Protective anti‐prion antibodies in human immunoglobulin repertoires

Abstract

Prion immunotherapy may hold great potential, but antibodies against certain PrP epitopes can be neurotoxic. Here, we identified > 6,000 PrP‐binding antibodies in a synthetic human Fab phage display library, 49 of which we characterized in detail. Antibodies directed against the flexible tail of PrP conferred neuroprotection against infectious prions. We then mined published repertoires of circulating B cells from healthy humans and found antibodies similar to the protective phage‐derived antibodies. When expressed recombinantly, these antibodies exhibited anti‐PrP reactivity. Furthermore, we surveyed 48,718 samples from 37,894 hospital patients for the presence of anti‐PrP IgGs and found 21 high‐titer individuals. The clinical files of these individuals did not reveal any enrichment of specific pathologies, suggesting that anti‐PrP autoimmunity is innocuous. The existence of anti‐prion antibodies in unbiased human immunological repertoires suggests that they might clear nascent prions early in life. Combined with the reported lack of such antibodies in carriers of disease‐associated PRNP mutations, this suggests a link to the low incidence of spontaneous prion diseases in human populations.

Soluble dimeric prion protein ligand activates Adgrg6 receptor but does not rescue early signs of demyelination in PrP-deficient mice

“…Under non-reducing conditions FT 2 Fc predominantly existed as a dimer (56 kD), whereas in the presence of reducing agents (DTT) it migrated as a monomer ( Fig 1B ). We confirmed the identity of the secreted protein by western blotting using either anti-mouse IgG antibodies or a monomeric antibody Fab fragment specifically targeting the KKRPK domain of FT [ 23 ] (Fab83). Both antibodies detected FT 2 Fc in cell culture supernatant as well as purified FT 2 Fc ( Fig 1B ).…”

“…Finally, we immunoprecipitated FT 2 Fc from cell culture supernatant using beads coupled with Fab83. Peptides derived from the linear sequence of mouse PrP were previously used to map the epitope of Fab83 [ 23 ]. FT 2 Fc could be eluted from the beads with a peptide competing for the Fab83 binding site (amino acids 23–34 of mouse PrP), but not with a non-competing peptide (amino acids 53–64) ( Fig 1D ).…”

“…The following primary antibodies were used for western blotting: phospho-AKT (1:1000, Cell Signaling Technologies, 4060S), AKT (1:1000, Cell Signaling Technologies, 4685S), GFAP (1:2000, Cell Signaling Technologies, 12389S), c-Jun (1:1000, 9165s), Egr2 (1:2000, Abcam, ab108399), Calnexin (1:2000, Enzo, ADI-SPA-865-D), Actin (1:10’000, Milipore, MAB1501R). In addition, we used an in-house produced Fab-fragment directed against the N-terminus of PrP [ 23 ] (Fab83, 6 μg/ml). The following horseradish peroxidase coupled secondary antibodies were used: anti-mouse IgG (1:10’000, Jackson Immuno Research, 115-035-003), anti-rabbit IgG (1:4000, Jackson Immuno Research, 111-035-003), anti-human Fab (1:7000, Sigma, A0293).…”

“…Immunoprecipitation (IP) of FT 2 Fc from cell culture supernatant was performed as previously described with minor modifications [ 23 ]. Briefly, sheep-anti mouse IgG paramagnetic beads (Dynal, 11201D) were coupled with anti-His mAb (Invitrogen, 37–2900) in coating buffer (PBS plus 0.1% immunoglobulin-free BSA) for 2 h at RT on a rotating wheel.…”

The ultrastructure of infectious L-type bovine spongiform encephalopathy prions constrains molecular models

“…The Fab fragments were selected from a phage display library. Fab 69 and Fab 29 react with epitopes within residues 100 GWG QGGTHGQW 110 and 227 TQYQRESQAYY 237 located at N-and C-terminal regions of the bovine prion protein, respectively [52]. The Fabs were expressed in E.coli (BL21(DE3)) as a His-tagged, soluble, and functional protein.…”