Detection of the GPI-anchorless prion protein fragment PrP226* in human brain.

BACKGROUND: The accumulation of the misfolded forms of cellular prion protein, i.e. prions (PrPSc), in the brain is one of the crucial characteristics of fatal neurodegenerative disorders, called transmissible spongiform encephalopathies (TSEs). Cellular prion protein is normally linked to the cell surface by the glycosylphosphatidylinositol (GPI) anchor. There is accumulating evidence that the GPI-anchorless prion protein may act as an accelerator of formation and propagation of prions. In the TSE affected human brain we have previously discovered a novel GPI-anchorless prion protein fragment, named PrP226*, which ends with the tyrosine 226. This fragment can be labeled specifically by the monoclonal antibody V5B2. METHODS: We developed a DELFIA based assay for quick and sensitive detection of the PrP226* fragment in human brain tissue homogenates. By calculating the ratio between the signals of native (N) and denatured (D) samples applied to the assay we were able to observe significant difference between 24 TSE affected brains and 10 control brains. The presence of PrP226* in brain tissue was confirmed by western blot. RESULTS: Our results demonstrate that PrP226* is present in small quantities in healthy human brain, whereas in degenerated brain it accumulates in prion aggregates, proportionally to PrPSc. Samples with high D/N ratio generally comprised more proteinase K resistant PrP, while no correlation was found between the quantity of PrP226* and standard classification of Creutzfeldt-Jakob disease (CJD). CONCLUSIONS: In the present study we show that the PrP226* fragment accumulates in prion aggregates and after being released from them by a denaturation procedure, could serve as a proteinase K digestion independent biomarker for human TSEs. The PrP226* assay described in this paper offers a tool to follow and study this unique anchorless PrP fragment in various parts of human brain and possibly also in other tissues and body fluids.

TSE diagnostics: recent advances in immunoassaying prions.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of rare fatal neurodegenerative diseases, affecting humans and animals. They are believed to be the consequence of the conversion of the cellular prion protein to its aggregation-prone, ß -sheet-rich isoform, named prion. Definite diagnosis of TSEs is determined post mortem. For this purpose, immunoassays for analyzing brain tissue have been developed. However, the ultimate goal of TSE diagnostics is an ante mortem test, which would be sensitive enough to detect prions in body fluids, that is, in blood, cerebrospinal fluid, or urine. Such a test would be of paramount importance also for screening of asymptomatic carriers of the disease with the aim of increasing food, drugs, and blood-derived products safety. In the present paper, we have reviewed recent advances in the development of immunoassays for the detection of prions.

Anti-idiotypic antibodies: a new approach in prion research.

BACKGROUND: In certain cases, anti-idiotypic antibodies that recognize an antigen-combining site of an antibody can mimic the structure and/or function of certain nominal antigens. This feature makes them particularly useful if conventional experimental approaches fail to fulfil expectations, especially when the molecule of interest is infectious, toxic or difficult to isolate and purify. We suggest the application of an anti-idiotype concept to the field of prion biology, with the aim of evoking a humoral immune response against the pathological isoform of the prion protein (PrPSc). Different ways to induce anti-idiotypic responses were studied in mice and chickens using various forms of V5B2, a PrPSc-specific monoclonal antibody we have described previously. RESULTS: The preparation of anti-idiotypic monoclonal antibodies was achieved with well-defined strategies of immunization, selection and subsequent characterization. Our results demonstrate that it is possible to induce a strong anti-idiotypic immune response against the V5B2 monoclonal antibody in both xenogeneic and syngeneic experimental systems. From the competition seen between polyclonal and monoclonal anti-idiotypic antibodies and the original immunogen, the P1 peptide, and even more importantly, the ultimate target antigen, PrPSc, we conclude that selected antibodies bind to the antigen-combining site of the V5B2 monoclonal antibody and might even resemble the PrPSc-specific epitope. The involvement of both antigen-combining sites in the interaction between V5B2 and the most promising monoclonal anti-idiotypic antibody was further supported by molecular docking. CONCLUSION: The results of the present study not only provide an example of the successful production of Ab2 monoclonal antibodies based on a well planned strategy for selection, but should also provide a new experimental approach that is applicable to the field of prion diseases.

Identification of an epitope on the recombinant bovine PrP that is able to elicit a prominent immune response in wild-type mice.

The main cause for the development of transmissible spongiform encephalopathies (TSE) is the conformational change of prion protein from the normal cellular isoform (PrP(C)) into the abnormal isoform, named prion (PrP(Sc)). The two isoforms have the same primary structure, and with PrP being highly conserved among different species, no immune response to PrP(Sc) has been observed in infected humans or other mammals so far. The problem of inducing immune response was encountered when producing monoclonal antibodies against PrP, therefore mice lacking a functional Prnp gene were predominantly used for the immunization. In the present paper we report that by immunizing wild-type BALB/c mice with chemically unmodified recombinant bovine PrP a potent humoral immune response was achieved. Furthermore, we were able to isolate the monoclonal antibody (mAb) E12/2 and few other mAbs, all reacting specifically with bovine and human PrP, but not with PrP from several other mammals. The epitope of mAb E12/2 is located at the C-terminal end of helix 1, with His155 being crucial for binding. It has been proven that mAb E12/2 is useful for human and bovine TSE research as well as for diagnostics. Our results show that there are sufficient structural differences between mouse and bovine PrP to provoke a prominent humoral immune response.

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.

Characterization of four new monoclonal antibodies against the distal N-terminal region of PrP(c).

Prion diseases are a group of fatal neurodegenerative disorders that affect humans and animals. They are characterized by the accumulation in the central nervous system of a pathological form of the host-encoded prion protein (PrP(C)). The prion protein is a membrane glycoprotein that consists of two domains: a globular, structured C-terminus and an unstructured N-terminus. The N-terminal part of the protein is involved in different functions in both health and disease. In the present work we discuss the production and biochemical characterization of a panel of four monoclonal antibodies (mAbs) against the distal N-terminus of PrP(C) using a well-established methodology based on the immunization of Prnp (0/0) mice. Additionally, we show their ability to block prion (PrP(Sc)) replication at nanomolar concentrations in a cell culture model of prion infection. These mAbs represent a promising tool for prion diagnostics and for studying the physiological role of the N-terminal domain of PrP(C).

Regional distribution of anchorless prion protein, PrP226*, in the human brain.

It was shown previously that truncated molecules of prion protein can be found in brains of patients with some types of transmissible spongiform encephalopathy. One such molecule, PrP226*, is a fragment of prion protein, truncated at Tyr226. It was found to be present in aggregates, from which it can be released using chaotropic salts. In this study we investigated the distribution of PrP226* in Creutzfeldt-Jakob disease affected human brain, employing the mAb V5B2, specifically recognizing this fragment. The results show that PrP226* is not evenly distributed among different regions of human brain. Among brain regions analyzed, the fragment was found most likely to be accumulated in the cerebellum. Its distribution correlates with the distribution of PrP(Sc).

Identification and characterisation of new Campylobacter group III phages of animal origin.

Campylobacter-specific bacteriophages (phages) are considered as an alternative intervention strategy to decrease the level of poultry contamination with Campylobacter, a leading cause of gastroenteritis worldwide. Eradication efficiency depends primarily on phage-host interaction mediated by phage tail-spike proteins and bacterial receptors. Here, this interaction was characterised using tail-spike gene sequence analysis, phage neutralisation by antiserum and host range analysis of newly isolated group III Campylobacter phages with 68 Campylobacter jejuni and Campylobacter coli strains. Three different groups of phages were obtained using antibody neutralisation assay, and they were further divided according to polymorphisms observed within tail fibre sequences and host range. Only moderate congruence was observed between these criteria with notable exception of two phages. The infection relied on capsule in all phages isolated, and flagella were found to influence phage propagation on agar plates, but not in broth. Their specificity was more C. jejuni oriented with tendency to lyse human isolates more efficiently. Additionally, natural resistance of C. jejuni to phages did not correlate with their antibiotic resistance patterns. These findings provide new insights into Campylobacter-phage interaction.

Chimeric flagellin as the self-adjuvanting antigen for the activation of immune response against Helicobacter pylori.

Helicobacter pylori infection can cause gastritis, peptic ulcer and can lead to gastric cancer. Lengthy antibiotic therapy does not protect the host against reinfection. H. pylori evolved to evade the recognition of the immune response by modifying several of its components whose orthologous proteins from other bacteria activate the innate immune response. Flagella are essential for the H. pylori effective colonization of human duodenum and stomach. TLR5, a member of the Toll-like receptor family, recognizes flagellin of most bacteria, such as Escherichia coli, but does not recognize the flagellin FlaA of H. pylori. We restored the ability of FlaA for the recognition by TLR5 by engineering a chimeric flagellin, in which both terminal segments of H. pylori flagellin were replaced by the corresponding segments from TLR5-activating E. coli flagellin. Recombinant chimeric flagellin folded correctly and was able to activate TLR5. Significantly increased serum IgG and IgA antibody responses were determined in mice vaccinated with chimeric flagellin in comparison to mice vaccinated with a control protein (FlaA) or negative control. Antibody titers remained high even 8 months after the last immunization. Antibodies were able to bind native flagellin from H. pylori lysate. Vaccination with chimeric flagellin provided mice with significant protection against H. pylori. The approach of chimeric flagellin can therefore generate effective immunogens that enable activation of innate and adaptive immune response and can be used to construct efficient vaccines against H. pylori or other flagellated bacteria that evade TLR5 recognition.

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.

Metabolic aspects of prion diseases: an overview.

Prion diseases are fatal neurodegenerative disorders that affect humans and other mammals. The hallmark of these diseases is the conformational change of the cellular prion protein (PrP(C)) to the misfolded protein capable of propagation and associated with neurodegenration, named prion (PrP(Sc)). In a strict sense, prion diseases are a consequence of aberrations in the metabolism of the cellular prion protein (PrP(C)). This brief review addresses current understanding of metabolic disturbances in prion disorders at the cellular, organ and organism level, selectively pointing out some relevant diagnostic and treatment options.

Oligomeric forms of peptide fragment PrP(214-226) in solution are preferentially recognized by PrP(Sc)-specific antibody.

A specific monoclonal antibody (mAb) V5B2 that discriminates between brain tissue of Creutzfeldt-Jakob disease patients and that from normal controls without proteinase K digestion has been prepared using a 13-residue synthetic peptide P1 from the primary structure of human PrP. In the light of the specific interaction between mAb V5B2 and the pathological isoform of PrP (PrP(Sc)), we investigated the solution behavior of antigen P1 and its interactions with mAb V5B2. Our results show that V5B2 recognizes epitope P1 in dimeric/oligomeric forms in solution and in the fibril-like aggregates, as well as in PrP(Sc) aggregates, and demonstrate that the specific epitope is present in all of these forms, but not in PrP(C).

Monoclonal antibody against a peptide of human prion protein discriminates between Creutzfeldt-Jacob's disease-affected and normal brain tissue.

Current methods for diagnosing transmissible spongiform encephalopathies rely on the degradation of the cellular prion protein (PrP(C)) and the subsequent detection of the protease-resistant remnant of the pathological prion isoform PrP(Sc) by antibodies that react with all forms of PrP. We report on a monoclonal antibody, V5B2, raised against a peptide from the C-terminal part of PrP, which recognizes an epitope specific to PrP(Sc). In cryostat sections from Creutzfeldt-Jacob's disease (CJD) patients' brains, V5B2 selectively labels various deposits of PrP(Sc) without any pretreatment for removal of PrP(C). V5B2 does not bind to non-CJD brain samples or to recombinant PrP, either in its native or denatured form. Specificity for PrP is confirmed by a sandwich enzyme-linked immunosorbent assay utilizing V5B2, which discriminates between CJD and normal samples without proteinase K treatment, and by immunoprecipitation from CJD brain homogenate. The PrP(Sc)-specific epitope is disrupted by denaturation. We conclude that the C-terminal part of PrP in disease-associated PrP(Sc) aggregates forms a structural epitope whose conformation is distinct from that of PrP(C).