Inhibition of Prion Propagation in

Inhibition of Prion Propagation in

Scrapie-infected Cell Lines using Mouse Monoclonal Antibodies against Prion Protein

Kazuyoshi Miyamoto^ Naoto Nakamura\ Noriyuki Nishida^, Takashi Yokoyama^, Masayoshi

^ and Hämo Matsuda^'^

Yokoyama^, Masayoshi Aosasa"^, Hiroyuki Horiuchi^'"^, Shuichi Furusawa^'

^Laboratory of Immunobiology, Department of Molecular and Applied Bioscience, Graduate School of Biosphere Science, Hiroshima University,

1-4-4 Kagamiyama, Higasi-Hiroshima 739-8528 Japan ^Department of Molecular Microbiology and Immunology, Graduate School of Medical Sciences, Nagasaki University ^National Institute of Animal Health, and

"^Hiroshima Prefectural Institute of Industrial Science and Technology

<e-mail> miyamoto@hiroshima-u.ac.jp

Abstract

Introduction

Monoclonal antibodies (mAbs) against prion protein (PrP) are useful for diagnosis of prion diseases as well as for research into the conformational change from the cellular isoform (PrP^) to the abnormal isoform (PrP^'^).

Recent reports indicate that some PrP-specific mAbs are able to inhibit prion propagation and exclude PrP^"" both in vitro and in vivo. However, no effective therapies against prion diseases currently exist and the mecha- nisms of prevention by PrP-specific antibodies are uncertain. Therefore, production and identification of PrP-specific mAbs for analysis of the PrP conformational change are important. In this study, we attempted to se- lect mouse mAbs that inhibit PrP conversion and present here two such in- hibitory mAbs.

Materials and Methods

In this study, five mouse mAbs specific for PrP generated by cell fusion using PrP^'^^ mice immunized with recombinant PrP were used and their specificity was characterized by ELISA and Western blotting analyses.

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To identify mAbs that inhibit conversion from PrP^ to PrP^^, an inhibition assay of prion propagation was performed using two scrapie-infected neuroblastoma cell lines, N2a/22L and N2a/Chandler. Cells treated with 10

|ig/ml of mAbs for 4 to 16 days were further cultured in the absence of mAbs for 4 to 16 days. After culture, PrP^^ in the cells was analyzed by Western blotting.

Results and Discussion

Of the five mAbs used, 3S9 and 22L/2H9 (2H9), which recognize both PrP^ and PrP^^, were identified as inhibitory mAbs. 3S9 recognized mouse and sheep PrPs, while the 2H9 recognized mouse and hamster PrPs.

Both mAbs inhibited the conversion of PrP^ to PrP^^ in the two

prion-infected cell lines used. These mAbs were also able to exclude

PrP^^ in these cells and inhibited accumulation of PrP^^ in PrP^^-expressing

cells. Interestingly, these mAbs recognized different PrP epitopes; 3S9

recognized residues 141-161, which includes the helix 1 region of PrP,

while 2H9 recognized residues 151-221. These results indicate that dif-

ferent site(s) from the helix 1 region may be also important for the PrP

conformational change and that the two mAbs selected may be useful for

analyzing the prevention mechanisms of prion propagation.