Clearance and prevention of prion infection in cell culture by anti‐PrP antibodies

Abstract

Prion diseases are transmissible and invariably fatal neurodegenerative disorders associated with a conformational transformation of the cellular prion protein (PrP C ) into a self-replicating and proteinase K (PK)-resistant conformer, scrapie PrP (PrP Sc ). Humoral immunity may significantly prolong the incubation period and even prevent disease in murine models of prionoses. However, the mechanism(s) of action of anti-PrP monoclonal antibodies (Mabs) remain(s) obscure. The murine neuroblastoma N2a cell line, infected with the 22L mouse-adapted scrapie strain, was used to screen a large library of Mabs with similar binding affinities to PrP, to identify those antibodies which could clear established infection and/or prevent infection de novo. Three Mabs were found capable of complete and persistent clearing of already-infected N2a cells of PrP Sc . These antibodies were 6D11 (generated to PK-resistant PrP Sc and detecting PrP residues 93-109), and 7H6 and 7A12, which were raised against recombinant PrP and react with neighbouring epitopes of PrP residues 130-140 and 143-155, respectively. Mabs were found to interact with PrP Sc formation both on the cell surface and after internalization in the cytosol. Treatment with Mabs was not associated with toxicity nor did it result in decreased expression of PrP C . Both preincubation of N2a cells with Mabs prior to exposure to 22L inoculum and preincubation of the inoculum with Mabs prior to infecting N2a cells resulted in a significant reduction in PrP Sc levels. Information provided in these studies is important for the rational design of humoral immune therapy for prion infection in animals and eventually in humans.

An N-terminal Polybasic Domain and Cell Surface Localization Are Required for Mutant Prion Protein Toxicity

“…Importantly, two of these regions (23-31 and 95-125) overlap with those that we have shown here to be critical for the functional activity of PrP C , as determined by the DBCA and patch clamp measurements. Ligands (GAGs and monoclonal antibodies) that bind to these two regions have been shown to inhibit PrP Sc formation in vitro and in vivo (16,68,69). Strikingly, we have found that some of these same ligands (e.g.…”

“…Western Blotting and Immunofluorescence Staining-Western blots were probed with anti-PrP antibodies 6D11 (16) or 6H4 (17) followed by goat anti-mouse IgG (Pierce) and developed with ECL (GE Healthcare). Some of the samples in Fig.…”

Neurotoxic Mutants of the Prion Protein Induce Spontaneous Ionic Currents in Cultured Cells

“…Western Blotting and Immunofluorescence Staining-Western blots were probed with anti-PrP antibody 6D11 (18) and goat anti-mouse IgG (Pierce) and developed with ECL (GE Healthcare). Samples for each blot were normalized for total protein following measurement with the BCA protein assay (Pierce).…”

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

“…Alternatively, PrP in S and P fractions was immunoprecipitated with 15B3 or motif-grafted antibodies, as described below. Western blots were developed with anti-PrP antibodies 6D11 (Pankiewicz et al 2006) or 8H4 (Zanusso et al 1998) using the enhanced chemiluminescent detection system (Amersham Biosciences, Piscataway, NJ, USA).…”