Susceptibility of cell substrates to PrPSc infection and safety control measures related to biological and biotherapeutical products

Concerns over the potential for infectious prion proteins to contaminate human biologics and biotherapeutics have been raised from time to time. Transmission of the pathogenic form of prion protein (PrPSc) through veterinary vaccines has been observed, yet no human case through the use of vaccine products has been reported. However, iatrogenic transmissions of PrPSc in humans through blood components, tissues, and growth hormone have been reported. These findings underscore the importance of reliable detection or diagnostic methods to prevent the transmission of prion diseases, given that the number of asymptomatic infected individuals remains unknown, the perceived incubation time for human prion diseases could be decades, and no cure of the diseases has been found yet. A variety of biochemical and molecular methods can selectively concentrate PrPSc to facilitate its detection in tissues and cells. Furthermore, some methods routinely used in the manufacturing process of biological products have been found to be effective in reducing PrPSc from the products. Questions remain unanswered as to the validation criteria of these methods, the minimal infectious dose of the PrPSc required to cause infection and the susceptibility of cells used in gene therapy or the manufacturing process of biological products to PrPSc infections. Here, we discuss some of these challenging issues.     

Susceptibility of cell substrates to PrPSc infection and safety control measures related to biological and biotherapeutical products

Concerns over the potential for infectious prion proteins to contaminate human biologics and biotherapeutics have been raised from time to time. Transmission of the pathogenic form of prion protein (PrP^Sc) through veterinary vaccines has been observed, yet no human case through the use of vaccine products has been reported. However, iatrogenic transmissions of PrP^Sc in humans through blood components, tissues and growth hormone have been reported. These findings underscore the importance of reliable detection or diagnostic methods to prevent the transmission of prion diseases, given that the number of asymptomatic infected individuals remains unknown, the perceived incubation time for human prion diseases could be decades, and no cure of the diseases has been found yet. A variety of biochemical and molecular methods can selectively concentrate PrP^Sc to facilitate its detection in tissues and cells. Furthermore, some methods routinely used in the manufacturing process of biological products have been found to be effective in reducing PrP^Sc from the products. Questions remain unanswered as to the validation criteria of these methods, the minimal infectious dose of the PrP^Sc required to cause infection and the susceptibility of cells used in gene therapy or the manufacturing process of biological products to PrP^Sc infections. Here, we discuss some of these challenging issues.Key words: prion, transmission, detection, tissue, blood transfusion, biologics, biotherapeutics, vaccine, cell substrates     

Cell-based immunotherapy of prion diseases by adoptive transfer of antigen-loaded dendritic cells or antigen-primed CD4+ T lymphocytes

Prion diseases are neurodegenerative conditions caused by the transconformation of a normal host glycoprotein, the cellular prion protein (PrPc) into a neurotoxic, self-aggregating conformer (PrPSc). TSEs are ineluctably fatal and no treatment is yet available. In principle, prion diseases could be attacked from different angles including: blocking conversion of PrPc into PrPSc, accelerating the clearance of amyloid deposits in peripheral tissues and brain, stopping prion progression in secondary lymphoid organs, reducing brain inflammation and promoting neuronal healing. There are many indications that adaptive and innate immunity might mediate those effects but so far, the achievements of immunointervention have not matched all expectations. Difficulties arise from the impossibility to diagnose TSE before substantial brain damage, poor accessibility of the CNS to immunological agents, deep immune tolerance to self-PrP, and short term effects of many immune interventions contrasting with the slow progression of TSEs. Here, we discuss two approaches, inspired from cancer immunotherapy, which might overcome some of those obstacles. One is vaccination with antigen-pulsed or antigen-transduced dendritic cells to bypass self-tolerance. The other one is the adoptive transfer of PrP-sensitized CD4+ T cells which can promote humoral, cell-mediated or regulatory responses, coordinate adaptive and innate immunity and have long lasting effects.     

Mechanistic insights into the cure of prion disease by novel antiprion compounds

Prion diseases are fatal neurodegenerative disorders. Identification of possible therapeutic tools is important in the search for a potential treatment for these diseases. Congo red is an azo dye that has been used for many years to detect abnormal prion protein in the brains of diseased patients or animals. Congo red has little therapeutic potential for the treatment of these diseases due to toxicity and poor permeation of the blood-brain barrier. We have prepared two Congo red derivatives, designed without these liabilities, with potent activity in cellular models of prion disease. One of these compounds cured cells of the transmissible agent. The mechanism of action of these compounds is possibly multifactorial. The high affinity of Congo red derivatives, including compounds that are ineffective and are effective at the cure of prion disease, for abnormally folded prion protein suggests that the amyloidophylic property of these derivatives is not as critical to the mechanism of action as other effects. Congo red derivatives that are effective at the cure of prion disease increased the degradation of abnormal PrP by the proteasome. Therefore, the principal mechanism of action of the Congo red analogues was to prevent inhibition of proteasomal activity by PrPSc.     

Chaperones that cure yeast artificial [PSI+] and their prion-specific effects

The [PSI(+)] nonsense-suppressor determinant of Saccharomyces cerevisiae results from the ability of Sup35 (eRF3) translation termination factor to undergo prion-like aggregation [1]. Although this process is autocatalytic, in vivo it depends on the chaperone Hsp104, whose lack or overexpression can cure [PSI(+)] [2]. Overproduction of the chaperone protein Ssb1 increased the [PSI(+)] curing by excess Hsp104, although it had no effect on its own, and excess chaperone protein Ssa1 protected [PSI(+)] against Hsp104 [3,4]. We used an artificial [PSI(+)(PS)] based on the Sup35 prion-forming domain from yeast Pichia methanolica [5] to find other prion-curing factors. Both [PSI(+)(PS)] and [PSI(+)] have prion 'strains', differing in their suppressor efficiency and mitotic stability. We show that [PSI(+)(PS)] and a 'weak' strain of [PSI(+)] can be cured by overexpression of chaperones Ssa1, Ssb1 and Ydj1. The ability of different chaperones to cure [PSI(+)(PS)] showed significant prion strain specificity, which could be related to variation in Sup35 prion structure. Our results imply that homologs of these chaperones may be active against mammalian prion and amyloid diseases.     

Copper-dependent functions for the prion protein

Prion diseases such as bovine spongiform encephalopathy and Creutzfeldt-Jakob disease are fatal neurodegenerative diseases. These diseases are characterized by the conversion of a normal cellular protein, the prion protein, to an abnormal isoform that is thought to be responsible for both pathogenesis in the disease and the infectious nature of the disease agent. Understanding the biology and metabolism of the normal prion protein is therefore important for understanding the nature of these diseases. This review presents evidence for the normal function of the cellular prion protein, which appears to depend on its ability to bind copper (Cu). There is now considerable evidence that the prion protein is an antioxidant. Once the prion protein binds Cu, it may have an activity like that of a superoxide dismutase. Conversion of the prion protein to an abnormal isoform might lead to a loss of antioxidant protection that could be responsible for neurodegeneration in the disease.     

Cell-based immunotherapy of prion diseases by adoptive transfer of antigen-loaded dendritic cells or antigen-primed CD4+ T lymphocytes

Prion diseases are neurodegenerative conditions caused by the transconformation of a normal host glycoprotein, the cellular prion protein (PrPc) into a neurotoxic, self-aggregating conformer (PrPSc). TSEs are ineluctably fatal and no treatment is yet available. In principle, prion diseases could be attacked from different angles including: blocking conversion of PrPc into PrPSc, accelerating the clearance of amyloid deposits in peripheral tissues and brain, stopping prion progression in secondary lymphoid organs, reducing brain inflammation and promoting neuronal healing. There are many indications that adaptive and innate immunity might mediate those effects but, so far, the achievements of immunointervention have not matched all expectations. Difficulties arise from the impossibility to diagnose TSE before substantial brain damage, poor accessibility of the CNS to immunological agents, deep immune tolerance to self-PrP and short term effects of many immune interventions contrasting with the slow progression of TSEs. Here, we discuss two approaches, inspired from cancer immunotherapy, which might overcome some of those obstacles. One is vaccination with antigen-pulsed or antigen-transduced dendritic cells to bypass self-tolerance. The other one is the adoptive transfer of PrP-sensitized CD4⁺ T cells which can promote humoral, cellmediated or regulatory responses, coordinate adaptive and innate immunity and have long lasting effects.Key words: prion, TSE, dendritic cells, CD4⁺ T cells, cellular immunotherapy, vaccination, adoptive cell transfer     

淫羊藿提取物对酵母朊病毒［PSI+］治愈的影响

研究6种菊科中药对酵母朊病毒[PSI^＋]治愈效果。通过在培养基内加入适量浓度中药成分的方式,利用表型检测方法在细胞水平上检测中药对酵母朊病毒的治愈。淫羊藿醇提物对朊病毒[PSI^＋]最佳治愈浓度为0．0975g/mL,治愈率可达到3％,而水提物没有治愈作用。淫羊藿醇提物对酵母朊病毒有一定的治愈作用。     

Concealment of epitope by reduction and alkylation in prion protein

Conversion of the cellular prion protein (PrP(C)) into its pathological isoform (PrP(Sc)), the key molecular event in the pathogenesis of prion diseases, is accompanied by a conformational transition of alpha-helix into beta-sheet structures involving alpha-helix 1 (alpha1) domain from residues 144 to 154 of the protein. Reduction and alkylation of PrP(C) have been found to inhibit the conversion of PrP(C) into PrP(Sc) in vitro. Here we report that while antibody affinity of epitopes in the N- and C-terminal domains remained unchanged, reduction and alkylation of the PrP molecule induced complete concealment of an epitope in alpha1 for anti-PrP antibody 6H4 that is able to cure prion infection in the cell model. Mass spectrometric analysis of recombinant PrP showed that the alkylation reaction takes place at reduced cysteines but no modification was observed in this cryptic epitope. Our study suggests that reduction and alkylation result in local or global rearrangement of PrP tertiary structure that is maintained in both liquid and solid phases. The implications in the conversion of PrP(C) into PrP(Sc) and the therapeutics of prion diseases are discussed.     

Promising developments bringing prion diseases closer to therapy and prophylaxis

Prion diseases are fatal, infectious, neurodegenerative disorders, and there are no available therapeutic or prophylactic regimens. The potential of immune system components in combating peripheral prion infection has long been underestimated, but recent studies have suggested that such molecules could be effective. For example, promising results have been reported from a passive vaccination study in prion-infected mice. In addition, elegant transgenic mouse studies have shown the inhibitory effect on prion propagation of a soluble immunoglobulin G (IgG)-like dimeric prion protein. This type of molecule might represent a new class of anti-prion compounds.     

Orally administered amyloidophilic compound is effective in prolonging the incubation periods of animals cerebrally infected with prion diseases in a prion strain-dependent manner

The establishment of effective therapeutic interventions for prion diseases is necessary. We report on a newly developed amyloidophilic compound that displays therapeutic efficacy when administered orally. This compound inhibited abnormal prion protein formation in prion-infected neuroblastoma cells in a prion strain-dependent manner: effectively for RML prion and marginally for 22L prion and Fukuoka-1 prion. When the highest dose (0.2% [wt/wt] in feed) was given orally to cerebrally RML prion-inoculated mice from inoculation until the terminal stage of disease, it extended the incubation periods by 2.3 times compared to the control. The compound exerted therapeutic efficacy in a prion strain-dependent manner such as that observed in the cell culture study: most effective for RML prion, less effective for 22L prion or Fukuoka-1 prion, and marginally effective for 263K prion. Its effectiveness depended on an earlier start of administration. The glycoform pattern of the abnormal prion protein in the treated mice was modified and showed predominance of the diglycosylated form, which resembled that of 263K prion, suggesting that diglycosylated forms of abnormal prion protein might be least sensitive or resistant to the compound. The mechanism of the prion strain-dependent effectiveness needs to be elucidated and managed. Nevertheless, the identification of an orally available amyloidophilic chemical encourages the pursuit of chemotherapy for prion diseases.     

Production of cattle lacking prion protein

Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrP(C), such as PrP(BSE) in bovine spongiform encephalopathy (BSE) in cattle and PrP(CJD) in Creutzfeldt-Jakob disease (CJD) in humans. Disruption of PrP(C) expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities. However, the impact of ablating PrP(C) function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrP(C)-deficient cattle produced by a sequential gene-targeting system. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification. PrP(C)-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins.     

Molecular advances in understanding inherited prion diseases

The prion diseases are neurodegenerative disorders that have attracted great interest because of the possible link between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (CTD) in humans. Possible transmission of these diseases has been linked to a single protein termed the prion protein. This protein is an abnormal isoform of a normal synaptic glycoprotein. The majority of prion diseases does not appear to be caused by transmission of an infectious agent but occur spontaneously with no known cause. The strongest supporting evidence that the prion protein is the causative agent in prion disease comes from specific inheritable forms of prion disease which are linked to single point mutations in the prion protein gene. Paradoxically, these point mutations, although autosomal dominant with 100% penetrance do not lead to disease until late in life. Molecular techniques are now being used extensively to determine how these point-mutations alter the prion protein’s normal structure and activity. This review deals with the latest insights into how inherited mutations in the prion protein gene lead to neurodegenerative disease.     

THERPA: A small molecule database related to prion protein regulation and prion diseases progression

Prion diseases are fatal neurodegenerative disorders that affect humans and animals. Although various small molecules have been evaluated for application in the treatment of prion diseases, none have been shown to be efficacious. Expanding our knowledge of these molecules is important for understanding of the complex mechanisms of prion diseases. To improve access to the scattered information on small molecules related to prion diseases, we built a database of therapeutic molecules associated with prion diseases (THERPA, therpa.pythonanywhere.com). THERPA includes 119 small molecules and their 283 relationships with prion diseases. THERPA is an interactive visual database and useful for improving search efficiency which can help researchers identify intrinsic small molecules that can be used for developing therapeutics for prion diseases.     

Prions and manganese: A maddening beast

The prion protein is well known because of its association with prion diseases. These diseases, which include variant CJD, are unusual because they are neurodegenerative diseases that can be transferred between individuals experimentally. The prion protein is also widely known as a copper binding protein. The binding of copper to the prion protein is possibly necessary for its normal cellular function. The prion protein has also been suggested to bind other metals, and among these, manganese. Despite over ten years of research on manganese and prion disease, this interaction has often been dismissed or at best seen as a poor cousin to the involvement of copper. However, recent data has shown that manganese could stabilise prions in the environment and that chelation therapy specifically aimed at manganese can extend the life of animals with prion disease. This article reviews the evidence for a link between prions and manganese.     

Prions at the crossroads: the need to identify the active TSE agent

Structural change in the cellular prion protein, PrPC to a ProteinaseK-resistant beta-sheet-rich insoluble form PrPSC and its accumulation have been considered to be central to the pathogenesis of the prion diseases (TSE). In a recent paper, Deleault et al have shown that specific endogenous RNA molecules can induce in vitro structural conversion of endogenous PrPC to PrPSC. Small highly structured synthetic RNAs can also induce this conversion process. However, recent in vivo results show that PrPSC is not directly involved in the prion pathogenesis. It is possible, however, that nucleic-acid-induced PrPSC associated with the inducer nucleic acid could be the components of the infectious agent.     

Prion protein reduces both oxidative and non-oxidative copper toxicity

The prion protein is a membrane tethered glycoprotein that binds copper. Conversion to an abnormal isoform is associated with neurodegenerative diseases known as prion diseases. Expression of the prion protein has been suggested to prevent cell death caused by oxidative stress. Using cell based models we investigated the potential of the prion protein to protect against copper toxicity. Although prion protein expression effectively protected neurones from copper toxicity, this protection was not necessarily associated with reduction in oxidative damage. We also showed that glycine and the prion protein could both protect neuronal cells from oxidative stress. Only the prion protein could protect these cells from the toxicity of copper. In contrast glycine increased copper toxicity without any apparent oxidative stress or lipid peroxidation. Mutational analysis showed that protection by the prion protein was dependent upon the copper binding octameric repeat region. Our findings demonstrate that copper toxicity can be independent of measured oxidative stress and that prion protein expression primarily protects against copper toxicity independently of the mechanism of cell death.     

Effects of post-translational modifications on prion protein aggregation and the propagation of scrapie-like characteristics in vitro

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are typically characterised by CNS accumulation of PrP(Sc), an aberrant conformer of a normal cellular protein PrP(C). It is thought PrP(Sc) is itself infectious and the causative agent of such diseases. To date, no chemical modifications of PrP(Sc), or a sub-population thereof, have been reported. In this study we have investigated whether chemical modification of amino acids within PrP might cause this protein to exhibit aberrant properties and whether these properties can be propagated onto unmodified prion protein. Of particular interest were post-translational modifications resulting from physiological conditions shown to be associated with TSE disease. Here we report that in vitro exposure of recombinant PrP to conditions that imitate the end effects of oxidative/nitrative stress in TSE-infected mouse brains cause the protein to adopt many of the physical characteristics of PrP(Sc). Most interestingly, these properties could be propagated onto unmodified PrP protein when the modified protein was used as a template. These data suggest that post-translational modifications of PrP might contribute to the initiation and/or propagation of prion protein-associated plaques in vivo during prion disease, thereby high-lighting novel biochemical pathways as possible therapeutic targets for these conditions.