R. Anthony Williamson

Antibodies inhibit prion propagation and clear cell cultures of prion infectivity.

Prions are the transmissible pathogenic agents responsible for diseases such as scrapie and bovine spongiform encephalopathy. In the favoured model of prion replication, direct interaction between the pathogenic prion protein (PrPSc) template and endogenous cellular prion protein (PrPC) is proposed to drive the formation of nascent infectious prions. Reagents specifically binding either prion-protein conformer may interrupt prion production by inhibiting this interaction. We examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with PrPSc. Here we show that antibodies binding cell-surface PrPC inhibit PrPSc formation in a dose-dependent manner. In cells treated with the most potent antibody, Fab D18, prion replication is abolished and pre-existing PrPSc is rapidly cleared, suggesting that this antibody may cure established infection. The potent activity of Fab D18 is associated with its ability to better recognize the total population of PrPC molecules on the cell surface, and with the location of its epitope on PrPC. Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrPC for drug targeting.

Measuring prions causing bovine spongiform encephalopathy or chronic wasting disease by immunoassays and transgenic mice

There is increasing concern over the extent to which bovine spongiform encephalopathy (BSE) prions have been transmitted to humans, as a result of the rising number of variant Creutzfeldt–Jakob disease (vCJD) cases. Toward preventing new transmissions, diagnostic tests for prions in livestock have been developed using the conformation-dependent immunoassay (CDI), which simultaneously measures specific antibody binding to denatured and native forms of the prion protein (PrP). We employed high-affinity recombinant antibody fragments (recFab) reacting with residues 95–105 of bovine (Bo) PrP for detection and another recFab that recognizes residues 132–156 for capture in the CDI. We report that the CDI is capable of measuring the disease-causing PrP isoform (PrPSc) in bovine brainstems with a sensitivity similar to that of end-point titrations in transgenic (Tg) mice expressing BoPrP. Prion titers were ∼107 ID50 units per gram of bovine brainstem when measured in Tg(BoPrP) mice, a figure ∼10 times greater than that determined by bioassay in cattle and ∼10,000× greater than in wild-type mice. We also report substantial differences in BoPrPSc levels in different areas of the obex region, where neuropathology has been consistently observed in cattle with BSE. The CDI was able to discriminate between PrPSc from BSE-infected cattle and Tg(BoPrP) mice as well as from chronic wasting disease (CWD)-infected deer and elk. Our findings argue that applying the CDI to livestock should considerably reduce human exposure to animal prions.

Strain-specified relative conformational stability of the scrapie prion protein

Studies of prion biology and diseases have elucidated several new concepts, but none was more heretical than the proposal that the biological properties that distinguish different prion strains are enciphered in the disease‐causing prion protein (PrPSc). To explore this postulate, we examined the properties of PrPSc from eight prion isolates that propagate in Syrian hamster (SHa). Using resistance to protease digestion as a marker for the undenatured protein, we examined the conformational stabilities of these PrPSc molecules. All eight isolates showed sigmoidal patterns of transition from native to denatured PrPSc as a function of increasing guanidine hydrochloride (GdnHCl) concentration. Half‐maximal denaturation occurred at a mean value of 1.48 M GdnHCl for the Sc237, HY, SHa(Me7), and MT‐C5 isolates, all of which have ∼75‐d incubation periods; a concentration of 1.08 M was found for the DY strain with a ∼170‐d incubation period and ∼1.25 M for the SHa(RML) and 139H isolates with ∼180‐d incubation periods. A mean value of 1.39 M GdnHCl for the Me7‐H strain with a ∼320‐d incubation period was found. Based on these results, the eight prion strains segregated into four distinct groups. Our results support the unorthodox proposal that distinct PrPSc conformers encipher the biological properties of prion strains.

Single-Cell Repertoire Analysis Demonstrates that Clonal Expansion Is a Prominent Feature of the B Cell Response in Multiple Sclerosis Cerebrospinal Fluid

Single-cell RT-PCR was used to sample CD19+ B cell repertoires in cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) or viral meningitis. Analysis of amplified Ab H and L chain products served to identify the rearranged germline segment and J segment, and to determine the degree of homology for the H and L chain sequence of individual B cells. The B cell repertoire of viral meningitis CSF was predominately polyclonal, whereas B cell clonal expansion was a prominent feature of the IgG repertoire in three of four MS patients. Two dominant clonal populations in one MS CSF accounted for ∼70% of the IgG H chain V regions sequenced, while the corresponding IgM repertoires were more heterogeneous. One clonal B cell population revealed multiple L chain rearrangements, raising the possibility of a role for receptor editing in shaping the B cell response in some MS patients. The most immediate implications of identifying rearranged Ig sequences in MS B cells is the potential to accurately recreate recombinant Abs from these overrepresented H and L chains that can be used to discover the relevant Ag(s) in MS.

A change in the conformation of prions accompanies the emergence of a new prion strain.

To investigate the role of the pathogenic prion protein (PrP(Sc)) in controlling susceptibility to foreign prions, two Syrian hamster (SHa) prion strains, Sc237 and DY, were transmitted to transgenic mice expressing chimeric SHa/mouse PrP genes, Tg(MH2M). First passage of SHa(Sc237) prions exhibited prolonged incubation times, diagnostic of a species barrier. PrP(Sc) of the new MH2M(Sc237) strain possessed different structural properties from those of SHa(Sc237), as demonstrated by relative conformational stability measurements. This change was accompanied by a disease phenotype different from the SHa(Sc237) strain. Conversely, transmission of SHa(DY) prions to Tg(MH2M) mice showed no species barrier, and the MH2M(DY) strain retained the conformational and disease-specific properties of SHa(DY). These results suggest a causal relationship between species barriers, changes in PrP(Sc) conformation, and the emergence of new prion strains.

Trafficking of prion proteins through a caveolae-mediated endosomal pathway

To understand the posttranslational conversion of the cellular prion protein (PrPC) to its pathologic conformation, it is important to define the intracellular trafficking pathway of PrPC within the endomembrane system. We studied the localization and internalization of PrPC in CHO cells using cryoimmunogold electron microscopy. At steady state, PrPC was enriched in caveolae both at the TGN and plasma membrane and in interconnecting chains of endocytic caveolae. Protein A–gold particles bound specifically to PrPC on live cells. These complexes were delivered via caveolae to the pericentriolar region and via nonclassical, caveolae-containing early endocytic structures to late endosomes/lysosomes, thereby bypassing the internalization pathway mediated by clathrin-coated vesicles. Endocytosed PrPC-containing caveolae were not directed to the ER and Golgi complex. Uptake of caveolae and degradation of PrPC was slow and sensitive to filipin. This caveolae-dependent endocytic pathway was not observed for several other glycosylphosphatidyl inositol (GPI)-anchored proteins. We propose that this nonclassical endocytic pathway is likely to determine the subcellular location of PrPC conversion.

Antibodies Produced by Clonally Expanded Plasma Cells in Multiple Sclerosis Cerebrospinal Fluid

Intrathecal IgG synthesis, persistence of bands of oligoclonal IgG, and memory B‐cell clonal expansion are well‐characterized features of the humoral response in multiple sclerosis (MS). Nevertheless, the target antigen of this response remains enigmatic.

Transfer of scrapie prion infectivity by cell contact in culture.

BACKGROUND When a cell is infected with scrapie prions, newly synthesized molecules of the prion protein PrP(C) are expressed at the cell surface and may subsequently be converted to the abnormal form PrP(Sc). In an experimental scrapie infection of an animal, the initial innoculum of PrP(Sc) is cleared relatively rapidly, and the subsequent propagation of the infection depends on the ability of infected cells to convert uninfected target cells to stable production of PrP(Sc). The mechanism of such cell-based infection is not understood. RESULTS We have established a system in dissociated cell culture in which scrapie-infected mouse SMB cells are able to stably convert genetically marked target cells by coculture. After coculture and rigorous removal of SMB cells, the target cells express PrP(Sc) and also incorporate [35S]methionine into PrP(Sc). The extent of conversion was sensitive to the ratio of the two cell types, and conversion by live SMB required 2500-fold less PrP(Sc) than conversion by a cell-free prion preparation. The conversion activity of SMB cells is not detectable in conditioned medium and apparently depends on close proximity or contact, as evidenced by culturing the SMB and target cells on neighboring but separate surfaces. SMB cells were killed by fixation in aldehydes, followed by washing, and were found to retain significant activity at conversion of target cells. CONCLUSIONS Cell-mediated infection of target cells in this culture system is effective and requires significantly less PrP(Sc) than infection by a prion preparation. Several lines of evidence indicate that it depends on cell contact, in particular, the activity of aldehyde-fixed infected cells.

Toward Molecular Dissection of PrPC-PrPSc Interactions

Direct interaction between endogenous cellular prion protein (PrPC) and misfolded, disease-associated (PrPSc) conformers is a key event in prion propagation, which precedes templated conversion of PrPC into nascent PrPSc and prion infectivity. Although almost none of the molecular details of this pivotal process are understood, the persistence of individual prion strains suggests that assembly of the prion replicative complex is mechanistically precise. To systematically map defined regions of PrPC sequence that bind tightly to PrPSc, we have generated a comprehensive panel of over 45 motif-grafted antibodies containing overlapping peptide grafts collectively spanning PrP residues 19–231. Grafted antibody binding experiments, performed under stringent conditions, clearly identified only three distinct and independent high affinity PrPSc recognition motifs. The first of these binding motifs lies at the very N-terminal region of the mature PrP molecule within PrP-(23–33); the second motif lies within PrP-(98–110); and the third is contained within PrP-(136–158). Mutational analyses of these PrPSc-binding regions revealed that reactivity of the 23–33 and 98–110 segments are largely dependent upon the presence of multiple positively charged amino acid residues. These studies yield new insight into critical peptidic components composing one side of the prion replicative interface.

Comparative Analysis of the CD19+ and CD138+ Cell Antibody Repertoires in the Cerebrospinal Fluid of Patients with Multiple Sclerosis

Increased amounts of intrathecally synthesized IgG and oligoclonal bands have long been recognized as a hallmark of multiple sclerosis (MS). B cells and plasma cells are components of the inflammatory infiltrates in both active and chronic MS lesions, and increased numbers of these cells are present in MS cerebrospinal fluid (CSF). Single-cell RT-PCR was used to analyze both the CD19+ B cell and CD138+ plasma cell populations in CSF of two patients with clinically definite MS and of one MS patient whose CSF was obtained after a clinically isolated syndrome, but before the second episode. Sequence analysis of amplified IgG V region sequences identified the rearranged germline segments, extent of somatic mutation, and clonal relationships within and between the two cell populations in the three MS patients. Expanded B cell and plasma cell clones were detected in each MS CSF and in all three patients the CD138+ IgG repertoire was more restricted. However, little if any significant sequence overlap was observed between the CD19+ and CD138+ repertoires of each donor. Detection of plasma cell clones by single-cell PCR will facilitate the in vitro production of recombinant Abs useful in identifying disease-relevant Ags.