The role of prion strain diversity in the development of successful therapeutic treatments

Abstract

Prions are a self-propagating misfolded conformation of a cellular protein. Prions are found in several eukaryotic organisms with mammalian prion diseases encompassing a wide range of disorders. The first recognized prion disease, the transmissible spongiform encephalopathies (TSEs), affect several species including humans. Alzheimer’s disease, synucleinopathies, and tauopathies share a similar mechanism of self-propagation of the prion form of the disease-specific protein reminiscent of the infection process of TSEs. Strain diversity in prion disease is characterized by differences in the phenotype of disease that is hypothesized to be encoded by strain-specific conformations of the prion form of the disease-specific protein. Prion therapeutics that target the prion form of the disease-specific protein can lead to the emergence of drug-resistant strains of prions, consistent with the hypothesis that prion strains exist as a dynamic mixture of a dominant strain in combination with minor substrains. To overcome this obstacle, therapies that reduce or eliminate the template of conversion are efficacious, may reverse neuropathology, and do not result in the emergence of drug resistance. Recent advancements in preclinical diagnosis of prion infection may allow for a combinational approach that treats the prion form and the precursor protein to effectively treat prion diseases.

1. Prion diseases

Prions are a self-propagating misfolded conformation of a cellular protein. The conversion of the normal isoform to the misfolded prion form occurs in a stepwise manner, but the exact mechanism is unknown.^1–4 Prions are found in several eukaryotic organisms. Yeast prions are a dominant, non-Mendelian form of epigenetic inheritance that causes a detectable phenotype. For example, the [URE3], [SWI+], and [GAR+] prions in Saccharomyces cerevisiae enhance growth on nutrient-poor sources, while the [PSI+] prion enables translational readthrough.^5–9 The prion state in yeast is dependent on a chromosomal gene and transient overproduction of the normal cellular protein promotes the prion state which can be reversibly cured.^8,10,11 The [Het-s] prion of Podospora anserina is infectious and has a necessary role in heterokaryon incompatibility, a process that restricts fusion of hyphae to genetically similar partners.^12–14 Prions found in yeast, fungi, and mammals can have advantageous roles. Functional prion-like proteins involved in memory, RNA metabolism, and immunity have been identified.^15–19 However, prions in mammals are most notably associated with neurodegenerative diseases.

Mammalian prion diseases encompass a wide range of disorders. The first recognized prion disease, the transmissible spongiform encephalopathies (TSEs), are characterized by long incubation periods followed by a relatively short duration of clinical signs that ultimately leads to death of the host.^1,20 TSEs occur in both domesticated populations of sheep and goats (scrapie), cattle (bovine spongiform encephalopathy, BSE), and mink (transmissible mink encephalopathy, TME). Chronic wasting disease (CWD) is unique among TSEs as it can affect both wild and domestic populations of cervids.^21–23 Camel prion disease (CPD) in domesticated dromedary camels was recently identified, but the geographic distribution and potential of transmission to wild populations is unknown.²⁴ Human TSEs can have infectious (Kuru), inherited (Gerstmann-Sträussler-Scheinker, GSS), or sporadic (Creutzfeldt-Jakob disease, CJD) etiologies, with sporadic being the most prevalent.²⁵ Iatrogenic transmission of PrP prions can occur via cadaveric human growth hormone (c-hGH), dura mater grafts, or corneal transplants.^26–29 TSE agent replication involves the misfolded infectious prion conformation of the prion protein, PrP^Sc, binding to the normal, cellular conformation, PrP^C, initiating further seeded conversion.

Alzheimer’s disease, synucleinopathies, and tauopathies are characterized by the misfolding and aggregation of amyloid-β (Aβ; a peptide derived from the host cellular amyloid precursor protein (APP)), α-synuclein protein (α-syn), and tau protein, respectively. Similar to TSEs, these protein misfolding diseases share a similar mechanism of self-propagation of the prion form of the disease-specific protein and cell to cell spreading of the host protein in the prion state.^30–39 Experimental injection of the prion form of the protein into animals that express the respective precursor protein can accelerate the pathogenesis of disease, reminiscent of the infection process of TSEs (or PrP prions).^34,40–46 Although these non-prion protein disorders have both inherited and sporadic etiologies, conflicting evidence for infectious transmission has been reported. Epidemiological studies concluded that a history of one or more blood transfusions was not associated with a higher risk of developing AD compared to age- and gender-matched controls for each patient case.^47,48 Injection of nonhuman primates with over 600 patient samples from individuals diagnosed with nonspongiform diseases (i.e., AD, PD, etc.) failed to transmit disease.⁴⁹ No cases of AD or PD were identified among recipients of c-hGH in the United States despite finding mild amounts of pathological Aβ, tau, and α-synuclein by immunohistochemical analysis of pituitary glands from neurodegenerative disease and control patients.⁵⁰ However, a recent study reported that recipients of c-hGH contaminated with PrP prions subsequently developed CJD with concurrent Aβ deposition in the brain, suggesting iatrogenic transmission of Aβ.⁵¹ Retrospective studies on the archived c-hGH samples identified significant amounts of tau, Aβ₄₂ and Aβ₄₀.⁵² Intracerebral injection of these c-hGH samples to transgenic mice expressing a human mutant of the amyloid precursor protein resulted in the development of brain pathology similar to the patients injected with the same material.⁵² This finding suggests, under certain conditions, iatrogenic transmission of AD is possible. Overall, the aforementioned neurodegenerative disorders share profound similarities at the biochemical and cellular level.

2. Prion strain diversity

Prion strains are operationally defined by heritable differences in the biological properties of disease under controlled agent and host parameters.^53–56 In yeast, prion strains are classified based on several traits that differ depending on the prion state (i.e., [PSI+] or [URE3]) but were initially distinguished by strength of phenotype (i.e., “strong” versus “weak”).^57–59 TSE strains are classified by differences in incubation period and neuropathology but can differ in their clinical signs and biochemical features of PrP^Sc.^60,61 Similar to TSEs, Aβ, tau, and ɑ-synuclein strains are defined by differences in abnormal protein deposition and pathology in the brain as well as protein conformation or seeding activity of the prion form of the respective protein.^{38,44,62–64} The relationship between the strain-specific biochemical features of the prion form of the protein and the phenotype of disease is poorly understood.

2.1. Prion strain diversity in yeast

Studies of yeast prion strain diversity have added much to the knowledge of prion strain diversity. Yeast prion strains differ in strength of phenotype, stability of propagation, toxicity/lethality, ability to overcome inter- or intraspecies transmission barriers, and biochemical and physical properties.^{57–59,65–67} Nuclear magnetic resonance (NMR) spectroscopy identified conformational differences between [PSI+] yeast prion strains, and atomic force microscopy (AFM) reveals strain-specific differences in yeast prion fibril morphology.⁶⁸ There are strong correlations between biochemical properties of the prion form of the protein and strain phenotype in yeast. The strength of the yeast prion phenotype corresponds with an increased fragility of protein aggregates.⁶⁹ Mechanistically, increased fragility of prion aggregates results in rapid generation of new free ends, accelerating prion formation (Fig. 1, step 5).⁶⁹

Fig. 1.

Mechanistic location of therapeutic targets for PrP prion diseases. Therapeutic targets: ❶ PrP^C post-translational processing, metabolism, cellular trafficking, or localization; ❷ PrP^C by binding or inducing conformational change that prevents PrP^C interaction; ❸ PrP^Sc by stabilization, redistribution; ❹ conversion process by blocking binding site on PrP^C, capping growth, inhibition of cofactor interactions, or ❺ promoting fragmentation or ❻ clearance from the host.

2.2. TSE strain diversity

Strain diversity is hypothesized to be encoded by strain-specific conformations of PrP^Sc.^{63,64,68,70–72} Strain-specific differences in the biochemical properties of PrP^Sc were first identified in mice.⁷³ These differences were later observed in hamsters infected with the hyper (HY) or drowsy (DY) strains of hamster-adapted transmissible mink encephalopathy (TME).⁷⁴ Detergent extraction and protease digestion of HY and DY PrP^Sc revealed strain-specific Western blot migration profiles suggesting strain-specific differences in the PK digestion site on PrP^Sc.⁷⁵ Edman protein sequencing of PrP^Sc from HY or DY TME-infected hamsters confirmed that PrP^Sc from these two strains had different PK cleavage sites.⁷⁰ Based on these observations, the authors proposed the hypothesis that strain-specific conformations of PrP^Sc encoded prion strain diversity.^70,75,76 Consistent with this hypothesis, the molecular weight of the proteinase K-resistant fragment of PrP^Sc after deglycosylation in fatal familial insomnia is 19 kDa while the same fragment was 21 kDa in sporadic and familial Creutzfeldt Jakob disease.⁷² When inoculated into mice, the strain-specific migration pattern of PrP^Sc from human prion strains was preserved supporting the hypothesis that strain-specific conformations of PrP^Sc is the basis of strain diversity.⁷²

The presence of cellular cofactors during prion conversion may be responsible for the maintenance of strain-specific, infectious conformations of PrP^Sc. Development of synthetic, in vitro-generated prions able to successfully infect wild-type rodents relied on cofactors such as polyanions and lipids.^77,78 Propagation of a recombinant prion strain in PMCA in the presence of both recombinant murine PrP and the lipid phosphatidylethanolamine (PE) retained infectivity.⁷⁹ Withdrawal of the cofactor (PE) led to the emergence of a protein-only strain, named OSU, that did not cause disease in mice suggesting cellular cofactors are required for infectivity. Additionally, in vitro PMCA propagation of three distinct prion strains with PE as the sole cofactor resulted in the three strains evolving into a single strain.⁷⁹ This work on the directed evolution of prions suggests that cellular cofactors are involved in prion strain diversity. Recent work has clarified the role of prion cofactors. Using a novel in vitro system where protein only PrP^Sc preparations, in the absence of PMCA adaptation, are directly infectious to animals, but only in the presence of co-factors.⁸⁰ Importantly, protein-only and cofactor generated PrP^Sc had identical strain properties indicating that the conformation of PrP^Sc is sufficient to encode prion strain information and that cellular co-factors are needed for prion propagation.⁸⁰ Overall, these data provide a unified model of prion infectivity where strain information is encoded by the structure of PrP^Sc and host cellular cofactors are necessary for prion conversion and subsequent infectivity.

Lesion profiles, cellular assays, and the biochemical properties of PrP^Sc are used to differentiate prion strains. The scrapie cell assay (SCA) and the cell panel assay (CPA) assess the relative susceptibility or resistance of murine neuroblastoma cell lines to infection by different prion strains.^81–83 The Western blot migration profile, conformational stability, and sensitivity to proteolytic digestion of PrP^Sc can be strain specific. It is unclear which, if any, of the strain-specific biochemical differences of PrP^Sc directly contributes the strain-specific phenotype of disease. For example, in murine PrP prions, decreased PrP^Sc conformational stability is positively correlated with a shorter incubation period, however, in humans and hamsters the inverse relationship between PrP^Sc conformational stability and incubation period was identified.^71,84–87

2.3. Aβ strain diversity

The heterogeneous clinical presentation of AD may be attributed to strain diversity.⁸⁸ The Arctic and Swedish APP mutations in humans result in distinct AD pathologies.^89–92 It was hypothesized that the two AD clinical disease outcomes were a result of two distinct Aβ strains. Conformational stability studies of Aβ from patient brains with Arctic AD and Swedish AD mutations established that aggregates of Aβ found in the Arctic AD patients were significantly more sensitive to denaturation with guanidine hydrochloride compared to Swedish AD.⁴⁴ Further, injecting transgenic mice expressing human APP containing the Swedish mutation with the Arctic or Swedish AD brain material resulted in two distinct phenotypes of disease that were maintained upon serial passage consistent with the predicted behavior of prion strains.⁴⁴ Luminescent conjugated oligothiophenes, a class of amyloid binding dyes, to investigate how the Aβ amyloid structure in familial AD differs from sporadic AD.⁹³ The emission spectra of luminescent conjugated oligothiophenes changes when bound to amyloid of differing structure. The emission spectra of luminescent conjugated oligothiophenes bound to Aβ some of the subtypes of familial and sporadic AD varied significantly, suggesting a variety of Aβ structures exist in different AD cases.⁹³ Solid state nuclear magnetic resonance imaging analysis of Aβ from patient tissue samples from PCA-AD or rp-AD indicated that Aβ from PCA-AD resembled typical AD with predominant Aβ40 fibril structure while rp-AD samples were composed of a cloud of variant Aβ40 structures.⁹⁴ Overall, this data is consistent with the hypothesis that Aβ structural variance contribute to the clinical and pathological heterogeneity of AD.

2.4. Tau strain diversity

Alzheimer’s disease (AD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), Pick’s disease (PiD), and argyrophilic grain disease (AGD) are unique tauopathies defined by the pattern of tau deposition in the brain along with distinct clinical phenotypes.⁹⁵ Transmission studies in wild-type and transgenic mouse models have established that tau extracts from various tauopathies transmit distinct tau neuropathology.^{38,64,96–98} For example, AD-tau variants can differentially seed tau pathology after intracranial injection of wild-type mice; the tau variants were determined to be structurally different by trypsin digests that resulted in three distinct trypsin resistant fragments.³⁸ Injecting wild-type mice with tau from AD, CBD, or PSP resulted in differences in tau conversion efficiency and cellular tropism in the CNS suggesting the three tau isolates are distinct prion strains.⁶⁴ Consistent with this hypothesis, tau fibrils from the tau strains showed varying resistance to Gdn-HCl denaturation with CBD-tau, AD-tau, and PSP-tau having the lowest to highest stability, respectively.⁶⁴ Two recombinant tau strains in HEK293 cells expressing the tau repeat domain that propagated conformationally distinct forms of tau that could be serially passaged in PS19 transgenic tau mice that retained the strain-specific conformation of tau.⁹⁹ Further, the HEK293 cell model was used to examine AD, CBD, and AGD patient samples, and each tauopathy induced unique tau inclusion morphology.⁹⁹ Consistent with this finding, 18 tau strains were identified based on differences in limited proteolysis and in vitro seeding activity of tau in combination with hippocampal tau pathology when transmitted to PS19 transgenic tau mice.¹⁰⁰ Taken together, these data support that tau structural variance may explain the distinct pathologies and clinical presentation of the tauopathies.

2.5. ɑ-synuclein strain diversity

Strain-specific prion forms of α-synuclein have been identified in synucleinopathies including Parkinson’s disease (PD), dementia with Lewy bodies, and multiple system atrophy (MSA). Synthetic α-synuclein fibrils generated under varying salt concentrations form distinct conformations with differences in seeding ability and toxicity in vivo and in vitro.^62,101,102 Recombinant fibrillar α-synuclein created under different laboratory conditions varied in ability to cross-seed tau aggregation in cell culture and in vivo.⁶³ Consistent with this observation, proteinase K digestion revealed differences in protein cleavage between the synthetic α-synuclein as well as two distinct α-synuclein digestion products isolated from Parkinson’s disease with dementia (PDD) patient brains.⁶³ Distinct digestion products are suggestive of two α-synuclein conformations reminiscent of prior observations in PrP prions.^{72,75,103–105} In vitro seeding assays utilizing a cell line that readily propagates MSA α-synuclein was not susceptible to α-synuclein from PD patient brains suggesting that PD contains a different strain of α-synuclein.¹⁰⁶ Brain material from PD and MSA patients has distinct seeding activity and cellular inclusion pathology in HEK293T cells, suggestive of distinct strain of α-synuclein.¹⁰⁷ Overall, α-synuclein strains share many features of PrP prion strains that include differences in biochemical features, neuropathology, incubation period, and clinical manifestation of disease.^38,62,63

3. Prion strains exist as a mixture

Multiple prion strains can co-exist in an individual host. In cases of sporadic Creutzfeldt-Jakob disease (sCJD), the polymorphism at codon 129 and different proteinase K cleavage patterns via Western blot classify disease as Type 1 (21 kDa) or 2 sCJD (19 kDa).¹⁰³ Type 1 and Type 2 sCJD PrP^Sc can co-exist in the CNS of the same patient.^108–116 It is suggested that up to 40% of sCJD cases contain more than one strain, however, the prevalence rate is controversial since methodological differences in strain typing and the region of the brain that is evaluated can both affect the outcome.^108–116 It is not well understood if strains of Aβ can co-exist in a single host, however, distinct strains of Aβ have been identified raising the possibility that sporadic AD may be a mixture of strains.⁴⁴ Mapping phospho-tau patterns of deposition in the hippocampal regions of individuals with various tauopathies established that the pattern of pathology is disease-specific with regional selectivity reminiscent of strain targeting in TSE’s.¹¹⁷ This same study determined that AD-related tau hippocampal deposition is greatly influenced by simultaneous argyrophilic grain disease (AGD), another tauopathy, suggesting that coexisting tau strains can create heterogeneous brain pathologies and overlapping clinical features highlighting the need for discriminatory methods of post-mortem diagnosis.¹¹⁷ Antibodies that distinguish AD tau from non-AD tau deposits of CBD, PSP, and Pick’s Disease (PiD) can discern the differential deposition in co-occurring pathologies.¹¹⁸ While ample experimental evidence suggests that different α-synucleinopathy strains exist, the coexistence of α-synucleinopathy strains has not been described.

Coexisting prion strains can interfere with each other. Strain interference is a phenomenon in which a slowly-converting blocking strain can delay or prevent the emergence of a quickly-converting strain. A number of parameters govern prion strain interference. Increasing the titer of the blocking strain results in a corresponding increase in the interference effect.^119–122 Importantly, prion conversion in a common population of cells is required for interference to occur as the strains compete for a limiting cellular resource, PrP^C.^123,124 Consistent with this hypothesis, co-infection of two low conversion efficiency prion strains results in each strain converting independently without interference that is due to PrP^C not becoming limiting.¹²⁵ Overall, strain interference is an important parameter in the emergence of a dominant strain from a mixture.

Currently, strain interference has not been reported for mammalian non-PrP prions, and further investigation is required to determine if this phenomenon is a common property of mammalian prions. In yeast, there is evidence that strain variants compete within a cell during and after mating, with “strong” variants outcompeting “weak” variants for the template of conversion.¹²⁶ Mechanistically, “weak” variants are characterized by quick growth but low frangibility, whereas “strong” variants grow slowly but are more easily fragmented, leading to increased free ends for growth and therefore a competitive advantage over “weak” variants.⁶⁹

4. Prion therapeutics

Therapeutics that target prion conversion can work via several mechanisms. Drugs can target PrP^C prior to its interaction with PrP^Sc by affecting the post-translational processing, metabolism, cellular trafficking, or localization of PrP^C (Fig. 1, step 1). Drugs that bind to or induce a conformational change in PrP^C can hinder the interaction between PrP^C and PrP^Sc (Fig. 1, step 2). Drugs can target PrP^Sc by stabilizing or redistributing PrP^Sc, interfering with the interaction between PrP^C and PrP^Sc (Fig. 1, step 3). The conversion of PrP^C to PrP^Sc can be targeted by drugs that block the PrP^Sc binding site on PrP^C, cap PrP^Sc aggregate growth, or inhibit cofactors that enable conversion (Fig. 1, step 4). Therapeutics that target clearance of PrP^Sc either increase fragmentation of PrP^Sc aggregates (Fig. 1, step 5) or promote clearance of PrP^Sc from cells (Fig. 1, step 6).

4.1. Treatments that target PrP^Sc

Several drugs that directly interact with PrP^Sc have anti-prion effects (Table 1 and Fig. 1). Congo red, an amyloid binding dye, was one of the first compounds identified to extend the incubation period of prion-infected animals and can reduce the formation of PrP^Sc in scrapie-infected mouse neuroblastoma cells (Sc⁺ MNB).¹²⁷ Congo red is hypothesized to directly bind and over-stabilize PrP^Sc, preventing further prion formation.^127–129 Oral administration of Compound B, an amyloidophilic compound, extends the incubation period of TSEs and is hypothesized to inhibit formation of PrP^Sc by directly binding PrP^Sc or by interacting with cofactors/chaperones necessary for prion conversion.^134,135 The diphenyl-pyrazole anle138b inhibits PrP^Sc amplification and reduces neurotoxicity by shifting PrP^Sc oligomers to a smaller size by reducing intermolecular hydrogen bonding and obstructing the formation of higher order oligomers, modulating the formation of toxic PrP^Sc aggregates.^136,137 Anle138b has anti-prion activity both in vitro and in vivo and substantially prolonged survival even when treatment began after onset of signs of prion disease.¹³⁷

Table 1.

Anti-prion therapeutics.

Drug	Type	Model	Efficacy	Suggested mechanism	Strain Specificity	References
PrPsc target
Congo Red	Sulfonated amyloid dye	Neuronal cell culture, hamster	Decreases PrPSc accumulation and inhibits propagation in culture, prolongs mean incubation period in vivo	Stabilizes PrPSc, inhibits GAG-PrP binding and potentially PrPC-PrPSc binding	127S, Sc237, PrP (27–30), 263K, 139H	127–131
LIN5001, LIN7002, LIN5044	Luminescent conjugated polythiophenes (LCPs)	Mouse	IV infusion prolongs survival, reduces infectivity	Aggregate hyperstabilization	RML6, 263K	132,133
Compound B	4-pyridinecarboxaldehyde, 2-[4-(5-oxazolyl)phenyl] hydrazone	Neuronal cell culture, mouse	Eliminates PrPSc in culture; prolongs incubation period/life, reduces PrP deposition, large quantity needed for efficacy	Binds to abnormal PrP, inhibits new formation of abnormal PrP	RML, 22L (marginally), Fukuoka-1 GSS, 263K (barely)	134,135
anle138b	3,5-diphenyl-pyrazole (DPP)	Neuronal cell culture, mouse	Antiprion activity in vitro and in vivo, substantial prolongation of survival (txt after clinical onset), good bioavailability, BBB penetration	Directly blocks PrPSc amplification, causes shift towards smaller PrPSc oligomer size, inhibits pathological aggregation (PrPSc specific)	RML, ME7, 301c, sCJD, vCJD, a-syn	136–138
U18666A	Cholesterol synthesis inhibitor	Neuronal cell culture	Significantly decreased PrP-res levels	Redistributes PrPSc to late endosome-lysosome, degradation of PrPSc in secondary lysosomes	22L	139
Pentosan Polysulfate (PPS)	Cysteine protease inhibitor	Neuronal cell culture, mouse, human	IV infusion prolonged survival even when administered after abnormal PrP deposition, rapidly and significantly decreased PrPSc levels, various efficacy in humans	May act as a competitive coreceptor (with endogenous GAG or other proteoglycans) for PrP at cell surface or causes fragmentation of PrPSc at the cell surface	263K, RML, Fukuoka-1 GSS, 22L, iCJD, vCJD	139–147
Heterologous protein	Hamster PrP	Neuronal cell culture, mouse	Decreases PrP-res in culture, slows disease progression, increases survival, significantly delays onset of clinical signs, decreases PrPSc accumulation in brain/spleen	Either binds to PrPSc creating a functionally impotent aggregate unable to produce additional PrPSc or binds and blocks the conversion site from PrPC	RML-Chandler	148–151
IND24, IND81, IND114338, IND125, IND126461	2-aminothiazoles	Neuronal cell culture, mouse	Strain-specific and administration time-dependent extension in survival, decreased & altered distribution of PrPSc in brain, can lead to drug resistance (e.g. IND24-resistant RML)	Inhibits formation of new PrPSc, reduces PrPSc load via drug-like mechanism, might promote clearance of PrPSc	RML, ME7, 22L, CWD	138,152–156
Amphotericin B	Fungizone (polyene antibiotic)	Neuronal cell culture, mouse, hamster, human	Administration time-dependent prolongation of incubation period, delays onset of clinical signs without improvement of neurological symptoms or prolongation of clinical disease, delays accumulation ofPrPSc in the brain	Potentially could: interact with PrPC/PrPSc, interfere with uptake of PrPSc into cells, target detergent-resistant microdomains (modify cell surface distribution and/or trafficking of PrP), regulate microglial/glial activation, directly bind and either “cap” amyloid growth or overstabilize PrPSc	263K, CJD	140,157–164
PrPC Target
771, active ASO 1, active ASO 2	Antisense oligonucleotide	Neuronal cell culture, mouse	Reduces PrPC/PrPSc in cell culture and in vivo, delays onset of clinical signs, slows disease progression, toxicity issues when administered after prion inoculation	Decreased PrPC levels slow accumulation of PrPSc	RML, potentially more since PrPC is the target	165,166
J1, J8, J20, J35	Chalcones	Neuronal cell culture	Decreases PrPSc levels, completely blocks PrP109–149 aggregation	Directly binds to PrPC (C-terminal), decreases amount of PrPC on cell surface (arrest in ER), may stabilize PrPC or bind seeding surface	RML, 22L, CWD, 263K, CJD	167,168
Y13, Y17	Oxadiazole	Neuronal cell culture	Could not totally block conversion but Increased lag phase for conversion; minor decrease in PrP-res formed in RT-QuIC	Direct binding to PrPC (stabilize PrPC or bind at seeding surface), potentially interacts with PrPC/PrPSc N-terminal region, decrease amount of PrPC on cell surface causing arrest in ER	RML, 263K, CJD	167,168
Fe(III)-TMPyP	Cationic tetrapyrrole	Neuronal cell culture, mouse	Inhibits replication in vitro/cell culture, prolongs survival, inhibits amplification of PrPSc, inhibits cytotoxic effects of delta CR PrP	Interacts with C-terminal domain of PrPC (Helix-3), acts as pharmacological chaperone for PrPC (decreases prion-induced misfolding)	RML, 22L, bank vole strains (Italian, UK (SCR1), and CH1641)	169,170
D18	Anti-recombinant PrP (29–231) antibody (residues 132–156; helix A) Fabs	Neuronal cell culture, mouse	Abolishes prion replication and clears existing PrPSc in cell culture (2 week txt), prolongs incubation period and decreased titer in mice	Binds to PrPC on cell surface and hinders docking of PrPSc/a cofactor needed for conversion	Scrapie	171
6H4	Anti-murine PrPC antibody (Mab and expressed as transgene)	Neuronal cell	Prevents infection of susceptible cells, cures chronically infected cultures, conferred anti-PrP titers (Prnp0/0 mice) w/o induction of autoimmune disease, no infectivity detected (Prnp+/0-6H4u mice) when challenged, prevents or drastically delays pathogenesis	Occludes PrPC at prion replication sites, captures and degrades (immune-mediated) incoming PrPSc inoculum, steric competition with template-directed refolding, interference with seeded PrPSc nucleation reaction	RML	172,173
ICSM 18	Isotype IgG1, anti-murine alpha-PrP antibody (residues 146–159)	Neuronal cell culture, mouse	Reduces splenic PrPSc levels, increases survival, passive transfer of Abs had no effect late in incubation period	Direct inhibition of PrPSc production, binding may bury “active residues” and stabilize helix 1	RML	174,175
110	Monoclonal anti-PrP antibody (PHGGGWG at aa 59–65 and aa 83–89; octarepeat region)	Neuronal cell culture	Reduced PrPSc accumulation (dose-dependent) in cell culture, long term txt increased PrPC levels	Retains PrPC on cell surface (may interfere with PrPC metabolism), mAb-PrPC binding inhibits PrPC-PrPSc interaction by occupying binding domain or through steric interference	Chandler (139A), RML	176
6D11	Monoclonal antibody, recognizes PrPC, PrPSc, and recPrP (epitope between 93–109)	Neuronal cell culture	Complete abrogation of PrPSc, no re-emergence for at least 14 days, (following removal of txt), pre-incubation prevents infection	May prevent PrPC-PrPSc interaction or interfere with binding auxiliary molecules needed for prion propagation	22L	177
7H6	Monoclonal Ab, recognizes residues 130–140	Neuronal cell culture	Complete abrogation of PrPSc, no re-emergence for at least 14 days, (following removal of txt)	Similar to 6D11?	22L	177
7A12	Monoclonal Ab, recognizes residues 143–155 (alpha-helix A)	Neuronal cell culture	Complete abrogation of PrPSc, no re-emergence for at least 14 days, (following removal of txt), weakest preventative effect	Similar to 6D11?	22L	177
SAF34	Monoclonal Ab, recognizes residues 59–89 (HuPrP)	Neuronal cell culture	Dose-dependent decrease of PrPSc levels, increased inhibition when used in conjunction with SAF61, re-emergence of PrPSc following removal of txt	Decreases half-life of PrPC, antibodies act on PrPC or PrPSc isoforms, antibodies prevent PrPC-PrPSc interaction	22L	178
SAF61	Monoclonal Ab, recognizes residues 144–152 (HuPrP)	Neuronal cell culture	Dose-dependent decrease of PrPSc levels, increased inhibition of PrPSc when used in conjunction with SAF34, no re-emergence ofPrPSc following removal oftxt	Decreases half-life of PrPC, antibodies act on PrPC or PrPSc isoforms, antibodies prevent PrPC-PrPSc interaction, increase clearance/degradation of PrPC, modulate cellular trafficking of PrP, induce conformational change exposing PrP to protease attacks	22L	178
PrioV3	Camelid anti-PrP antibody (residues between 171 and 190, YYR motif)	Neuronal cell culture, endo-thelial cells, mouse	Crosses BBB in vitro and in vivo, abrogates PrPSc replication in cells, permanent depletion of PrPSc in cells, marked inhibition ofPrPSc accumulation in spleen	Alters PrPC expression (direct neutralizing effect on PrPC/PrPSc), could block PrPC incorporation into infectious PrPSc	RML	179,180
44B1	Monoclonal antibody	Neuronal cell culture	Rapidly and significantly decreased PrPSc levels	Retains PrPC on cell surface as an antigen-antibody complex, interferes with internalization and trafficking of PrPC to endocytic compartments	22L	139
POM2	Anti-PrP antibody to flexible tail of PrPC	Mouse COCS	Prevented prion-mediated neurodegeneration but prion titer was not decreased	Induces shift in distribution ofPrPSc moieties without affecting overall quantity	RML	181
LD7, JZ107	Phenethyl piperidine	Neuronal cell culture	Reduced PrPSc by 50%, permanently cured RML-infected cells (1 month exposure), protects against dendritic spine loss in presence of PrPSc	Might interact with PrPC in a cellular context, perhaps in conjunction with other cell-surface receptors, may interact with PrPC substrate	RML, 22L	182
Alprenolol hydrochloride	B-adrenergic blocker	Neuronal cell culture, mouse	Reduced levels of PrPSc in cells and brains of infected mice, inhibited PrPSc accumulation and spongiform changes but did not prolong survival	May inhibit via interaction with PrPC	Fukuoka-1	183
R12, R24, R12-A-R12	RNA aptamer	Neuronal cell culture	Significantly reduces PrPSc levels in cell culture	Tightly binds to and stabilizes PrPC, blocking conversion to PrPSc	Fukuoka-1	184,185
DE10, DC2, EB8, EF2	Monoclonal antibodies to N-terminal region of PrPSc	Neuronal cell culture	Promoted complete clearance ofPrPSc in cell culture	Binds to a region on PrPC that PrPSc also binds, blocking PrPC-PrPSc interaction	RML	186
PrP C /PrP Sc
Chlorpromazine (CPZ)	Phenothiazine	Neuronal cell culture	No to modest effect on PrPSc accumulation, inhibits prion replication in cells but not in vitro	Decreases PrPC at cell surface by inhibiting clathrin-mediated endocytosis, redistributes PrPSc to late endosome-lysosome	sheep scrapie 127S, Sc237, Type 1 CJD, RML, 22L	129,139,187
Quinacrine	Lysosomotropic agent	Neuronal cell culture, mouse, humans	Inhibits cell growth at concentrations greater than 2.0 μM, no prolongation effect in mice and humans, may lead to drug resistance	Competitive inhibition of GAG-PrP interaction, unfolding of PrP-res, destablilization of PrP-res conformation, induces conformational change that disfavors PrPSc conformation	Scrapie, 263K, vCJD, sCJD, iatrogenic CJD, genetic CJD	140,188–193
E-64d	Cysteine protease inhibitor	Neuronal cell culture, mouse	No toxicity to cell growth at [ ] up to 100 μM, no prolongation effect	Competitive inhibition of GAG-PrP interaction, unfolding of PrP-res, destabilization of PrP-res conformation	Scrapie, 263K	140,190
MS-8209	Amphotericin B derivative	Neuronal cell culture, mouse, hamster	Prolongs incubation period (dose- and timing dependent), delays onset of PrP-res/GFAP accumulation, vacuolation, spongiosis, and astrogliosis in brain, variable efficacy across prion species	Potentially affects conversion, may interact with astrocyte lysosomal system and limit propagation of PrP at inoculation site	Sheep scrapie 127S, 139A, Sc237 (weaker with 139H), type 1 CJD, C506M3 (similar to ME7), 263K	129,194–200
31C6	Monoclonal anti-PrP antibody (residues 143–149), IgG1	Neuronal cell culture, mouse	Reduces PrPSc in cell culture (dose-dependent), no re-emergence following txt, mAb txt at 120 dpi increased survival (not statistically significant), slowed weight loss, disease progression, and accumulation ofPrPSc in brain	Direct inhibition of PrPC-PrPSc interaction by occupying binding domains, could interfere with PrPC metabolism by retaining PrPC on cell surface	Chandler (139A), RML	176,201
Doxycycline	Antibiotic	Neuronal cell culture, human	Variably affected PrP-res accumulation in culture, variable prolongation of survival, reduction in widespread & severe lesions (early txt)	Destabilize abnormal PrP, could operate at cell level by modulating formation of PrP aggregates	CJD (sporadic and genetic–E200K or V210I), sCJDMM1, sCJDVV2a, vCJDMM2b, iCJDMM1	202–206
Other
GSK2606414	Protein kinase RNA-like ER kinase (PERK) inhibitor	Mouse	Oral treatment reversed cognitive deficits and prevented clinical disease, effective pre-and post-symptomatic, halted progression in spongiform degeneration, protected from neuronal loss	Prevents activation of UPR branch that mediates prion neurotoxicity by inhibiting PERK	RML	207

Cellular redistribution of PrP^Sc can inhibit prion formation. Chlorpromazine (CPZ), an antipsychotic, redistributes PrP^Sc from the early endosomal/endocytic recycling pathway to the late endosomal/lysosomal pathway resulting in an inhibition of prion conversion in prion-infected N2a cells; however, CPZ was ineffective in vivo.¹³⁹ A cholesterol synthesis inhibitor, U18666A, can redistribute PrP^Sc from the early endosomal/endocytic recycling pathway to the late endosomal/lysosomal pathway, causing an increase in PrP^Sc degradation that corresponds to a decrease in total PrP^Sc in prion-infected N2a cells.¹³⁹

Increasing degradation of PrP^Sc can ameliorate prion infection (Fig. 1, step 6). The tyrosine kinase inhibitor STI571 decreases the half-life of PrP^Sc in prion-infected ScN2a cells via interaction with the tyrosine kinase c-Abl without affecting the cellular location or trafficking of PrP^Sc.²⁰⁸ Several drugs increase degradation of PrP^Sc by inducing autophagy. A-12 and A-14, derivatives of the antitumor drug celecoxib, can reduce or eliminate PrP^Sc in prion-infected neuronal cells lines by stimulating autophagy.²⁰⁹ Lithium, which is used to treat depression, reduces PrP^Sc in prion-infected neuronal and non-neuronal cells cultures by inducing autophagy.²¹⁰

Pentosan polysulfate (PPS), a cysteine protease inhibitor, induces a reduction in prion conversion by causing fragmentation of PrP^Sc at the cell surface or utilizing endogenous glycosaminoglycans (GAGs) to competitively interfere with the binding of PrP^C to PrP^Sc.^139,140 Treatment of prion-infected mice with PPS decreased PrP^Sc accumulation and prolonged survival compared to untreated controls even when administered after the onset of PrP^Sc deposition in the CNS.^139,140 While effective in rodents, treatment of human prion diseases with PPS has varied results. In some patients treated with intravenous PPS, there is a significant increase in survival compared with untreated patients^141–143; however, there are several cases indicating treatment is not effective at extending survival or ameliorating clinical features of disease.^144–146

Treatment of prion disease with heterologous prion proteins (HetPrP) can interfere with the formation of PrP^Sc in both Sc⁺MNB cells and in vivo.^148–151 Treatment of RML-infected mice with hamster HetPrP significantly delayed onset of clinical symptoms and significantly decreased PrP^Sc accumulation in brain and spleen.¹⁵⁰ Mechanistically, it is hypothesized that HetPrP either incorporates itself into a growing PrP^Sc aggregate and, unable to be converted to PrP^Sc by the host species PrP^Sc, hinders conversion or binds directly to the conversion site on PrP^Sc blocking the PrP^C conversion site.¹⁴⁸ Overall, drugs that target PrP^Sc that are effective in prion-infected cell cultures and animals are ineffective in human clinical trials. The lack of efficacy in human clinical trials could be partly due to timing of treatment initiation. In prion-infected rodents, anti-prion drugs administered prior to or shortly after prion infection have the highest efficacy, with administration at the onset of clinical disease being less effective. In human prion disease, treatment is typically not begun until after onset of clinical signs, decreasing treatment efficacy.²¹¹ In an attempt to overcome this, an ongoing study is treating subjects at risk for developing fatal familial insomnia with doxycycline.²¹²

4.2. Strain-specific efficacy of anti-prion treatments

Treatment of prion-infected animals or cell cultures with anti-prion drugs can lead to the emergence of drug resistant prion strains. Swainsonine inhibits the processing of asparagine-linked glycoproteins by impeding the action of Golgi α-mannosidase II as well as lysosomal mannosidase.^213–215 Treatment of prion-infected neuroblastoma-derived R33 cells with swainsonine can result in the emergence of swainsonine-resistant prions.²¹⁶ Importantly, PrP^Sc from swainsonine-resistant and swainsonine-sensitive prions from cell culture have different PrP^Sc conformational stabilities suggesting they are distinct prion strains.^216,217 Treatment of prion-infected cells with swainsonine selected for a drug resistant substrain, but when the inhibitor is removed, the susceptible substrain reverted to a drug sensitive population in cell culture.²¹⁸ Additionally, passage of swainsonine-resistant RML prions from AMO10 to PK1 cells in the presence of swainsonine resulted in the selection of swainsonine-dependent prion variants with an increased efficiency to propagate in the presence of the drug.²¹⁹ However, passaging the swainsonine-dependent prions in the absence of drug resulted in reversion to a swainsonine-sensitive prion population in cells.²¹⁹

RML-infected ScN2a cells treated with the 2-aminothiazole IND24 resulted in the emergence of an IND24-resistant prion strain (RML [IND24]) that had a different host cell range, biochemical and neurological features compared to RML-infected mice treated with vehicle (RML [V]).¹⁵² In addition to IND24, this drug-resistant prion strain was also resistant to the anti-prion drugs Compound B and quinacrine.¹⁵² This suggests that the mechanism of resistance between the three anti-prion compounds is similar. Susceptibility of the RML[IND24] strain to IND24 could be restored when passaged in the absence of IND24 resulting in the reversion of the RML[IND24] phenotype to the RML[V] phenotype in mice.¹⁵²

The mechanism underlying the emergence of drug-resistant prion strains is not known. Anti-prion treatments can be strain specific.^152,157 One hypothesis is that compounds effective against the most predominant, but not all, strains in a mixture can allow for the emergence of a drug resistant strain. Prion strains are thought to exist as a dynamic mixture of substrains, or mutant spectra.^220,221 In this scenario, the dominant strain suppresses the emergence of substrains and, once the dominant strain is removed by the anti-prion drug, the suppressive effect is diminished allowing for emergence of a preexisting drug resistant strain. Studies of strain interference have established the conditions and mechanism of how a dominant strain can suppress the emergence of a minor strain consistent with this hypothesis.^{121,123,221–223} Selective removal of a dominant strain from a mixture using strain-specific differences in the stability of PrP^Sc can allow for the emergence of a highly pathogenic substrain from a mixture, providing additional support for this hypothesis.²²⁴ Alternatively, it is possible that anti-prion compounds directly interact with PrP^Sc altering its conformation (i.e., mutation) and therefore strain properties. Anti-prion compounds can bind to PrP^Sc consistent with this hypothesis, however, RML[IND24] remained resistant to IND24 for up to 20 passages in CAD5 cells in the absence of IND24 suggesting that IND24 interaction with PrP^Sc is not required for the resistant phenotype.¹⁵² Overall, it is unclear if the emergence of drug resistant prion strains is due to selection of a preexisting mutant strain from the mutant spectra and/or if the anti-prion drug acts as a prion mutagen to generate the drug resistant strain.

Maintenance of a drug resistant phenotype may have a high fitness cost. Passage of drug resistant prion strains in the absence of the anti-prion drug can result in rapid reversion to the original drug sensitive strain. This suggests that the drug resistant prion strain has relatively poor fitness for the host. In the absence of drug, the mutant spectra that arises from prion conversion produces mutants that have increased fitness for the host. This observation raises several questions. First, reversion of the drug resistant strain in the drug-free environment leads to the emergence of the original drug sensitive strain. It is unclear why the drug resistant strain would revert to the parent strain as opposed to another strain if generation of the mutant spectra is random. This suggests that the drug resistant strain may contain a “memory” that favors reversion to the original strain. Second, treatment of a host with an anti-prion drug can extend the incubation period of disease without the emergence of a drug resistant strain. The mechanism behind this observation is unknown. It is possible that certain strains can still retain sufficient suppressive effect of substrains during anti-prion treatment. The relative distribution of the mutant spectra in any given isolate may differ depending on its passage history. For example, biologically cloned strains may have a more limited mutant spectra compared to uncloned or field isolates. Finally, highly stable class I strains may have a more limited mutant spectra compared to unstable class III strains.²²⁵ Overall, very little is known about how the interaction between the host and the strain influences prion strain fitness.

The emergence of drug resistant prions is a significant barrier to the development of effective treatments of prion diseases that target the prion form of the protein. Treatments that reduce or eliminate the abundance or the ability of the conversion template to form the prion form of the protein may offer an alternative strategy for anti-prion therapies.

4.3. Treatments that target PrP^C

Prion conversion is dependent on the presence of the template of conversion (Fig. 1, step 2). Genetic ablation of PrP in mice results in complete resistance to prion infection with little known detriment to development or function.^226,227 Similarly, ablation of PrP^C in cattle does not result in major functional or immunological deficits and tissue from these animals does not support prion conversion.²²⁸ In a conditional PrP^C knockout model, transgenic mice express PrP^C until approximately 12 weeks of age before undergoing gene-directed depletion of PrP^C.²²⁹ When these mice are challenged with RML prions at 3–4 weeks of age, prion infection was established, but the depletion of PrP^C at 12 weeks halted progression to the clinical phase of prion disease and reversed spongiosis.²²⁹ Overall, these studies provide evidence that elimination of PrP^C, even after the establishment of prion infection, has potential as a therapeutic strategy.

Antisense oligonucleotides (ASOs) can modulate expression of PrP^C. Sequence-specific ASOs lower levels of PrP^C and PrP^Sc in ScN2a cells.¹⁶⁵ When sequence specific ASOs were administered to mice via either i.p. or intracerebroventricular (ICV) routes, ASOs were well tolerated and resulted in a decrease in Prnp mRNA levels in the brain compared to PBS negative controls.^165,166 When ASOs were administered prophylactically or at time of inoculation with the RML prion strain, there was diminished PrP^C expression and a corresponding reduction in PrP^Sc in the brains of ASO-treated mice compared to PBS controls. Reduction of PrP^Sc levels correlated to a prolongation of the incubation period of disease, but animals ultimately succumbed to prion disease.^165,166 When ASOs are administered just prior to the onset of clinical signs of disease, this resulted in many of the animals rapidly succumbing to the toxic effect of ASO administration.¹⁶⁶ Encouragingly, the mice that survived the ASO treatment had a delayed onset of clinical disease as well as an extended clinical duration of disease that was three times longer than saline treated controls.¹⁶⁶ Overall, ASOs can delay the onset of prion disease whether given prophylactically or after the establishment of infection and, one issues of toxicity are addressed, may be an attractive method of depressing PrP^C expression to treat prion diseases.

Treatments that alter the expression of PrP^C on the cell surface can reduce prion conversion. For example, a group of chalcones and oxadiazoles can directly bind to PrP^C and decrease its cell surface expression arresting PrP^C trafficking to the endoplasmic reticulum resulting in a decrease in PrP^Sc abundance and aggregation in prion-infected N2a cells.^167,168 Alternatively, some of these compounds prevent conversion of PrP^C to PrP^Sc by binding to and stabilizing PrP^C or directly binding to PrP^C at the seeding surface.^168,184,185

Alteration of post translational processing of PrP^C can inhibit prion formation. PrP^C can undergo either α- or β-endoproteolytic cleavage resulting in C1 and C2 fragments, respectively.²³⁰ The C2 amino acid sequence approximately aligns with that of the PrP^Sc protease resistant core, and has been found in abundance in prion infected brains.^231–237 The production of proteolytic cleavage products with varying pathogenic propensities is similar to what is observed of amyloid precursor protein, where APP cleavage is central to the development of AD.²³⁸ Processing of APP by the enzymes β-secretase and γ-secretase leads to production of the peptides Aβ-40 and Aβ-42. While Aβ-40 has neuroprotective qualities,^239,240 Aβ-42 has been implicated in AD pathogenesis.^241,242 Endopeptidase cleavage of α-synuclein results in aggregation and toxicity that ultimately causes PD.²⁴³ Pharmacological alteration of cellular proteolytic cleavage of the precursor proteins is an attractive druggable target. For example, a drug that preferentially inhibits the production of the C2 fragment would be a viable anti-prion treatment as the unfavorable PrP^C cleavage product (C2) would be sequestered while the C1 fragment would be preserved and able to continue to perform a protective function.²⁴⁴ The metalloprotease, ADAM10, plays a role PrP^C shedding from the cell surface, but has also been suggested to contribute the α-cleavage of PrP^C.^{232,245–249} Inhibition of ADAM10 resulted in an earlier onset of prion disease and slower spread to other brain regions in mice as a result of PrP^C sequestration, suggesting ADAM10 may cleave newly formed PrP^Sc from the surface of infected cells.^247,250 These data suggest that a therapeutic that increases PrP^C shedding, instead of inhibiting post-translational processing of PrP^C, may be more effective at dampening disease progression.

Monoclonal antibodies (mAb) directed against PrP^C can inhibit prion conversion. Several mAbs (D18, 6H4, ICSM 18, 31C6, etc.) are able to abrogate prion conversion and cure prion-infected N2a cells.^{171,172,176–180,251} In prion-infected N2a cells, the inhibition of prion conversion depends on the binding affinity between the mAb and PrP^C at the cell surface, with the most potent inhibitors often binding the ɑ-helical domain of PrP.^174,176,177 Inhibition of PrP^Sc formation by antibodies in cell culture has been proposed to occur via several mechanisms. The antibodies D18 and 6D11 can abolish prion conversion by binding to PrP^C at the cell surface and hindering PrP^Sc-templated conversion.^171,177 Similarly, treatment of prion-infected N2a cells with mAb 44B1 retains PrP^C at the cell surface in an antigen–antibody complex rendering it unavailable for conversion resulting in a significant decrease of PrP^Sc levels.¹³⁹ A group of N-terminal monoclonal antibodies (DE10, DC2, EB8, and EF2) are hypothesized to directly bind to a region on PrP^C that has been shown to tightly bind PrP^Sc, effectively blocking the PrP^C-PrP^Sc interaction.¹⁸⁶ Antibodies that disrupt PrP^C metabolism or trafficking either by preventing internalization of PrP^C or reducing the half-life of PrP^C, result in a decrease in PrP^Sc in cell culture models.^176,178,251 Although the aforementioned antibodies were reported to have no effect on cell growth or induce cellular toxicity, there are reports of anti-PrP antibodies (e.g., POM1) that are neurotoxic.^{181,252–255} Anti-PrP antibodies that are toxic tend to bind epitopes in the globular domain of PrP and may trigger similar pathogenic cell signaling pathways as prion disease.^181,254

Monoclonal antibodies directed against PrP^C can alter prion pathogenesis and delay the onset of clinical signs of prion infection. ICSM 18, when administered to mice either intraperitoneal (i.p.) or i.c. 7 or 30 days after i.p. inoculation with RML was found to decrease PrP^Sc levels in the spleen and delay the onset of clinical signs of prion infection compared to untreated controls.¹⁷⁵ However, treatment was ineffective when administered at the onset of clinical signs or if mice were i.c. inoculated with RML.¹⁷⁵ Treatment of mice infected with the Chandler strain of murine-adapted scrapie with mAb 31C6 120 dpi led to an increase in the incubation period, a decrease in PrP^Sc levels, and delayed progression of neuropathological lesions in the cerebellum.²⁰¹ Creating PrP^0/0 mice expressing the 6H4 antibody as a transgene (Prnp^0/0-6H4μ) led to these mice producing anti-PrP titers in sera.¹⁷³ Crossbreeding of Prnp^0/0-6H4μ mice with C57BL/6 mice led to re-introduction of one or two PrP alleles, generating Prnp^+/0-6H4μ and Prnp^+/+-6H4μ mice, respectively, which developed anti-PrP serum levels.¹⁷³ Following intraperitoneal inoculation of the RML strain of scrapie, Prnp^+/0 6H4μ mice lacked deposition of PrP^Sc in the spleen or the brain, whereas Prnp^+/0 mice had deposition in both tissues.¹⁷³ Expression of the 6H4 antibody as a transgene drastically delayed prion pathogenesis in mice.¹⁷³ Overall, anti-PrP antibodies can reduce or eliminate PrP^Sc formation in cells and animals and provides proof of principle of the efficacy of this approach.

Vaccines that result in host induced production of antibodies that recognize PrP^Sc or PrP^C have been attempted. Vaccination of mice or white-tailed deer with attenuated Salmonella expressing PrP resulted in significant prolongation of onset of clinical disease in the vaccinated group with complete protection in a portion of the animals as determined by the absence of PrP^Sc deposition; however, these studies involved a relatively small sample size.^256,257 Vaccines consisting of peptides derived from the primary amino acid sequence of the prion protein resulted in a humoral immune response to all peptides and delayed the onset of clinical signs of prion infection compared to control prion-infected mice.²⁵⁸ Other studies have focused on protein epitopes specific to PrP^Sc with strong induction of antibody responses after vaccination of mice, sheep, and white-tailed deer, strengthening the possibility of an effective PrP^Sc-specific vaccine.^259–262 In contrast to the previous studies, vaccinating elk to induce a antibody response against PrP^Sc resulted in a significant shortening of incubation period of CWD.²⁶³ The acceleration in disease onset could be attributed to antibody-mediated misfolding of PrP^C or by promoting the uptake of PrP^Sc in the gut.²⁶³ Since PrP^C is a host-encoded protein, development of an effective vaccine directed immune response is impeded by host tolerance. While many approaches have aimed to overcome tolerance, there are negative consequences of inducing an antibody response to a host-encoded cellular protein. The effects of antibodies directed at the globular domain of PrP^C results in varying degrees of neurotoxicity while other PrP^C-specific antibodies lead to apoptosis and improper signaling cascades.^181,253,264 Vaccination strategies have successfully induced strong humoral and mucosal immune responses in multiple animal models; however, more work is required to evaluate the most effective vaccination strategy to prevent disease onset while avoiding toxicity.

5. Future prospects

Development of ultra-sensitive methods of PrP^Sc detection have greatly improved and may lead to routine detection of prion infection in asymptomatic individuals. Protein misfolding cyclic amplification (PMCA) and real time quaking induced conversion (RT-QuIC) can detect a single infectious prion particle.^265,266 Both in vitro methods can detect PrP^Sc in urine, feces, blood, saliva, and cerebrospinal fluid (CSF).^267–272 PMCA detects PrP^Sc from vCJD patient blood and urine samples, while RT-QuIC is currently being used for the diagnosis of sCJD from CSF and nasal mucosa swab samples.^{267,269–271,273} Advancements in in vitro techniques have enabled reliable preclinical detection in experimental prion disease. PMCA can detect prions in the blood or skin from pre-clinical prion-infected mice and hamsters.^274–276 The sensitivity of RT-QuIC can detect prions in the blood of prion-infected preclinical hamsters and deer at timepoints as early as 5 days and 1 month post inoculation, respectively.^277,278 Like PrP^Sc, Aβ, tau, and α-synuclein are present in biological fluids making these in vitro techniques promising for preclinical diagnosis.^279–283 Overall, preclinical diagnosis of prion diseases will provide a larger window for therapeutic treatments and consequently a greater likelihood of success. Challenging this is the presence of multiple prion strains. Strain-specific efficacy of anti-prion drugs or vaccines can result in emergence of resistant strains that may be more pathogenic or have a different host range compared to the original dominant strain. These challenges are reminiscent of those faced by bacterial, fungal, and viral drug development and a successful anti-prion therapy may require a multidrug approach. The unique nature of prions, however, provides an additional therapeutic target, namely the precursor protein. Treatments that target the prion precursor protein can inhibit prion formation and may reverse pathological changes. A combinational approach that treats the prion form and the precursor protein will likely be needed to effectively treat the broad spectrum of prion diseases.