Production of monoclonal antibodies to the prion protein and their characterization

Antibodies to the prion protein (PrP), particularly, monoclonal antibodies, are necessary tools in the diagnostics and study of prion diseases and potential means of their immunotherapy. For the production of monoclonal antibodies, BALB/c mice were immunized by a recombinant bovine PrP. Three stable hybridomas producing antibodies of IgM class were prepared. The antibodies were bound to PrP in a solid-phase enzyme immunoassay and immunoblotting. The epitope mapping accomplished with the use of synthetic peptides showed that an epitope located in region 25–36 of PrP corresponds to one antibody, and epitopes located in region 222–229, to the other two. The antibodies to fragment 222–229 purified by affinity chromatography recognized with a high specificity conglomerates of a pathogenic prion in the brain tissue of cows suffering from spongiform encephalopathy. Thus, in nontransgenic mice, PrP-specific monoclonal antibodies were produced, useful in studies and diagnostics of prion diseases.     

Sensitive detection of aggregated prion protein via proximity ligation

The DNA assisted solid-phase proximity ligation assay (SP-PLA) provides a unique opportunity to specifically detect prion protein (PrP) aggregates by investigating the collocation of 3 or more copies of the specific protein. We have developed an SP-PLA that can detect PrP aggregates in brain homogenates from infected hamsters even after a 10⁷-fold dilution. In contrast, brain homogenate from uninfected animals did not generate a detectable signal at 100-fold higher concentration. Using either of the 2 monoclonal anti-PrP antibodies, 3F4 and 6H4, we successfully detected low concentrations of aggregated PrP. The presented results provide a proof of concept that this method might be an interesting tool in the development of diagnostic approaches of prion diseases.     

Non-infectious aggregates of the prion protein react with several PrPSc-directed antibodies

The key event in the pathogenesis of prion diseases is the conformational conversion of the normal prion protein (PrP) (PrP^C) into an infectious, aggregated isoform (PrP^Sc) that has a high content of β-sheet. Historically, a great deal of effort has been devoted to developing antibodies that specifically recognize PrP^Sc but not PrP^C, as such antibodies would have enormous diagnostic and experimental value. A mouse monoclonal IgM antibody (designated 15B3) and three PrP motif-grafted monoclonal antibodies (referred to as IgG 19–33, 89–112, and 136–158) have been previously reported to react specifically with infectious PrP^Sc but not PrP^C. In this study, we extend the characterization of these four antibodies by testing their ability to immunoprecipitate and immunostain infectious and non-infectious aggregates of wild-type, mutant, and recombinant PrP. We find that 15B3 as well as the motif-grafted antibodies recognize multiple types of aggregated PrP, both infectious and non-infectious, including forms found in brain, in transfected cells, and induced in vitro from purified recombinant protein. These antibodies are exquisitely selective for aggregated PrP, and do not react with soluble PrP even when present in vast excess. Our results suggest that 15B3 and the motif-grafted antibodies recognize structural features common to both infectious and non-infectious aggregates of PrP. Our study extends the utility of these antibodies for diagnostic and experimental purposes, and it provides new insight into the structural changes that accompany PrP oligomerization and prion propagation.     

Immunoreactivity of the monoclonal antibody F89/160.1.5 for the human prion protein

Monoclonal antibodies (Mab) to the prion protein (PrP) have been critical to the neuropathological characterisation of PrP-related diseases in human and animals. Although PrP is highly evolutionary conserved, there is some sequence divergence among species. We have analysed the F89/160.1.5 Mab raised against the bovine prion protein for immunoreactivity with the human prion protein. The antibody recognised the IHFG epitope of the prion protein. An analysis of the Swiss Prot database confirmed conservation of the epitope in humans. Further immunohistochemical (IHC) analysis showed a highly sensitive (final concentration 55 ng/ml) and specific antibody for prion detection in humans. The observed immunoreactivity of the prion protein did not differ from that observed after staining with the well-known 3F4 (Senetek) monoclonal antibody.     

Disease-associated prion protein elicits immunoglobulin M responses in vivo

Prion diseases such as Creutzfeldt-Jakob disease are believed to result from the misfolding of a widely expressed normal cellular prion protein, PrPc. The resulting disease-associated isoforms, PrP(Sc), have much higher beta-sheet content, are insoluble in detergents, and acquire relative resistance to proteases. Although known to be highly aggregated and to form amyloid fibrils, the molecular architecture of PrP9Sc) is poorly understood. To date, it has been impossible to elicit antibodies to native PrP(Sc) that are capable of recognizing PrP(Sc) without denaturation, even in Pm-P(o/o) mice that are intolerant of it. Here we demonstrate that antibodies for native PrPc and PrP(Sc) can be produced by immunization of Pm-P(o/o) mice with partially purified PrPc and PrP(Sc) adsorbed to immunomagnetic particles using high-affinity anti-PrP monoclonal antibodies (mAbs). Interestingly, the polyclonal response to PrP(Sc) was predominantly of the immunoglobulin M (IgM) isotype, unlike the immunoglobulin G (IgG) responses elicited by PrP(c) or by recombinant PrP adsorbed or not to immunomagnetic particles, presumably reflecting the polymeric structure of disease-associated prion protein. Although heat-denatured PrP(Sc) elicited more diverse antibodies with the revelation of C-terminal epitopes, remarkably, these were also predominantly IgM suggesting that the increasing immunogenicity, acquisition of protease sensitivity, and reduction in infectivity induced by heat are not associated with dissociation of the PrP molecules in the diseased-associated protein. Adsorbing native proteins to immunomagnetic particles may have general applicability for raising polyclonal or monoclonal antibodies to any native protein, without attempting laborious purification steps that might affect protein conformation.     

Toluidine blue-O staining of prion protein deposits

Prion diseases or transmissible spongiform encephalopathies are a group of fatal neurodegenerative diseases caused by an abnormal form of prion protein (PrP(sc)). In this study, we developed a sensitive histochemical detection of PrP(sc) deposits in a Gertsmann-Str?ussler-Scheinker disease (GSS) patient using toluidine blue-O staining, a specific reagent to stain mucins and mucopolysaccharides. Detection of prion deposits correlated with immunohistochemistry using anti-prion antibodies. Control assays were performed using amyloid-beta (Abeta) plaques from Alzheimer's disease (AD) brains. Our results demonstrated that toluidine blue-O staining allowed to recognize 69.1+/-2.6% of the total plaques recognized by the anti-prion antibody. Furthermore, in the 15 studied brain regions from the GSS patient, toluidine blue-O revealed the same recognition pattern as anti-prion labeling. Toluidine blue-O stained specifically the prion deposits but not the Abeta plaques in AD brains. The specificity of the technique was confirmed in a Creutzfeldt-Jakob disease brain. This method opens several possibilities for postmortem diagnoses. Our results also suggest the relevance of specific post-translational modifications of PrP(sc), identified by toluidine blue-O, that might participate in the transformation of PrP(c) to PrP(sc).     

Aggregation of prion protein with insertion mutations is proportional to the number of inserts

Mutation in the prion gene, PRNP, accounts for approx. 10-15% of human prion diseases. However, little is known about the mechanisms by which a mutant prion protein (PrP) causes disease. We compared the biochemical properties of a wild-type human prion protein, rPrP(C) (recombinant wild-type PrP), which has five octapeptide-repeats, with two recombinant human prion proteins with insertion mutations, one with three more octapeptide repeats, rPrP(8OR), and the other with five more octapeptide repeats, rPrP(10OR). We found that the insertion mutant proteins are more prone to aggregate, and the degree and kinetics of aggregation are proportional to the number of inserts. The octapeptide-repeat and alpha-helix 1 regions are important in aggregate formation, because aggregation is inhibited with monoclonal antibodies that are specific for epitopes in these regions. We also showed that a small amount of mutant protein could enhance the formation of mixed aggregates that are composed of mutant protein and wild-type rPrP(C). Accordingly, rPrP(10OR) is also more efficient in promoting the aggregation of rPrP(C) than rPrP(8OR). These findings provide a biochemical explanation for the clinical observations that the severity of the disease in patients with insertion mutations is proportional to the number of inserts, and thus have implications for the pathogenesis of inherited human prion disease.     

Immunomodulation of the human prion peptide 106-126 aggregation

Site-directed monoclonal antibodies (mAbs) may interact with their antigens, leading to stabilization, refolding, and suppression of aggregation. In the following study, we show that mAbs raised against the peptide 106-126 of human prion protein (PrP 106-126) modulate the conformational changes occurring in the peptide exposed to aggregation conditions. MAbs 3-11 and 2-40 prevent PrP 106-126's fibrillar aggregation, disaggregates already formed aggregates, and inhibits the peptide's neurotoxic effect on the PC12 cells system, while mAb 3F4 has no protective effect. We suggest that there are key positions within the PrP 106-126 molecule where unfolding is initiated and their locking with specific antibodies may maintain the prion peptide native structure, reverse the aggregated peptide conformation, and lead to rearrangements involved in the essential feature of prion diseases.     

A single prion protein peptide can elicit a panel of isoform specific monoclonal antibodies

The main step in the pathogenesis of transmissible spongiform encephalopathies (TSE) is the conformational change of the normal cellular prion protein (PrP(C)) into the abnormal isoform, named prion (PrP(Sc)). Since PrP is a highly conserved protein, the production of monoclonal antibodies (mAbs) of high specificity and affinity to PrP is a difficult task. In the present study we show that it is possible to overcome the unresponsiveness of the immune system by immunizing wild-type BALB/c mice with a 13 amino acid PrP peptide from the C-terminal part of PrP, bound to the keyhole limpet hemocyanin (KLH). Immunization induced predominantly anti-PrP(Sc) humoral immune response. Furthermore, we were able to obtain a panel of mAbs of IgG class specific for different non-self-conformations of PrP, with anti-PrP(Sc)-specific mAbs being the most abundant.     

A pitfall in diagnosis of human prion diseases using detection of protease-resistant prion protein in urine. Contamination with bacterial outer membrane proteins

Because a definite diagnosis of prion diseases relies on the detection of the abnormal isoform of prion protein (PrPSc), it has been urgently necessary to establish a non-invasive diagnostic test to detect PrPSc in human prion diseases. To evaluate diagnostic usefulness and reliability of the detection of protease-resistant prion protein in urine, we extensively analyzed proteinase K (PK)-resistant proteins in patients affected with prion diseases and control subjects by Western blot, a coupled liquid chromatography and mass spectrometry analysis, and N-terminal sequence analysis. The PK-resistant signal migrating around 32 kDa previously reported by Shaked et al. (Shaked, G. M., Shaked, Y., Kariv-Inbal, Z., Halimi, M., Avraham, I., and Gabizon, R. (2001) J. Biol. Chem. 276, 31479-31482) was not observed in this study. Instead, discrete protein bands with an apparent molecular mass of approximately 37 kDa were detected in the urine of many patients affected with prion diseases and two diseased controls. Although these proteins also gave strong signals in the Western blot using a variety of anti-PrP antibodies as a primary antibody, we found that the signals were still detectable by incubation of secondary antibodies alone, i.e. in the absence of the primary anti-PrP antibodies. Mass spectrometry and N-terminal protein sequencing analysis revealed that the majority of the PK-resistant 37-kDa proteins in the urine of patients were outer membrane proteins (OMPs) of the Enterobacterial species. OMPs isolated from these bacteria were resistant to PK and the PK-resistant OMPs from the Enterobacterial species migrated around 37 kDa on SDS-PAGE. Furthermore, nonspecific binding of OMPs to antibodies could be mistaken for PrPSc. These findings caution that bacterial contamination can affect the immunological detection of prion protein. Therefore, the presence of Enterobacterial species should be excluded in the immunological tests for PrPSc in clinical samples, in particular, urine.     

Specific binding of the pathogenic prion isoform: development and characterization of a humanized single-chain variable antibody fragment

Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent.     

PrP(Sc)-specific antibodies with the ability to immunodetect prion oligomers

The development of antibodies with binding capacity towards soluble oligomeric forms of PrPSc recognised in the aggregation process in early stage of the disease would be of paramount importance in diagnosing prion diseases before extensive neuropathology has ensued. As blood transfusion appears to be efficient in the transmission of the infectious prion agent, there is an urgent need to develop reagents that would specifically recognize oligomeric forms of the abnormally folded prion protein, PrPSc.To that end, we show that anti-PrP monoclonal antibodies (called PRIOC mAbs) derived from mice immunised with native PrP-coated microbeads are able to immunodetect oligomers/multimers of PrPSc. Oligomer-specific immunoreactivity displayed by these PRIOC mAbs was demonstrated as large aggregates of immunoreactive deposits in prion-permissive neuroblastoma cell lines but not in equivalent non-infected or prn-p(0/0) cell lines. In contrast, an anti-monomer PrP antibody displayed diffuse immunoreactivity restricted to the cell membrane. Furthermore, our PRIOC mAbs did not display any binding with monomeric recombinant and cellular prion proteins but strongly detected PrPSc oligomers as shown by a newly developed sensitive and specific ELISA. Finally, PrioC antibodies were also able to bind soluble oligomers formed of Aβ and α-synuclein. These findings demonstrate the potential use of anti-prion antibodies that bind PrPSc oligomers, recognised in early stage of the disease, for the diagnosis of prion diseases in blood and other body fluids.     

Prion protein polymerisation triggered by manganese-generated prion protein seeds

Prion diseases are neurodegenerative diseases that can be transmitted between individuals. The exact cause of these diseases remains unknown. However, one of the key events associates with the disease is the aggregation of a cellular protein, the prion protein. The mechanism of this is still unclear. However, it is likely that the aggregation is trigged by a seeding mechanism in which an oligomer of the prion protein is able to catalyse polymerisation of further prion protein into larger aggregates. We have developed a model of this process using an oligomeric species generated from recombinant protein by exposure to manganese. On fractionation of the seeding species, we estimated that the smallest size the oligomer would be is an octomer. We analysed the catalytic mechanism of the seeding oligomer and its interaction with substrate. Different domains of the protein are necessary for the seeding ability of the prion protein as opposed to those required for it to form a substrate for the polymerisation reaction. Prion seeds formed from different sheep alleles are able to reproduce the characteristics of scrapie in terms of resistance to disease. However, we were also able to generate prion seed from chicken PrP a species where no prion disease is known. Our findings provide an insight into the aggregation process of the prion protein and its potential relation to disease progress.     

PrPSc binding antibodies are potent inhibitors of prion replication in cell lines

Conversion of the cellular alpha-helical prion protein (PrP(C)) into a disease-associated isoform (PrP(Sc)) is central to the pathogenesis of prion diseases. Molecules targeting either normal or disease-associated isoforms may be of therapeutic interest, and the antibodies binding PrP(C) have been shown to inhibit prion accumulation in vitro. Here we investigate whether antibodies that additionally target disease-associated isoforms such as PrP(Sc) inhibit prion replication in ovine PrP-inducible scrapie-infected Rov cells. We conclude from these experiments that antibodies exclusively binding PrP(C) were relatively inefficient inhibitors of ScRov cell PrP(Sc) accumulation compared with antibodies that additionally targeted disease-associated PrP isoforms. Although the mechanism by which these monoclonal antibodies inhibit prion replication is unclear, some of the data suggest that antibodies might actively increase PrP(Sc) turnover. Thus antibodies that bind to both normal and disease-associated isoforms represent very promising anti-prion agents.     

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.     

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.     

Isolation of bovine follicular dendritic cells allows the demonstration of a particular cellular prion protein

As interaction of cellular prion protein (PrPc) and the infectious agent (PrPres) appears to be a crucial pathogenic step promoted by homology, variation in PrPc isoforms on bovine immune cells may explain the absence of infectivity in most bovine lymph organs. In this study, we examined PrPc expression in bovine lymph organs (tonsils and lymph nodes) and on isolated follicular dendritic cells (FDCs). We used a panel of different monoclonal antibodies (MoAbs) raised against different epitopes of prion protein. Two MoAbs recognise amino acids 79-92 (SAF 34 and SAF 32 Mo-Abs); the 6H4 antibody reacts with a specific peptide comprising the 144-152 amino acids, and the 12F10 MoAb recognises the sequence 142-160. After immunolabelling of frozen sections of lymph organs with 6H4 or 12F10 MoAbs, we detected cellular prion protein in germinal centres. However, using the SAF 34 or SAF 32 antibodies, PrPc was revealed outside the lymphoid tissues. No PrPc was observed in the germinal centres. Therefore, we adapted the method of FDC isolation, making it suitable for the study of PrPc expression on their surface. Using electron microscopy, the presence of PrPc on the surface of FDCs was demonstrated only with 6H4 MoAb. These results suggest that bovine follicular dendritic cells express a particular form of prion protein. Either the N-terminal part of PrPc is cleaved or the accessibility of the specific epitope (79-92) of SAF 34 MoAb is abolished by interaction with other molecules. This particular isoform of PrPc on bovine FDCs might be related to the apparent absence of infectivity in lymph organs in cattle affected by bovine spongiform encephalopathy.     

The role of the prion protein membrane anchor in prion infection

In transmissible spongiform encephalopathies (TSE or prion diseases) such as sheep scrapie, bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease, normally soluble and protease-sensitive prion protein (PrP-sen or PrPC) is converted to an abnormal, insoluble and protease-resistant form termed PrP-res or PrPSc. PrP-res/PrPSc is believed to be the main component of the prion, the infectious agent of the TSE/prion diseases. Its precursor, PrP-sen, is anchored to the cell surface at the C-terminus by a co-translationally added glycophosphatidyl-inositol (GPI) membrane anchor which can be cleaved by the enzyme phosphatidyl-inositol specific phospholipase (PIPLC). The GPI anchor is also present in PrP-res, but is inaccessible to PIPLC digestion suggesting that conformational changes in PrP associated with PrP-res formation have blocked the PIPLC cleavage site. Although the GPI anchor is present in both PrP-sen and PrP-res, its precise role in TSE diseases remains unclear primarily because there are data to suggest that it both is and is not necessary for PrP-res formation and prion infection.     

Induction of immune response by synthetic fragments of the bovine prion protein and their analogues in mice of various lines

The antibodies to the bovine prion protein were produced by immunizing mice of three lines with five synthetic fragments of the protein and their six analogues. The analogues contained the amino acid substitutions that, according to theoretical calculation, should lead to an increase in the immunogenic activity of peptides. All the peptides, except for one, induced the formation of antibodies. All the sera containing the antipeptide antibodies were tested by an immunohistochemical method. The sera that were effectively bound to the brain preparations from the bovine with spongiform encephalopathy were identified; it was shown that they do not interact with the preparations of normal brain. Therefore, it was shown that the immunization of mice with the synthetic fragments of a prion protein helps obtain specific antibodies suitable for the study and diagnostics of prion diseases.     

Generation of monoclonal antibodies against human prion proteins in PrP0/0 mice.

BACKGROUND: Prion diseases belong to a group of neurodegenerative disorders affecting humans and animals. The human diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). The pathogenic mechanisms of the prion diseases are not yet understood. Monoclonal antibodies provide valuable tools in the diagnosis, as well as in the basic research, of several diseases; however, monospecific antisera or monoclonal antibodies (mAbs) against human prion proteins were, until now, not available. MATERIALS AND METHODS: We have developed an immunization protocol based on nucleic acid injection into nontolerant PrP0/0 mice. DNA or RNA coding for different human prion proteins including the mutated sequences associated with CJD, GSS, and FFI were injected into muscle tissue. Mice were primarily inoculated with DNA plasmids encoding the prion protein (PRNP) gene and boosted either with DNA, RNA, or recombinant Semliki Forest Virus particles expressing PRNP. Hybridomas were then prepared. RESULTS: Different mAbs against human prion proteins were obtained, and their binding behavior was analyzed by peptide enzyme-linked immunosorbent assay, Western blot, immunofluorescence, and immunoprecipitation. Their cross-reactivity with prion protein from other species was also determined. Our mAbs are directed against four different linear epitopes and may also recognize discontinuous regions of the native prion protein. CONCLUSIONS: These antibodies should allow us to address questions concerning the nature of the prion protein as well as the initiation and progression of prion diseases. Moreover, these mAbs can now be used for the diagnosis of prion diseases of humans and animals.