A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions.

Prions are usually quantified by bioassays based on intracerebral inoculation of mice that are slow, imprecise, and costly. We have isolated neuroblastoma N2a sublines highly susceptible to mouse prions, as evidenced by accumulation of infectivity and the scrapie form of prion protein (PrPSc), and developed quantitative in vitro assays for prion infectivity. In the scrapie cell (SC) assay, susceptible N2a cells are exposed to prion-containing samples for 3 days, grown to confluence, and split 1:10 three times, and the proportion of PrPSc-containing cells is determined with automated counting equipment. In a log/log plot, the dose-response is linear over two logs of prion concentrations. The SC assay is about as sensitive as the mouse bioassay, 10 times faster, >2 orders of magnitude less expensive, and suitable for robotization. SC assays performed in a more time-consuming end point titration format extend the sensitivity and show that infectivity titers measured in tissue culture and in the mouse are similar.

CD4+ T cell-mediated immunity against prion proteins.

The prion protein (PrP(C)) is essential for susceptibility to transmissible spongiform encephalopathies. A specific conformer of this protein (PrP(Sc)) is, according to the 'protein only' hypothesis, the principal or only component of the infectious agent, designated prion. Transmission of prions between species is often inefficient, resulting in low attack rates and/or prolonged incubation times and is ascribed to a 'species barrier' caused by differences in the amino acid sequence of PrP between recipient and donor. In this report, we demonstrate that these differences in amino acid sequence result in presentation of distinct peptides on major histocompatibility complex class II molecules. These peptides result in activation of specific CD4+ T cells which leads to the induction of an effective immune response against foreign PrP as demonstrated by antibody production. Therefore, CD4+ T cells represent a crucial component of the immune system to distinguish between foreign and self PrP.

Two new proteases in the MHC class I processing pathway.

The proteasome generates exact major histocompatibility complex (MHC) class I ligands as well as NH2-terminal-extended precursor peptides. The proteases responsible for the final NH2-terminal trimming of the precursor peptides had, until now, not been determined. By using specific selective criteria we purified two cytosolic proteolytic activities, puromycin-sensitive aminopeptidase and bleomycin hydrolase. These proteases could remove NH2-terminal amino acids from the vesicular stomatitis virus nucleoprotein cytotoxic T cell epitope 52-59 (RGYVYQGL) resulting, in combination with proteasomes, in the generation of the correct epitope. Our data provide evidence for the existence of redundant systems acting downstream of the proteasome in the antigen-processing pathway for MHC class I molecules.

Generation of the vesicular stomatitis virus nucleoprotein cytotoxic T lymphocyte epitope requires proteasome-dependent and -independent proteolytic activities.

The proteasome is involved in the generation of most of the MHC class I antigenic epitopes. However, it is not known if the proteasome generates the exact cytotoxic T lymphocyte (CTL) epitope or only epitope precursors which require further modification by additional proteases. Digestion of the extended vesicular stomatitis virus nucleoprotein epitope 52-59 (RGYVYQGL) by the 20S proteasome in vitro shows that the proteasome is capable of generating the correct C terminus but not the exact N terminus of the CTL epitope. This finding suggests that proteolytic activity in addition to the proteasome is required for generation of the CTL epitope. By using the proteasome inhibitor lactacystin we were able to confirm this finding in vivo. Lactacystin prevented the processing of N- and C-terminally extended epitopes, whereas the processing of only N-terminally extended epitopes was unaffected. Thus, the proteasome is necessary and sufficient for the generation of the exact C terminus of this CTL epitope, whereas the exact N terminus seems to be generated by a different protease.