Assessment of the risk of mutations in the PrP gene associated with familial Creutzfeldt-Jakob disease: implications for the safety of cell substrates used to prepare vaccines and other biologics. Background: Agents of transmissible spongiform encephalopathies (TSE) must be considered as potential adventitious agents in biologics. There have been precedents when vaccines accidentally prepared from tissues of scrapie-infected sheep caused scrapie outbreaks involving hundreds of immunized animals. Emergence of new-variant Creutzfeldt-Jakob disease (vCJD) and its probable link to bovine spongiform encephalopathy (BSE) made this an important issue for human vaccines and other biologics as well. The risk is best addressed by careful selection of source materials used to produce biologics. However, there is another hypothetical mechanism by which human biologics produced in cell cultures can become contaminated with TSE agents. According to the most widely held opinion (the "prion" hypothesis, not universally accepted), TSE agents can emerge de novo by aberrant folding of a normal protein produced by a cellular gene--the prion protein or PrP. Such transition from a normal protein to its infectious form (associated with an increase in beta-pleated-sheet content, loss of solubility in detergent solutions and markedly increased resistance to digestion by the enzyme proteinase K) is thought to be facilitated by any of a number of point mutations or reduplications in the PrP-encoding gene. These mutations are consistently found in one of the two PrP genes of patients with various familial forms of CJD, which is inherited as an autosomal dominant trait of variable penetrance depending on the specific mutation involved. Since cell cultures used as substrates to prepare biologics are serially propagated in vitro in extremely large numbers, there is a theoretical but troubling possibility that spontaneous mutation in the PrP gene might result in contamination of vaccines with infectious protein. Although this scenario is purely hypothetical, it cannot be discounted until experimental evaluation of infectivity of the mutant PrP gene products is performed. Rationale and experimental design: To prove that spontaneous PrP gene mutations that may occur in candidate cell substrates pose no risk for vaccine recipients, it is proposed to engineer Vero cells (and possibly other candidate cell substrates later) to express recombinant PrP genes bearing one or the other of the two point mutations most often found in patients with familial CJD (fCJD)and propagated until large numbers of cells are available. Cells will then be sequenced to confirm the continued presence of the fCJD-associated mutations and tested for the presence of the protease-resistant PrP that typically appears in tissues of humans and animals with TSEs. To determine whether actual infectivity has appeared during serial passaging of the cells bearing the fCJD-associated mutations, extracts from large numbers of these cells will be inoculated intracerebrally into squirrel monkeys--the most sensitive validated model for detection of human TSE agents; monkeys will be observed for at least five years for evidence of TSE. (If an adequate number of transgenic mice [see previous project] are available, cells may be assayed for infectivity in mice as well.) Funding for this part of the project has been granted by NIAID. Furthermore, even if the mutant proteins are not themselves infectious, their presence might increase susceptibility of cell substrates to infections with a TSE agent present in the environment. Therefore, should additional funds be obtained, the same recombinant cultures may be exposed to small amounts of reference CJD agent, propagated for a number of passages sufficient to eliminate all of the remaining original inoculum, and then assayed for genetic stability, protease-resistant PrP and infectivity as described above. Two results are possible. If no monkey develops disease, then we may conclude that, under conditions that highly favored replication of the TSE agent, no infectivity appeared, implying that the risk for recipients of biologics prepared in the unmutated cell substrate must be exceedingly small. But if any monkey develops a TSE, then the substrate must be considered to pose a potential threat. (The result might also provide at least circumstantial support for or against the "prion" hypothesis of spontaneous self-propagating changes in protein folding as the basis of TSE infectivity.)