Real-time quaking-induced conversion (RT-QuIC) is an assay in which disease-associated prion protein (PrP) initiates a rapid conformational transition in recombinant PrP (recPrP), resulting in the formation of amyloid that can be monitored in real time using the dye thioflavin T. It therefore has potential advantages over analogous cell-free PrP conversion assays such as protein misfolding cyclic amplification (PMCA). The QuIC assay and the related amyloid seeding assay have been developed largely using rodent-passaged sheep scrapie strains. Given the potential RT-QuIC has for Creutzfeldt-Jakob disease (CJD) research and human prion test development, this study characterized the behaviour of a range of CJD brain specimens with hamster and human recPrP in the RT-QuIC assay. The results showed that RT-QuIC is a rapid, sensitive and specific test for the form of abnormal PrP found in the most commonly occurring forms of sporadic CJD. The assay appeared to be largely independent of species-related sequence differences between human and hamster recPrP and of the methionine/valine polymorphism at codon 129 of the human PrP gene. However, with the same conditions and substrate, the assay was less efficient in detecting the abnormal PrP that characterizes variant CJD brain. Comparison of these QuIC results with those previously obtained using PMCA suggested that these two seemingly similar assays differ in important respects.
The molecular basis of the three major alleles ( Fy a / Fy b / Fy ) of the Duffy (FY) blood group system has recently been established but the Fy x phenotype associated with weak expression of the Fy b and other FY antigens is poorly understood. In the Fy x genes of five unrelated British and Swedish donors with the Fy(a+b+ weak ) phenotype we found two missense mutations predicting amino acid changes Arg89Cys and Ala100Thr in the FY glycoprotein. The same mutations were found in two Fy(a−b+ weak ) samples from individuals of Swedish and Algerian origin. Their red blood cells showed a marked decrease in Fy b , Fy3 and Fy6 expression measured by routine serology and flow cytometry. The rare FY genotypes Fy x Fy x and Fy x Fy were confirmed by family studies and DNA sequencing. Screening by allele‐specific primer PCR (ASP‐PCR) for these mutations among 100 Caucasian and 100 Black random blood donors indicated allele frequencies of 2.5% and 0%, respectively. Ala100Thr alone was present in 33% of the Caucasians (but none of the Blacks) with no weakening of FY expression. A novel allele at the FY locus associated with the Fy x phenotype was studied. Mistyping of this weak Fy b antigen in clinical transfusion medicine may lead to delayed haemolytic transfusion reactions in immunized patients. A potential role for genomic typing is proposed.
A murine monoclonal antibody of specificity anti‐Lu b was produced. Immunoblotting of the electrophoretically separated components of membranes from Lu(b+) red cells with the monoclonal antibody identified two glycoproteins of relative molecular mass 85 and 78 kd, respectively. The expression of Lu b antigenic activity on these glycoprotein components was shown to be dependent on the presence of one or more N ‐glycosidically linked oligosaccharides and on the presence of disulphide bonding.
Current cerebrospinal fluid (CSF) tests for sporadic Creutzfeldt-Jakob disease (sCJD) are based on the detection of surrogate markers of neuronal damage such as CSF 14-3-3, which are not specific for sCJD. A number of prion protein conversion assays have been developed, including real time quaking-induced conversion (RT-QuIC). The objective of this study is to investigate whether CSF RT-QuIC analysis could be used as a diagnostic test in sCJD.An exploratory study was undertaken that analyzed 108 CSF samples from patients with neuropathologically confirmed sCJD or from control patients. Of the 108 CSF samples, 56 were from sCJD patients (30 female, 26 male; aged 31-84 years; mean age, 62.3 ± 13.5 years), and 52 were from control patients (26 female, 26 male; aged 43-84 years; mean age, 67.8 ± 10.4 years). A confirmatory group of 118 patients was subsequently examined that consisted of 67 cases of neuropathologically confirmed sCJD (33 female, 34 male; aged 39-82 years; mean age, 67.5 ± 9.0 years) and 51 control cases (26 female, 25 male; aged 36-87 years; mean age, 63.5 ± 11.6 years).The exploratory study showed that RT-QuIC analysis had a sensitivity of 91% and a specificity of 98% for the diagnosis of sCJD. These results were confirmed in the confirmatory study, which showed that CSF RT-QuIC analysis had a sensitivity and specificity of 87% and 100%, respectively.This study shows that CSF RT-QuIC analysis has the potential to be a more specific diagnostic test for sCJD than current CSF tests.
BACKGROUND The P‐Capt prion reduction filter (MacoPharma) removes prion infectivity in model systems. This independent evaluation assesses prion removal from endogenously infected animal blood, using CE‐marked P‐Capt filters, and replicates the proposed use of the filter within the UK Blood Services. STUDY DESIGN AND METHODS Two units of blood, generated from 263K scrapie–infected hamsters, were processed using leukoreduction filters (LXT‐quadruple, MacoPharma). Approximately 100 mL of the removed plasma was added back to the red blood cells (RBCs) and the blood was filtered through a P‐Capt filter. Samples of unfiltered whole blood, the prion filter input (RBCs plus plasma and SAGM [RBCPS]), and prion‐filtered leukoreduced blood (PFB) were injected intracranially into hamsters. Clinical symptoms were monitored for 500 ± 1 day, and brains were assessed for spongiosis and prion protein deposit. RESULTS In Filtration Run 1, none of the 50 challenged animals were diagnosed with scrapie after inoculation with the RBCPS fraction, while two of 190 hamsters injected with PFB were infected. In Filtration Run 2, one of 49 animals injected with RBCPS and two of 193 hamsters injected with PFB were infected. Run 1 reduced the infectious dose (ID) by 1.467 log (>1.187 log and <0.280 log for leukoreduction and prion filtration, respectively). Run 2 reduced prion infectivity by 1.424 log (1.127 and 0.297 log, respectively). Residual infectivity was estimated at 0.212 ± 0.149 IDs/mL (Run 1) and 0.208 ± 0.147 IDs/mL (Run 2). CONCLUSION Leukoreduction removed the majority of infectivity from 263K scrapie hamster blood. The P‐Capt filter removed a proportion of the remaining infectivity, but residual infectivity was observed in two independent processes.
Conversion of the cellular α-helical prion protein (PrPC) into a disease-associated isoform (PrPSc) 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 PrPC have been shown to inhibit prion accumulation in vitro. Here we investigate whether antibodies that additionally target disease-associated isoforms such as PrPSc inhibit prion replication in ovine PrP-inducible scrapie-infected Rov cells. We conclude from these experiments that antibodies exclusively binding PrPC were relatively inefficient inhibitors of ScRov cell PrPSc 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 PrPSc turnover. Thus antibodies that bind to both normal and disease-associated isoforms represent very promising anti-prion agents. Conversion of the cellular α-helical prion protein (PrPC) into a disease-associated isoform (PrPSc) 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 PrPC have been shown to inhibit prion accumulation in vitro. Here we investigate whether antibodies that additionally target disease-associated isoforms such as PrPSc inhibit prion replication in ovine PrP-inducible scrapie-infected Rov cells. We conclude from these experiments that antibodies exclusively binding PrPC were relatively inefficient inhibitors of ScRov cell PrPSc 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 PrPSc turnover. Thus antibodies that bind to both normal and disease-associated isoforms represent very promising anti-prion agents. Prion diseases are fatal neurodegenerative disorders that include scrapie in sheep and goats, bovine spongiform encephalopathy in cattle, and Creutzfeldt-Jakob disease in humans. The recent emergence of a new human prion disease, variant Creutzfeldt-Jakob disease, almost certainly resulting from the human consumption of bovine spongiform encephalopathy-infected material (1Collinge J. Lancet. 1999; 354: 317-323Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar) is a major public health and safety issue (2Ghani A.C. Ferguson N.M. Donnelly C.A. Anderson R.M. Nature. 2000; 406: 583-584Crossref PubMed Scopus (179) Google Scholar, 3Hill A.F. Joiner S. Linehan J. Desbruslais M. Lantos P. Collinge J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10248-10253Crossref PubMed Scopus (258) Google Scholar). The infectious agent or prion is mainly composed of PrPSc, 1The abbreviations used are: PrPSc, disease-associated isoform of prion protein; PrPC, cellular prion protein; mAb, monoclonal antibody; DS500, dextran sulfate 500; PBS, phosphate-buffered saline. 1The abbreviations used are: PrPSc, disease-associated isoform of prion protein; PrPC, cellular prion protein; mAb, monoclonal antibody; DS500, dextran sulfate 500; PBS, phosphate-buffered saline. a detergent-insoluble and partially protease-resistant isoform of the host-encoded cellular prion protein, PrPC (4Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4014) Google Scholar). According to the protein-only hypothesis, in the course of prion infection, α-helical PrPC is refolded without post-translational modification into β-sheet-rich PrPSc, initially in the presence of exogenous PrPSc and then by an autocatalytic process (5Clarke A.R. Jackson G.S. Collinge J. Philos. Trans. R. Soc. Lond-Biol. Sci. 2001; 356: 185-195Crossref PubMed Scopus (34) Google Scholar). Currently, no treatment of prion disease is effective once neurological illness has developed, but any molecule able to interact with either one or both isoforms could potentially delay or even cure the disease (6Mallucci G. Dickinson A. Linehan J. Klohn P.C. Brandner S. Collinge J. Science. 2003; 302: 871-874Crossref PubMed Scopus (585) Google Scholar). Recent reports indicate that anti-PrP monoclonal antibodies (mAbs) efficiently inhibit PrPSc accumulation in ScN2a mouse neuroblastoma cells (7Enari M. Flechsig E. Weissmann C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9295-9299Crossref PubMed Scopus (373) Google Scholar, 8Peretz D. Williamson R.A. Kaneko K. Vergara J. Leclerc E. Schmitt-Ulms G. Mehlhorn I.R. Legname G. Wormald M.R. Rudd P.M. Dwek R.A. Burton D.R. Prusiner S.B. Nature. 2001; 412: 739-743Crossref PubMed Scopus (465) Google Scholar) and in infected transgenic mice engineered to produce one of these mAbs, 6H4 (9Heppner F.L. Musahl C. Arrighi I. Klein M.A. Rulicke T. Oesch B. Zinkernagel R.M. Kalinke U. Aguzzi A. Science. 2001; 294: 178-182Crossref PubMed Scopus (307) Google Scholar). Their efficiency was related directly to their epitope and to their affinity for PrPC (10Korth C. Stierli B. Streit P. Moser M. Schaller O. Fischer R. SchulzSchaeffer W. Kretzschmar H. Raeber A. Braun U. Ehrensperger F. Hornemann S. Glockshuber R. Riek R. Billeter M. Wuthrich K. Oesch B. Nature. 1997; 390: 74-77Crossref PubMed Scopus (537) Google Scholar, 11Williamson R.A. Peretz D. Pinilla C. Ball H. Bastidas R.B. Rozenshteyn R. Houghten R.A. Prusiner S.B. Burton D.R. J. Virol. 1998; 72: 9413-9418Crossref PubMed Google Scholar). We also have recently reported that mAbs effectively suppress systemic prion replication in vivo (12White A.R. Enever P. Tayebi M. Mushens R. Linehan J. Brandner S. Anstee D. Collinge J. Hawke S. Nature. 2003; 422: 80-83Crossref PubMed Scopus (412) Google Scholar). In that study, two mAbs with differential affinity for normal and disease-associated isoforms of prion protein were used: ICSM 18, which almost exclusively binds to PrPC, and ICSM 35, which efficiently binds to both normal and disease-associated isoforms. Both mAbs were efficient equally at delaying the onset of prion disease in the treated mice, but it was unclear whether the additional targeting of PrPSc by ICSM 35 played a role in controlling prion replication. In this study, we have used a larger panel of mAbs raised in PrPC null mice (Prnp0/0) against the α and β isoforms of human recombinant PrP (13Jackson G.S. Hosszu L.L. Power A. Hill A.F. Kenney J. Saibil H. Craven C.J. Waltho J.P. Clarke A.R. Collinge J. Science. 1999; 283: 1935-1937Crossref PubMed Scopus (361) Google Scholar) to inhibit prion replication in scrapieinfected epithelial Rov (ScRov) cells (14Vilette D. Andreoletti O. Archer F. Madelaine M.F. Vilotte J.L. Lehmann S. Laude H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4055-4059Crossref PubMed Scopus (180) Google Scholar). Rov cell PrPC expression is inducible by doxycycline, thereby allowing the clearance of PrPSc to be studied in the absence of its PrPC substrate. Here we show that mAbs that additionally target disease-associated isoforms of PrP block PrPSc accumulation much more efficiently than mAbs recognizing PrPC alone. We also found that the inhibition by several mAbs was similar and even greater than turning off the production of the PrPC substrate, suggesting antibody-mediated enhancement of the proteolysis of intracellular PrPSc. Production of Monoclonal Antibodies—The panel of ICSM mAbs was produced as described previously (15Beringue V. Mallinson G. Kaisar M. Tayebi M. Sattar Z. Jackson G. Anstee D. Collinge J. Hawke S. Brain. 2003; 126: 2065-2073Crossref PubMed Scopus (110) Google Scholar). They were affinity-purified from hybridoma culture supernatant over the protein A or G matrix (Äkta Prime, Amersham Biosciences), filter-sterilized, and stored at 4 °C. Treatment of ScRov Cells with the ICSM Antibodies—ScRov cells were grown in 24-well plates as described previously (14Vilette D. Andreoletti O. Archer F. Madelaine M.F. Vilotte J.L. Lehmann S. Laude H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4055-4059Crossref PubMed Scopus (180) Google Scholar). Doxycycline (1 μg/ml) was present in the culture medium unless mentioned. A triplicate of ScRov cells was treated with the ICSM mAbs once a week just after splitting. The controls were either left untreated or treated with isotype control mAb (15Beringue V. Mallinson G. Kaisar M. Tayebi M. Sattar Z. Jackson G. Anstee D. Collinge J. Hawke S. Brain. 2003; 126: 2065-2073Crossref PubMed Scopus (110) Google Scholar, 16Avent N. Judson P.A. Parsons S.F. Mallinson G. Anstee D.J. Tanner M.J. Evans P.R. Hodges E. Maciver A.G. Holmes C. Biochem. J. 1988; 251: 499-505Crossref PubMed Scopus (79) Google Scholar). In one experiment, the cells were exposed to similar concentrations of dextran sulfate 500 (DS500, Sigma). One quarter of the cells was passaged weekly, and the residual cells (typically ∼24 × 104 cells or 80 μg of proteins) were pelleted, lysed in lysis buffer (20 mm Tris-HCl, pH 7.5, 1% Nonidet P40, and 0.5% sodium deoxycholate), and stored at –80 °C for subsequent analysis. PrPSc was extracted from 40 μg of protein by 100 μg/ml proteinase K for 1 h at 37 °C. The protein was then denatured with 3 volumes of Laemmli buffer (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) for 5 min at 100 °C before an additional concentration of the protein with cold acetone. Typically, 15 μg of proteins (i.e. the equivalent of 45 × 103 cells) were used for Western blots (see below). Immunoprecipitation—Scrapie-infected and -uninfected Rov cells were washed three times in cold PBS and scraped in cold lysis buffer. A mixture of protease inhibitors (Roche Applied Science) in addition to 5 mm phenylmethylsulfonyl fluoride was added prior to (Rov cells) or after proteinase K treatment (ScRov cells) at 50 μg/ml for 1 h at 37 °C. Lysates then were incubated with 10 μg/ml purified mAbs in lysis buffer for 2 h at 4 °C on a rotator. Negative controls omitted the capture mAb or used the relevant isotype control (15Beringue V. Mallinson G. Kaisar M. Tayebi M. Sattar Z. Jackson G. Anstee D. Collinge J. Hawke S. Brain. 2003; 126: 2065-2073Crossref PubMed Scopus (110) Google Scholar, 16Avent N. Judson P.A. Parsons S.F. Mallinson G. Anstee D.J. Tanner M.J. Evans P.R. Hodges E. Maciver A.G. Holmes C. Biochem. J. 1988; 251: 499-505Crossref PubMed Scopus (79) Google Scholar). The immune complexes were adsorbed overnight onto protein G-agarose beads (Roche Applied Science) at 4 °C on a rotator. The beads were washed 4–5 times according to the manufacturer's instructions. They were re-suspended in Laemmli buffer (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar), heated at 100 °C for 5 min, and pelleted at 12,000 × g to detach/denature the bound protein. The supernatant was analyzed by Western blot (see below). For live cell immunoprecipitation, Rov cells were incubated overnight with 10 μg/ml ICSM mAbs in the culture medium. The protocol then was similar with the exception that the lysates were incubated directly with protein G-agarose beads. The bound fraction was compared with the total levels of PrPC, which corresponds to the unbound fraction when immunoprecipitation is performed with the relevant isotype controls. This fraction was precipitated by cold acetone, re-suspended in Laemmli buffer, and analyzed by Western blot. Immunofluorescence—Rov and ScRov cells were incubated overnight on slides with 10 μg/ml ICSM mAbs. Controls were left untreated or treated with isotype control (15Beringue V. Mallinson G. Kaisar M. Tayebi M. Sattar Z. Jackson G. Anstee D. Collinge J. Hawke S. Brain. 2003; 126: 2065-2073Crossref PubMed Scopus (110) Google Scholar, 16Avent N. Judson P.A. Parsons S.F. Mallinson G. Anstee D.J. Tanner M.J. Evans P.R. Hodges E. Maciver A.G. Holmes C. Biochem. J. 1988; 251: 499-505Crossref PubMed Scopus (79) Google Scholar). Cells were washed twice with cold PBS and fixed in 4% paraformaldehyde for 30 min. After three washes in PBS, cells were permeabilized with 0.5% Triton X-100 for 5 min. After several washes, the cells were incubated for 1 h with a 1/400 dilution of an isothiocyanate-conjugated anti-mouse IgG mAb (P.A.R.I.S, Paris, France) in 5% milk in PBS. After three washes in PBS, slides were mounted in antifading solution (Dabko) and kept in the dark at 4 °C until microscopic analysis with the Nikon fluorescent microscope. Western Blotting—Samples were run on 12% polyacrylamide Criterion gels (Bio-Rad) or 12% NuPAGE gels (Invitrogen), electrotransferred onto polyvinylidene difluoride membranes (Millipore), and immunoblotted with 0.1–0.2 μg/ml of the biotinylated anti-PrP antibody, ICSM 18. Immunoreactivity was visualized with an enhanced chemiluminescence kit on autoradiographic films (ECL+, Amersham Biosciences). Densitometric analyses of the films were performed with the program NIH Image (Waine Rasband, National Institutes of Health) as described previously (18Beringue V. Adjou K.T. Lamoury F. Maignien T. Deslys J.P. Race R. Dormont D. J. Virol. 2000; 74: 5432-5440Crossref PubMed Scopus (68) Google Scholar). The amount of PrPSc was estimated by comparison with a dilution scale of sheep scrapie PrPSc prepared in similar conditions and at the same time. Monoclonal Antibodies Raised against β-PrP Inhibit More Efficiently PrPSc Accumulation in ScRov Cells—In initial studies, we assessed how efficiently ICSM 4, 17, 18, and 19 raised against α-PrP and ICSM 35 raised against β-PrP inhibited prion replication (Table I). ICSM 18 and ICSM 17 recognize residues 146–159 and 140–159 of murine PrP. ICSM 35 recognizes residues 96 and 109 on the N-terminal region of PrP27–30. The epitopes of ICSM 4 and 19 are not definable with overlapping synthetic peptides and may be conformation-dependent (Table I) (15Beringue V. Mallinson G. Kaisar M. Tayebi M. Sattar Z. Jackson G. Anstee D. Collinge J. Hawke S. Brain. 2003; 126: 2065-2073Crossref PubMed Scopus (110) Google Scholar). Triplicate cultures of ScRov cells were treated with mAb concentrations ranging from 10 ng/ml to 10 μg/ml. The treatment was renewed once a week when the cells were split, as we identified by ELISA that the concentration in the culture supernatant fell only by 50% over each treatment period. Cells were collected each week, and the level of PrPSc was assessed by Western blot. ScRov cells also were treated with various concentrations of the anti-prion drug DS500, known to potently inhibit prion replication in other scrapie-infected cell lines (19Caughey B. Raymond G.J. J. Virol. 1993; 67: 643-650Crossref PubMed Google Scholar). As expected, DS500 rapidly inhibited PrPSc accumulation in a dose-dependent manner with a 50% inhibitory concentration (IC50) of 110 ng/ml (0.22 nm) after 3 weeks treatment (Fig. 1, a and b). When compared with DS500, mAb ICSM 35 (raised against β-PrP) was a more potent inhibitor (IC50, 4 ng/ml or 0.03 nm) (Fig. 1, a and b). Among the mAbs raised against α-PrP, ICSM 19 and ICSM 18 were ∼100 and 1500 times less efficient (IC50 of 360 ng/ml (3.30 nm) and 5 μg/ml (45.5 nm), respectively) (Fig. 1a). 1 μg/ml ICSM 19 strongly inhibited PrPSc accumulation after 6 weeks of treatment, whereas ICSM 18 at this dose could not prevent PrPSc accumulation (Fig. 1b). ICSM 4 and 17 were unable to inhibit PrPSc accumulation, even after prolonged treatment with 10 μg/ml (Table I). None of the mAbs modified PrPC expression regardless of the dose or length of treatment used (data not shown), indicating that they did not exhibit any toxicity for the cells. After 6 weeks treatment with 1 μg/ml DS500, ICSM 35, or ICSM 19, PrPSc levels were lowered 100–1000-fold (Fig. 1b). Interestingly, when the inhibitor was washed off, PrPSc reaccumulated (Fig. 1, b and c). Cells treated with ICSM 19, and DS500 reached control values 6 weeks after the treatment was stopped. At this point, those treated with ICSM 35 still accumulated ∼5 times less PrPSc than the controls (Fig. 1, b and c). This confirmed that ICSM 35 was a more potent inhibitor than ICSM 19 and DS500.Table IInhibition of ScRov PrPScaccumulation by ICSM monoclonal antibodiesICSMImmunogenaα and β refer to human recombinant α- and β-PrP91-231.EpitopebResidue numbering refers to mouse prion protein sequence.ImmunoreactivitydEstimated by at least three independent immunoprecipitation experiments and compared with the affinity obtained with ICSM 35.InhibitioneObserved at the 10 μg/ml dose after 3 weeks of treatment.PrPcC, conformational epitope.PrPSc% ± S.D.4αC37 ± 80-6αC27 ± 140-7αC26 ± 170-17α140-15926 ± 110-18α146-159183 ± 84 ± 1+19αC91 ± 260++41αC162 ± 444-44αC58 ± 373-35β96-109100100+++++37β96-109109 ± 8190 ± 7++++++42β96-10994 ± 2290 ± 4+++++a α and β refer to human recombinant α- and β-PrP91-231.b Residue numbering refers to mouse prion protein sequence.c C, conformational epitope.d Estimated by at least three independent immunoprecipitation experiments and compared with the affinity obtained with ICSM 35.e Observed at the 10 μg/ml dose after 3 weeks of treatment. Open table in a new tab PrPC expression in Rov cells is controlled by doxycycline (14Vilette D. Andreoletti O. Archer F. Madelaine M.F. Vilotte J.L. Lehmann S. Laude H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4055-4059Crossref PubMed Scopus (180) Google Scholar), allowing a comparison of the efficiency of antibody-mediated PrPSc clearance to turning off the PrPC promoter, the latter representing the maximal inhibition achievable by pure PrPC targeting. ScRov cells were treated with ICSM 19 (raised against α-PrP), ICSM 35 (raised against β-PrP), or control mAbs (each at 10 μg/ml) in the presence or absence of doxycycline. With doxycycline removed from the culture medium, the PrPSc half-life was 3 ± 0.2 days (Fig. 2). This was similar to treatment with ICSM 35 (Fig. 2, PrPSc half-life of 3.5 ± 0.5 days). Interestingly, both mechanisms of inhibition were synergistic, because ICSM 35 added to the cells in which doxycycline has just been removed induced an even faster clearance of PrPSc (Fig. 2, half-life 2.1 ± 0.3 day; p < 0.05; Mann-Whitney U test). In contrast, inhibition by ICSM 19 was much slower than turning off the PrPC substrate (PrPSc half-life 16 ± 4.4 days) (Fig. 2). Two other mAbs raised against β-PrP were similarly potent. In fact PrPSc clearance with ICSM 37 was significantly faster than with ICSM 35 and even faster than turning off the PrPC promoter (Fig. 3; p < 0.05; Mann-Whitney U test). Inhibition induced by ICSM 42 was similar to ICSM 35 (Table I and data not shown). In contrast, other mAbs raised against α-PrP (ICSM 6, 7, 41, and 44) failed to reduce PrPSc levels even after 3 weeks treatment with 10 μg/ml (Table I). Monoclonal Antibodies Raised against β-PrP-immunoprecipitated ScRov Cell PrPSc—One possible explanation for the differences observed in efficacy between the mAbs tested was that they differentially recognized normal and disease-associated isoforms of PrP. Therefore, we immunoprecipitated PrPC and the protease-resistant core of PrPSc (PrP27–30) from Rov and ScRov cells lysates. All of the mAbs reacted with PrPC, ICSM 18 exhibiting about twice the affinity of any mAbs raised against β-PrP (Table I and Fig. 4a, left panel). In stark contrast, only mAbs raised against β-PrP reacted strongly with PrP27–30, ICSM 37 immunoprecipitating 2-fold more PrPSc than ICSM 35 and 42 at an equivalent concentration (Table I and Fig. 4a, right panel). We then pulsed uninfected Rov cells with ICSM 18 or ICSM 19 (10 μg/ml overnight) and immunoprecipitated the PrPC-bound fraction after thoroughly washing the cells. Interestingly, ICSM 18 and ICSM 19 bound 62 ± 3 and 75 ± 10% PrPC molecules, whereas ICSM 35 and 37 bound, respectively, only 14 ± 3 and 33 ± 4% (Fig. 4, b and c). Similar experiments on ScRov cells were inconclusive, as we frequently observed non-specific binding of PrPSc to the beads used to bring down the antigen/antibody complexes (data not shown). However ICSM 35 and 37 do bind PrPSc in living cells. Using immunofluorescence microscopy, we found dotlike intracellular staining in ScRov cells (as indicated by an arrow) but not in Rov cells (Fig. 4d). This was similar to the staining of fixed ScRov cells with these mAbs after guanidium thiocyanate denaturation, 2V. Beringue and F. Archer, unpublished data. a treatment known to increase specifically PrPSc immunoreactivity (20Taraboulos A. Serban D. Prusiner S.B. J. Cell Biol. 1990; 110: 2117-2132Crossref PubMed Scopus (228) Google Scholar, 21Archer F. Bachelin C. Andreoletti O. Besnard N. Perrot G. Langevin C. Le Dur A. Vilette D. Baron-Van Evercooren A. Vilotte J.L. Laude H. J. Virol. 2004; 78: 482-490Crossref PubMed Scopus (72) Google Scholar). In contrast, we were unable to observe any differences in the binding of ICSM 18 and 19 between Rov and ScRov cells (Fig. 4d). Overall, these experiments indicate that mAbs raised against β-PrP bind endogenous intracellular PrPSc. Given that the transformation of normal cellular prion protein is central to the pathogenesis of prion disease, it is not surprising that most of the available therapeutic strategies target either normal or disease-related isoforms. However, success has been limited when translating methods that were shown to be effective in vitro to animal models and patients (22Brown P. Neurology. 2002; 58: 1720-1725Crossref PubMed Scopus (47) Google Scholar). Studies in neuroblastoma cells clearly indicate that targeting PrPC either by cleaving it from the cell surface with phosphatidylinositol-specific phospholipase C or stabilizing it with monoclonal antibodies inhibits prion replication very efficiently (7Enari M. Flechsig E. Weissmann C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9295-9299Crossref PubMed Scopus (373) Google Scholar, 8Peretz D. Williamson R.A. Kaneko K. Vergara J. Leclerc E. Schmitt-Ulms G. Mehlhorn I.R. Legname G. Wormald M.R. Rudd P.M. Dwek R.A. Burton D.R. Prusiner S.B. Nature. 2001; 412: 739-743Crossref PubMed Scopus (465) Google Scholar, 23Caughey B. Raymond G.J. J. Biol. Chem. 1991; 266: 18217-18223Abstract Full Text PDF PubMed Google Scholar). In our system, turning off the ovine PrP promoter completely abrogates prion replication (this study and Ref. 14Vilette D. Andreoletti O. Archer F. Madelaine M.F. Vilotte J.L. Lehmann S. Laude H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4055-4059Crossref PubMed Scopus (180) Google Scholar), analogous to the situation in PrP null mice that do not support prion replication (24Büeler H. Aguzzi A. Sailer A. Greiner R.A. Autenried P. Aguet M. Weissmann C. Cell. 1993; 73: 1339-1347Abstract Full Text PDF PubMed Scopus (1791) Google Scholar). However, not all PrPC-binding antibodies have inhibitory effects (Table I). Peretz et al. (8Peretz D. Williamson R.A. Kaneko K. Vergara J. Leclerc E. Schmitt-Ulms G. Mehlhorn I.R. Legname G. Wormald M.R. Rudd P.M. Dwek R.A. Burton D.R. Prusiner S.B. Nature. 2001; 412: 739-743Crossref PubMed Scopus (465) Google Scholar) elegantly show that artificially engineered Fabs were most potent when they targeted helix 1, a region to which ICSM 18 and mAb 6H4 bind (10Korth C. Stierli B. Streit P. Moser M. Schaller O. Fischer R. SchulzSchaeffer W. Kretzschmar H. Raeber A. Braun U. Ehrensperger F. Hornemann S. Glockshuber R. Riek R. Billeter M. Wuthrich K. Oesch B. Nature. 1997; 390: 74-77Crossref PubMed Scopus (537) Google Scholar, 15Beringue V. Mallinson G. Kaisar M. Tayebi M. Sattar Z. Jackson G. Anstee D. Collinge J. Hawke S. Brain. 2003; 126: 2065-2073Crossref PubMed Scopus (110) Google Scholar). Fabs binding the 90–109 region also inhibited prion replication (8Peretz D. Williamson R.A. Kaneko K. Vergara J. Leclerc E. Schmitt-Ulms G. Mehlhorn I.R. Legname G. Wormald M.R. Rudd P.M. Dwek R.A. Burton D.R. Prusiner S.B. Nature. 2001; 412: 739-743Crossref PubMed Scopus (465) Google Scholar), although interestingly, these artificially engineered antibodies do not recognize native PrPSc (11Williamson R.A. Peretz D. Pinilla C. Ball H. Bastidas R.B. Rozenshteyn R. Houghten R.A. Prusiner S.B. Burton D.R. J. Virol. 1998; 72: 9413-9418Crossref PubMed Google Scholar), unlike our mAbs raised against β-PrP in which the 90–109 region is immunodominant (this study). 3V. Beringue, G. Mallinson, M. Kaisar, M. Tayebi, and S. Hawke, unpublished observations. It is of some concern for the general applicability of cell lines as systems for screening anti-prion agents that prion replication in ScRov cells was inhibited inefficiently by mAbs binding helix 1 such as ICSM 18 that clearly have a very high affinity for PrPC and very efficiently inhibit the replication of mouse prions in ScN2a 3V. Beringue, G. Mallinson, M. Kaisar, M. Tayebi, and S. Hawke, unpublished observations. cells and in mice (12White A.R. Enever P. Tayebi M. Mushens R. Linehan J. Brandner S. Anstee D. Collinge J. Hawke S. Nature. 2003; 422: 80-83Crossref PubMed Scopus (412) Google Scholar). Although targeting PrPC exclusively is clearly an effective strategy in some cell lines, failure to inhibit infectious prions may allow the conversion to recur once the PrPC-binding inhibitor is removed. Thus infection is merely suppressed and not eradicated. Previous work indicates that ScN2a cells are curable with anti-PrPC antibodies (7Enari M. Flechsig E. Weissmann C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9295-9299Crossref PubMed Scopus (373) Google Scholar, 8Peretz D. Williamson R.A. Kaneko K. Vergara J. Leclerc E. Schmitt-Ulms G. Mehlhorn I.R. Legname G. Wormald M.R. Rudd P.M. Dwek R.A. Burton D.R. Prusiner S.B. Nature. 2001; 412: 739-743Crossref PubMed Scopus (465) Google Scholar), but clearly this does not apply to all cells capable of supporting prion replication as we show here. Perhaps this is mainly due to the level of PrPC.Itis worth noting that the clone successfully treated by mAb 6H4 expressed very low levels of PrPC (7Enari M. Flechsig E. Weissmann C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9295-9299Crossref PubMed Scopus (373) Google Scholar) compared with Rov cells that express similar levels of PrPC to those found in sheep brain (14Vilette D. Andreoletti O. Archer F. Madelaine M.F. Vilotte J.L. Lehmann S. Laude H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4055-4059Crossref PubMed Scopus (180) Google Scholar). One might anticipate that differential sensitivity to anti-prion agents may exist similarly in vivo, and continuous suppression with high concentrations of inhibitor may be required given that PrPC is widely expressed in variable amounts and rapidly turned over at the cell surface (25Bendheim P.E. Brown H.R. Rudelli R.D. Scala L.J. Goller N.L. Wen G.Y. Kascsak R.J. Cashman N.R. Bolton D.C. Neurology. 1992; 42: 149-156Crossref PubMed Google Scholar, 26Taraboulos A. Raeber A.J. Borchelt D.R. Serban D. Prusiner S.B. Mol. Biol. Cell. 1992; 3: 851-863Crossref PubMed Scopus (234) Google Scholar). Such a strategy may be employed with caution during the neuroinvasion course of the disease, because the administration of high doses of mAbs with high affinity for PrPC within the central nervous system may trigger neuronal apoptosis in vivo (27Solforosi L. Criado J.R. McGavern D.B. Wirz S. Sanchez-Alavez M. Sugama S. DeGiorgio L.A. Volpe B.T. Wiseman E. Abalos G. Masliah E. Gilden D. Oldstone M.B. Conti B. Williamson R.A. Science. 2004; 303: 1514-1516Crossref PubMed Scopus (308) Google Scholar). An alternative strategy is to target both PrPC and PrPSc, thereby blocking the incorporation of PrPC into propagating prions and additionally capping the infectious template. In these experiments, it was striking that the ability of each mAb to suppress prion replication correlated so well with its affinity for PrPSc but not for PrPC (Table I). Therefore it seems reasonable to suggest that PrPSc binding plays the major effector role, but differences between species and/or strains cannot be excluded despite helix I being highly conserved between species (28Oesch B. Westaway D. Prusiner S.B. Curr. Top. Microbiol. Immunol. 1991; 172: 109-124PubMed Google Scholar). The most potent mAbs bound significantly less PrPC than ISCM 18, and immunofluorescence confirmed that the mAbs raised against β-PrP bound disease-associated prion protein. In fact these studies correlated very well with the indirect immunoprecipitation. Thus mAbs exhibiting high affinity for PrPSc by immunoprecipitation stained intracellular organelles and inhibited prion replication very efficiently. Clearly, unless mAbs completely specific for PrPSc are used, it will not be possible to conclusively prove that PrPSc binding is crucial. It is possible, for example, that ovine PrPC is stabilized most efficiently by interactions at the 90–109 region. In any case, PrPC binding may be necessary for the mAbs to be internalized and/or presented to intracellular PrPSc. Currently, we are attempting to characterize mAbs that bind PrPSc exclusively and mAbs that bind to the N-terminal portion of PrP27–30 but that have low affinity for PrPSc (such as mAb 3F4) (29Kascsak R.J. Rubenstein R. Merz P.A. Tonna-DeMasi M. Fersko R. Carp R.I. Wisniewski H.M. Diringer H. J. Virol. 1987; 61: 3688-3693Crossref PubMed Google Scholar). However, taken together, we suggest that additional targeting of PrPSc may improve the efficacy of anti-prion agents. How could the mAbs interact with PrPSc? This may be direct if the PrPC conversion occurred at the cell surface (23Caughey B. Raymond G.J. J. Biol. Chem. 1991; 266: 18217-18223Abstract Full Text PDF PubMed Google Scholar). We have shown by immunofluorescence that our mAbs are internalized in contrast with recent studies in human cells (30Paitel E. Alves Da Costa C. Vilette D. Grassi J. Checler F. J. Neurochem. 2002; 83: 1208-1214Crossref PubMed Scopus (62) Google Scholar). If conversion occurred in endosomes, the antibodies may be internalized via PrPC and then bind to PrPSc whether or not they were released from PrPC. We did not find by immunofluorescence Fc receptors at the Rov cell surface, making Fc-mediated internalization of the mAbs unlikely (data not shown). Finally, the Rov cell system allowed a comparison of the kinetics of PrPSc inhibition to be studied in the absence of the PrPC substrate. Again, supporting a direct role for PrPSc binding, we found that several mAbs inhibited as rapidly as repressing PrPC expression and that one mAb, ICSM 37, was even more rapid, suggesting that it enhanced the breakdown of PrPSc. Interestingly, PrPSc was not released from the cells into the supernatant (data not shown). Perhaps mAb binding facilitates the intracellular clearance of PrPSc. Pulse-chase experiments are planned to study this intriguing phenomenon in greater detail. How does the work described here apply to our recent in vivo data (12White A.R. Enever P. Tayebi M. Mushens R. Linehan J. Brandner S. Anstee D. Collinge J. Hawke S. Nature. 2003; 422: 80-83Crossref PubMed Scopus (412) Google Scholar)? We have recently shown that both ICSM 18 and 35 effectively inhibit prion replication in vivo. In these experiments, it was not determined by which mechanism prion replication was inhibited or whether the infection was eradicated or merely suppressed. The fact that spleen PrPSc levels were lowered more efficiently with ICSM 18 compared with ICSM 35 and that the former mAb binds to native mouse PrPSc weakly suggests that targeting PrPC was the predominant inhibitory mechanism. Yet ICSM 35 was equally efficient at delaying the onset of clinical scrapie, and it is possible that the dose response curves for clinical effectiveness may not mirror those for PrPSc levels in the spleen. Isotype differences may also be relevant. Unfortunately, the short half-life of antibody fragments precludes a direct comparison of the variable regions without elaborate genetic engineering. Two reports using ScN2a cells have suggested that prion-infected cultures could be cured after long term treatment with antibodies (7Enari M. Flechsig E. Weissmann C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9295-9299Crossref PubMed Scopus (373) Google Scholar, 8Peretz D. Williamson R.A. Kaneko K. Vergara J. Leclerc E. Schmitt-Ulms G. Mehlhorn I.R. Legname G. Wormald M.R. Rudd P.M. Dwek R.A. Burton D.R. Prusiner S.B. Nature. 2001; 412: 739-743Crossref PubMed Scopus (465) Google Scholar). However, the ScN2a cells have levels of infectivity that are much lower than the ScRov cells (31Sabuncu E. Petit S. Le Dur A. Lan Lai T. Vilotte J.L. Laude H. Vilette D. J. Virol. 2003; 77: 2696-2700Crossref PubMed Scopus (51) Google Scholar) and therefore may be easier to cure. We showed in one of our experiments that mAb treatment of ScRov cells resulted in a 100–1000-fold decrease in PrPSc levels (Fig. 1). However, replication re-started when the treatment was stopped, an effect noted with both antibodies and DS500. This finding indicates that a cellular reservoir of PrPSc remained untouched by the antibodies or DS500 providing a template for conversion to recur. It is tempting to speculate that aggregates of PrPSc may be involved. Several sites of post-exposure therapeutic intervention could be envisaged in prion diseases. One comprises the central nervous system, and in vitro investigations in ScN2a cells have shown that mAbs might also in theory prevent PrPSc accumulation in neurons (7Enari M. Flechsig E. Weissmann C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9295-9299Crossref PubMed Scopus (373) Google Scholar, 8Peretz D. Williamson R.A. Kaneko K. Vergara J. Leclerc E. Schmitt-Ulms G. Mehlhorn I.R. Legname G. Wormald M.R. Rudd P.M. Dwek R.A. Burton D.R. Prusiner S.B. Nature. 2001; 412: 739-743Crossref PubMed Scopus (465) Google Scholar). Our study has shown that mAbs also were able to prevent PrPSc production in an epithelial cell line. These cells may play an important role in the spread of the infection in periphery (32Heppner F.L. Christ A.D. Klein M.A. Prinz M. Fried M. Kraehenbuhl J.P. Aguzzi A. Nat. Med. 2001; 7: 976-977Crossref PubMed Scopus (187) Google Scholar). In addition, the engineering of transgenic mice to produce anti-prion mAbs (9Heppner F.L. Musahl C. Arrighi I. Klein M.A. Rulicke T. Oesch B. Zinkernagel R.M. Kalinke U. Aguzzi A. Science. 2001; 294: 178-182Crossref PubMed Scopus (307) Google Scholar) or passive immunization (12White A.R. Enever P. Tayebi M. Mushens R. Linehan J. Brandner S. Anstee D. Collinge J. Hawke S. Nature. 2003; 422: 80-83Crossref PubMed Scopus (412) Google Scholar) have shown that they could prevent the generation of infectivity in the spleen when these mice were infected with scrapie. Therefore, antibodies may target different pathways of prion pathogenesis encompassing the transit from the site of infection, its early propagation in periphery and more lately in the central nervous system, and thus be an efficient therapy regardless of the incubation stage in the infected individuals. We thank R. Young for graphic editing.
The supply of blood, blood products and components in the UK, as elsewhere, is safe, although there is no cause for complacency. Use of blood, blood products and components is not without risk of morbidity and mortality. Transfusion-transmitted infections (TTIs) continue to occur and may severely affect the health and welfare of recipients. As indicated by recent and current inquiries, public interest in these TTIs is huge. The risk of TTI can be mitigated but not abolished. Measures to reduce risk include screening of donors, testing of donations and, where appropriate, treatment of donations. The introduction of newer screening tests might identify some infectious donations but come at a cost, which could exceed a justifiable limit. Thus, the recognition, detection, reporting and investigation of cases of possible TTIs need to be improved. Recipients of blood should understand that, although transfusion in the UK is safe, it is not free of risk and so should be provided with full information so that properly informed consent can be given.
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