;**76**:2097–2101.

doi:10.1099/0022‐1317‐74‐6‐1025

scrapie. Veterinary Record 1988;**123**:5–8. doi:10.1136/vr.123.1.5

Sciences of the United States of America. 1990;**87**(7):2476–80.

Cytogenetics and Cell Genetics. 1998;**81**:202–4. doi:10.1159/0000 15030

**17**:75–105.

172 Prion - An Overview

[69] Ianella P, McManus CM, Caetano AR, Paiva SR. *PRNP* Haplotype and genotype fre‐ quencies in Brazilian sheep: Issues for conservation and breeding programs. Research in Veterinary Science. 2012;**93**(1):219–25. doi:10.1016/j.rvsc.2011.06.025

[56] Thorgeirsdottir S, Sigurdarson S, Thorisson HM, Georgsson G, Palsdottir A. PrP gene polymorphism and natural scrapie in Icelandic Sheep. Journal of General Virology.

[57] Billinis C, Psychas V, Leontides L, Spyrou V, Argyroudis S, Vlemmas I, Leontides S, Sklaviadis T, Papadopoulos O. Prion protein gene polymorphisms in healthy and scrapie‐affected sheep in Greece. The Journal of General Virology. 2004;**85**:547–54.

[58] Harrington NP, O'Rourke KI, Feng Y, Rendulich J, Difruscio C, Balachandran A. Prion genotypes of scrapie‐infected Canadian Sheep 1998‐2008. Canadian Journal of Veterinary

[59] Gibbs CJ, Gajdusek DC. Transmission of scrapie to the cynomolgus monkey (*Macaca*

[60] Bruce M, Chree A, McConnell I, Foster J, Pearson G, Fraser H. Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences.

[61] Kimberlin RH, Walker CA. Evidence that the transmission of one source of scrapie agent to hamsters involves separation of agent strains from a mixture. Journal of General

[62] Bruce ME, Will RG, Ironside JW, McConnell I, Drummond D, Suttie A, McCardle L, Chree A, Hope J, Birkett C, Cousens S, Fraser H, Bostock CJ. Transmissions to mice indi‐ cate that 'new variant' CJD is caused by the BSE agent. Nature. 1997;**389**(6650):498–501.

[63] Nathanson N, Wilesmith J, Griot C, Bovine spongiform encephalopathy (BSE): Causes and consequences of a common source epidemic. American Journal of Epidemiology.

[64] Comoy EE, Mikol J, Luccantoni‐Freire S, Correia E, Lescoutra‐Etchegaray N, Durand V, Dehen C, Andreoletti O, Casalone C, Richt JA, Greenlee JJ, Baron T,.Benestad SL, Brown P, Deslys J‐P. 2015. Transmission of scrapie prions to primate after an extended silent

incubation period. Nature. 2015;**5**;11573 Scientific Reports doi:10.1038/srep11573

[66] Commission Decision, "Laying down minimum requirements for the establishment of breeding programmes for resistance to transmissible spongiform encephalopathies in

[67] EFSA. Scientific opinion on the scrapie situation in the EU after 10 years of monitoring and control in sheep and goats. EFSA Journal, 2014;**12**(7). doi:10.2903/j.efsa. 2014.3781

[68] Hunter N, Cairns D. Scrapie‐free merino and poll dorset sheep from Australia and New Zealand have normal frequencies of scrapie‐susceptible PrP genotypes. Journal of

[65] DEFRA. National Scrapie Plan for Great Britaint. 2001;1 (July):1–28.

General Virology. 1998;**79**:2079–82. doi:10.1099/0022‐1317‐79‐8‐2079

*fascicularis*). Nature. 1972;**236**(5341):73–74. doi:10.1038/236073a0

Virology. 1978;**39**(3):487–96. doi:10.1099/0022‐1317‐39‐3‐487

1994;**343**(1306):405–11. doi:10.1098/rstb.1994.0036

1997;**145**(11):959–69. doi:10.1093/aje/kwi188

sheep." Official Journal 2003/100/E:L41–45.

1999;**80**:2527–34.

174 Prion - An Overview

doi:10.1038/39057

doi:10.1099/vir.0.19520‐0

Research. 2010;**74**(3):228–232.

[92] Zhou RY, Li XL, Li LH, Wang HY, Lü JG. Polymorphism of the *PRNP* gene in the main breeds of indigenous Chinese goats. Archives of Virology. 2008;**153**:979–82. doi:10.1007/ s00705‐008‐0074‐1

[82] Vaccari G, Panagiotidis CH, Acin C, Peletto S, Barillet F, Acutis P, Bossers A, Langeveld J, van Keulen L, Sklaviadis T, Badiola JJ, Andreéoletti O, Groschup MH, Agrimi U, Foster J, Goldmann W. State‐of‐the‐art review of goat TSE in the European Union, with spe‐ cial emphasis on *PRNP* genetics and epidemiology. Veterinary Research. 2009;**40**(5):48.

[83] Vaccari G, Di Bari MA, Morelli L, Nonno R, Chiappini B, Antonucci G, Marcon S, Esposito E, Fazzi P, Palazzini N, Troiano P, Petrella A, Di Guardo G, Agrimi U. Identification of an allelic variant of the goat PrP gene associated with resistance to scrapie. Journal of

[84] Acutis PL, Colussi S, Santagada G, Laurenza C, Maniaci MG, Riina MV, Peletto S, Goldmann W, Bossers A, Caramelli M, Cristoferi I, Maione S, Sacchi P, Rasero R. Genetic variability of the *PRNP* gene in goat breeds from northern and southern Italy. Journal of

Applied Microbiology. 2008;**104**(6):1782–89. doi:10.1111/j.1365‐2672.2007.03703.x

[85] Goldmann W, Perucchini M, Smith A, Hunter N. Genetic variability of the PrP gene in a

[86] Goldmann W, Ryan K, Stewart P, Parnham D, Xicohtencatl R, Fernandez N, Saunders G, Windl O, González L, Bossers A, Foster J. Caprine prion gene polymorphisms are associ‐ ated with decreased incidence of classical scrapie in goat herds in the United Kingdom.

[87] Serrano C, Hammouchi M, Benomar A, Lyahyai J, Ranera B, Acín C, el Hamidi M, Monzón M, Badiola JJ, Tligui N, Zaragoza P, Martín‐Burriel I. *PRNP* haplotype distribution in Moroccan goats. Animal Genetics. 2009;**40**(4):565–68. doi:10.1111/j.1365‐2052.2009.01873.x

[88] Goldmann W, Martin T, Foster J, Hughes S, Smith G, Hughes K, Dawson M, Hunter N. Novel polymorphisms in the caprine PrP gene: A codon 142 mutation associated with scrapie incubation period. Journal of General Virology. 1996;**77**:2885–91. doi:10.1099/00

[89] Barillet F, Mariat D, Amigues Y, Faugeras R, Caillat H, Moazami‐Goudarzi K, Rupp R, Babilliot JM, Lacroux C, Lugan S, Schelcher F, Chartier C, Corbière F, Andréoletti O, Perrin‐Chauvineau C. Identification of seven haplotypes of the caprine PrP gene at codons 127, 142, 154, 211, 222 and 240 in French alpine and saanen breeds and their asso‐ ciation with classical scrapie. Journal of General Virology. 2009;**90**:769–76. doi:10.1099/

[90] White SN, Reynolds JO, Waldron DF. Extended scrapie incubation time in goats singly heterozygous for *PRNP* S146 or K222. Gene. 2012;**501**:49–51. doi:10.1016/j.

[91] Wopfner F, Weidenhöfer G, Schneider R, von Brunn A, Gilch S, Schwarz TF, Werner T, Schätzl HM. Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein. Journal of Molecular Biology. 1999;**289**(5):1163–78.

General Virology. 2006;**87**:1395–1402. doi:10.1099/vir.0.81485‐0

goat herd in the UK. Veterinary Record. 2004;**155**:177–178.

Veterinary Research. 2011;**42**:110. doi:10.1186/1297‐9716‐42‐110

doi:10.1051/vetres/2009031

176 Prion - An Overview

22‐1317‐77‐11‐2885

vir.0.006114‐0

gene.2012.03.068

doi:10.1006/jmbi.1999.2831

implications for transmissible spongiform encephalopathy surveillance. Journal of Veterinary Diagnostic Investigation. 2007;**19**(1):2–8. doi:10.1177/10406387070 1900102

[113] Will RG, Ironside JW, Zeidler M, Estibeiro, Cousens SN, Smith PG, Alperovitch A, Poser S, Pocchiari M, Hofman M. A new variant of Creutzfeldt‐Jakob disease in the UK. Lancet. 1996;**347**(9006):921–25. doi:10.5555/uri:pii:S0140673696914129

implications for transmissible spongiform encephalopathy surveillance. Journal of Veterinary Diagnostic Investigation. 2007;**19**(1):2–8. doi:10.1177/10406387070 1900102

[103] Colussi S, Vaccari G, Maurella C, Bona C, Lorenzetti R, Troiano P, Casalinuovo F, Di Sarno A, Maniaci MG, Zuccon F, Nonno R, Casalone C, Mazza M, Ru G, Caramelli M, Agrimi U, Acutis PL. Histidine at codon 154 of the prion protein gene is a risk factor for Nor98 scrapie in goats. Journal of General Virology 2008;**89**:3173–76. doi:10.1099/

[104] Mitchell GB, O'Rourke KI, Harrington NP, Soutyrine A, Simmons MM, Dudas S, Zhuang D, Laude H, Balachandran A. Identification of atypical scrapie in Canadian Sheep. Journal of Veterinary Diagnostic Investigation. 2010;**22**(3):408–11.

[105] Kittelberger R, Chaplin MJ, Simmons MM, Ramirez‐Villaescusa A, McIntyre L, MacDiarmid SC, Hannah MJ, Jenner J, Bueno R, Bayliss D, Black H, Pigott CJ, O'Keefe JS. Atypical scrapie/Nor98 in a sheep from New Zealand. Journal of Veterinary Diagnostic

[106] Moum T, Olsaker I, Hopp P, Moldal T, Valheim M, Moum T, Benestad SL. Polymorphisms at codons 141 and 154 in the ovine prion protein gene are associated with scrapie Nor98

cases. Journal of General Virology. 2005;**86**(1):231–35. doi:10.1099/vir.0.80437‐0 [107] Lühken G1, Buschmann A, Brandt H, Eiden M, Groschup MH, Erhardt G. Epidemiological and genetical differences between classical and atypical scrapie cases.

[108] Moreno CR, Moazami‐Goudarzi K, Laurent P, Cazeau G, Andreoletti O, Chadi S, Elsen JM, Calavas D. Which prp haplotypes in a French sheep population are the most sus‐ ceptible to atypical scrapie? Archives of Virology. 2007;**152**(6):1229–32. doi:10.1007/

[109] Arsac JN, Andreoletti O, Bilheude J‐M, Lacroux C, Benestad SL, Taron T. Similar Biochemical signatures and prion protein genotypes in atypical scrapie and Nor98 cases, France and Norway. Emerging Infectious Diseases. 2007;**13**(1):58–65. doi:10.3201/

[110] Wilesmith JW. Bovine Spongiform encephalopathy and related Diseases: An epi‐ demiological overview. New Zealand Veterinary Journal. 1994;**42**(1):1–8. doi:

[111] Ghani AC, Donnelly CA, Ferguson NM, Anderson RM. The transmission dynam‐ ics of BSE and vCJD. Comptes Rendus Biologies. 2002;**325**(1):37–47. doi:10.1016/

[112] WHO "World Health Organization." World Health Organization WHO/CDS/CS (WHO Consultation on public health and animal TSEs epidemiology, risk and research

Investigation. 2010:**22**(6):863–75. doi:10.1177/10406387100220060

Veterinary Research. 2007;**38**(1):65–80. doi:10.1006/geno.1993.1006

vir.0.2008/004150‐0

178 Prion - An Overview

s00705‐007‐0956‐7

eid1301.060393

10.1080/00480169.1994.35774

S1631‐0691(02)01389‐6

requirements: 2000;1–47.

doi:10.1177/104063871002200310

[138] Oztabak K, Ozkan E, Soysal I, Paya I, Un C. Detection of prion gene promoter and intron1 indel polymorphisms in Anatolian water buffalo (*Bubalus bubalis*). Journal of Animal Breeding and Genetics. 2009;**126**(6):463–67. doi:10.1111/j.1439‐0388.2009.00821.x

[126] Biacabe AG, Laplanche JL, Ryder S, Baron T. Distinct molecular phenotypes in bovine prion diseases. EMBO Reports. 2004;**5**;110–15. doi:10.1038/sj.embor.7400054

[127] Casalone C, Zanusso G, Acutis P, Ferrari S, Capucci L, Tagliavini F, Monaco S, Caramelli M. Identification of a second bovine amyloidotic spongiform encephalopa‐ thy: Molecular similarities with sporadic Creutzfeldt‐Jakob disease. Proceedings of the National Academy of Sciences of the United States of America. 2004;**101**(9):3065–70.

[128] Heaton MP, Keele JW, Harhay GP, Richt JA, Koohmaraie M, Wheeler TL, Shackelford SD, Casas E, King DA, Sonstegard TS, Van Tassell CP, Neibergs HL, Chase CC Jr, Kalbfleisch TS, Smith TP, Clawson ML, Laegreid WW. Prevalence of the prion protein gene E211K variant in U.S. Cattle. BMC Veterinary Research. 2008;**4**:25.

[129] Richt JA, Mark Hall S. BSE with case associated prion protein gene mutation. PLoS

[130] Nicholson EM, Brunelle BW, Richt JA, Kehrli ME Jr, Greenlee JJ. Identification of a heri‐ table polymorphism in bovine *PRNP* associated with genetic transmissible spongiform encephalopathy: Evidence of heritable BSE. PloS One. 2008;**3**(8):e2912. doi:10.1371/

[131] Clawson ML, Richt JA, Baron T, Biacabe AG, Czub S, Heaton MP, Smith TP, Laegreid WW. Association of a bovine prion gene haplotype with atypical BSE. PloS One.

[132] Gurgul A, Polak MP, Larska M, Słota E. *PRNP* and SPRN genes polymorphism in atyp‐ ical bovine spongiform encephalopathy cases diagnosed in Polish Cattle. Journal of

[133] Greenlee JJ, Smith JD, West Greenlee MH, Nicholson EM. Clinical and pathologic fea‐ tures of H‐type bovine spongiform encephalopathy associated with e211k prion pro‐ tein polymorphism. PloS One. 2012;**7**(6):e38678. doi:10.1371/journal.pone. 0038678 [134] Anonymous. Agriculture statistics at regional level. [Internet]. 2015. http://ec.europa. eu/eurostat/statistics‐explained/index.php/Agriculture\_statistics\_at\_ regional\_level#

[135] Ru G, Maurella C, Maroni Ponti A, Ingravalle F, Caramelli M. Epidemiological study of the decline of BSE in Italy. Veterinary Record. 2007;**161**(15):511–14. doi:10.1136/

[136] Morley RS, Chen S, Rheault N. Assessment of the risk factors related to bovine spongi‐ form encephalopathy. Revue scientifique et Technique. 2003;**22**(1):157–78.

[137] Di Guardo G. BSE in buffaloes. The Veterinary Record. 2014;174(19):485. doi:10.1136/

Applied Genetics. 2012;**53**(3):337–42. doi:10.1007/s13353‐012‐0102‐4

Further\_Eurostat\_information [Accessed: 21.02.2015].

Pathogens. 2008;**4**(9):e1000156. doi:10.1371/journal.ppat.1000156

doi:10.1073/pnas.0305777101