A novel anti-prion protein monoclonal antibody and its single-chain fragment variable derivative with ability to inhibit abnormal prion protein accumulation in cultured cells.

mAbs T1 and T2 were established by immunizing PrP gene ablated mice with recombinant MoPrP of residues 121-231. Both mAbs were cross-reactive with PrP from hamster, sheep, cattle and deer. A linear epitope of mAb T1 was identified at residues 137-143 of MoPrP and buried in PrP(C) expressed on the cell surface. mAb T1 showed no inhibitory effect on accumulation of PrP(Sc) in cultured scrapie-infected neuroblastoma (ScN2a) cells. In contrast, mAb T2 recognized a discontinuous epitope ranged on, or structured by, residues 132-217 and this epitope was exposed on the cell surface PrP(C). mAb T2 showed an excellent inhibitory effect on PrP(Sc) accumulation in vitro at a 50% inhibitory concentration of 0.02 microg/ml (0.14 nM). The scFv form of mAb T2 (scFv T2) was secreted in neuroblastoma (N2a58) cell cultures by transfection through eukaryotic secretion vector. Coculturing of ScN2a cells with scFv T2-producing N2a58 cells induced a clear inhibitory effect on PrP(Sc) accumulation, suggesting that scFv T2 could potentially be an immunotherapeutic tool for prion diseases by inhibition of PrP(Sc) accumulation.

Humanized knock-in mice expressing chimeric prion protein showed varied susceptibility to different human prions.

Mice to which human prions efficiently transmit in short incubation periods are valuable not only as research tools of human prions but also as reliable diagnostic tools. We recently produced a line of knock-in mouse expressing a unique human-mouse chimeric PrP (Ki-ChM mouse), which has mouse-specific residues practically only at the C-terminal part after posttranslational modification, and here we attempted transmission of various human prions to assess the susceptibility profile of the mouse. Susceptibility varied considerably depending on prions inoculated: highly susceptible to MM1 and MV1 types of sporadic Creutzfeldt-Jakob disease (CJD), developing disease within approximately 150 days, familial CJD with M232R mutation, and dura graft-associated CJD (dCJD) without amyloid plaque; less susceptible to MM2-type sporadic CJD and variant CJD, with some mice lacking any sign of transmission; and totally resistant to VV2 type sporadic CJD and dCJD with amyloid plaque. The rather short incubation time achieved by Ki-ChM mice suggests new approaches to produce mice that develop prion disease with very short incubation periods. We compared the characteristic susceptibility profile of Ki-ChM with those of other precedent transgenic mice and discussed, including the prospects in developing genetically engineered mice susceptible to human prions.

Association of an 11-12 kDa protease-resistant prion protein fragment with subtypes of dura graft-associated Creutzfeldt-Jakob disease and other prion diseases.

Creutzfeldt-Jakob disease can develop in subjects given a cadaveric dura mater graft (dCJD). This disease has a phenotypic heterogeneity despite the lack of genetic variation. Numerous plaque-type prion protein (PrP) deposits are found in the brain of some but not all subjects; hence, there may be two subtypes of this clinical entity. To validate dCJD subtypes further, we carried out a larger-scale clinicopathological analysis and typing of protease-resistant PrP (PrP(Sc)) in dCJD cases. Cases with plaque-type PrP deposits (p-dCJD) were shown to be distinct from those without PrP plaques (np-dCJD), from several clinicopathological aspects. Analysis of PrP(Sc) revealed that, while the major PrP(Sc) species from both subtypes was of 21 kDa after deglycosylation (type 1 PrP(Sc)), a C-terminal PrP fragment of 11-12 kDa (fPrP11-12) was associated with np-dCJD but not with p-dCJD. The disease type-specific association of fPrP11-12 was also observed in subjects with other prion diseases. An fPrP11-12-like C-terminal PrP fragment was detected in brain lysates from patients associated with fPrP11-12, but not from patients or normal subjects unassociated with fPrP11-12. Results indicated that fPrP was produced by CJD-associated processes in vivo. The present data provide several lines of evidence that support the need for subtyping of dCJD and contribute to the understanding of the processing of disease-specific PrP species. The unique relationship of fPrP11-12 with CJD phenotype supports the view that the phenotypic heterogeneity of CJD is related to the formation of different types of disease-specific PrP and fragments thereof.

[Prion protein structure and its relationships with pathogenesis].

In order to explore the structural domains of prion protein (PrP) that are required for the isoform conversion, prion formation and neurodegenerative effects, we designed a series of PrP deletion mutants and studied, using prion-infected cultured cells and transgemic (Tg) mice, 1) if these mutants can be converted to the abnormal isoform, 2) constitute prions, and 3) cause neurodegeneration when converted and accumulated in mouse brains. We discovered that a mutant PrP with deletions at the N-terminus and the middle portion retained all the three abilities. The molecule named PrP106 was composed of 106 amino acids, nearly a half of 208 composing wild type PrP. The abnormal isoform of PrP106 (PrPSc106) that had been purified from prion-infected Tg mice proved to share, with the abnormal isoform of wild type PrP (PrPSc), unique properties such as high beta-sheet content and propensity to form fibrous aggregates. These findings rationalized the structural analysis of PrPSc106 in comparison with PrPSc. The structure of 2D crystals of the two abnormal PrP isoforms was studied using electron-microscopy, and the data helped to make the 3D structural model of abnormal PrP isoform. Studies with Tg mice expressing other mutant PrPs are currently under way.

Follicular dendritic cell of the knock-in mouse provides a new bioassay for human prions.

Infectious prion diseases initiate infection within lymphoid organs where prion infectivity accumulates during the early stages of peripheral infection. In a mouse-adapted prion infection, an abnormal isoform (PrP(Sc)) of prion protein (PrP) accumulates in follicular dendritic cells within lymphoid organs. Human prions, however, did not cause an accumulation of PrP(Sc) in the wild type mice. Here, we report that knock-in mouse expressing humanized chimeric PrP demonstrated PrP(Sc) accumulations in follicular dendritic cells following human prion infections, including variant Creutzfeldt-Jakob disease. The accumulated PrP(Sc) consisted of recombinant PrP, but not of the inoculated human PrP. These accumulations were detectable in the spleens of all mice examined 30 days post-inoculation. Infectivity of the spleen was also evident. Conversion of humanized PrP in the spleen provides a rapid and sensitive bioassay method to uncover the infectivity of human prions. This model should facilitate the prevention of infectious prion diseases.