Assessing Prion Infectivity of Human Urine in Sporadic Creutzfeldt-Jakob Disease

Silvio Notari¹, Liuting Qing¹, Maurizio Pocchiari, Ayuna Dagdanova, Kristin Hatcher, Arend Dogterom, Jose F. Groisman, Ib Bo Lumholtz, Maria Puopolo, Corinne Lasmezas, Shu G. Chen, Qingzhong Kong, and Pierluigi Gambetti

Author affiliations: Case Western Reserve University, Cleveland, Ohio, USA (S. Notari, L. Qing, A. Dagdanova, K. Hatcher, S.G. Chen, Q. Kong, P. Gambetti); Istituto Superiore di Sanità, Rome, Italy (M. Pocchiari, M. Puopolo); Ferring Pharmaceuticals, Hvidore, Denmark (A. Dogterom); Instituto Massone, Buenos Aires, Argentina (J.F. Groisman); BL Consult ApS, Copenhagen, Denmark (I.B. Lumholtz); The Scripps Research Institute, Jupiter, Florida, USA (C. Lasmezas)

Abstract

Prion diseases are neurodegenerative conditions associated with a misfolded and infectious protein, scrapie prion protein (PrP^Sc). PrP^Sc propagate prion diseases within and between species and thus pose risks to public health. Prion infectivity or PrP^Sc presence has been demonstrated in urine of experimentally infected animals, but there are no recent studies of urine from patients with Creutzfeldt-Jakob disease (CJD). We performed bioassays in transgenic mice expressing human PrP to assess prion infectivity in urine from patients affected by a common subtype of sporadic CJD, sCJDMM1. We tested raw urine and 100-fold concentrated and dialyzed urine and assessed the sensitivity of the bioassay along with the effect of concentration and dialysis on prion infectivity. Intracerebral inoculation of transgenic mice with urine from 3 sCJDMM1 patients failed to demonstrate prion disease transmission, indicating that prion infectivity in urine from sCJDMM1 patients is either not present or is <0.38 infectious units/mL.

Prion diseases, a group of neurodegenerative disorders affecting humans and animals, have received considerable attention largely because of their intriguing pathogenetic mechanism and the threat they pose to public health because of their insidious infectivity. Despite their heterogeneity, all classic prion diseases are characterized by the presence of an abnormal isoform of the normal cellular prion protein (PrP^C), which predominantly accumulates in the central nervous system (1). The abnormal isoform, identified as scrapie PrP or PrP^Sc, is thought to form from a posttranslational change in conformation of PrP^C. Prion diseases can also be transmitted by an infectious mechanism because exogenous PrP^Sc can impose its conformation on to the host’s PrP^C through a PrP^Sc-templated conversion process (1).

The most common form of human prion disease, Creutzfeldt-Jakob disease (CJD), can be sporadic, inherited, or acquired by infection; the sporadic form alone accounts for the great majority of all the cases of CJD (2). Following protease digestion, the unglycosylated form of PrP^Sc exhibits the electrophoretic mobilities of either 21 kDa or 19 kDa (3). These two PrP^Sc isoforms, named PrP^Sc types 1 and 2, respectively, along with the methionine/valine polymorphism at codon 129 of the PrP gene, led to the current classification of sporadic CJD (sCJD) in 5 phenotypically distinct subtypes (2,3). The sCJDMM1 subtype (affecting persons homozygous for methionine at codon 129 and carrying the PrP^Sc type 1) accounts for ≈70% of all cases of sCJD and unquestionably is the most prevalent type of human prion disease (2,3).
PrP^Sc is generally considered the major, if not the sole, component of the infectious agent in prion diseases (1,4,5). The presence of PrP^Sc, prion infectivity, or both has been found in several tissues and organs outside the central nervous system in prion-affected humans and animals (6–10). These findings have caused mounting concerns regarding the risk of human transmission of the disease from a variety of sources, including the consumption of prion contaminated meat and other animal products, the use of contaminated surgical instruments and medicinal products, and the exposure to waste from infected humans and animals. In this context, body fluids such as saliva, milk, and urine have received particular attention as they may support horizontal transmission and environmental contamination, which in turn may contribute to the propagation of ovine scrapie and chronic wasting disease (CWD) of cervids. Indeed, saliva has been reported to be a source of prion in scrapie-infected sheep and CWD-infected deer (11–14), whereas milk has been found to contain prions in scrapie infected sheep (15,16).

The detection of PrP^Sc by immunoblotting has been previously reported in urine of prion-affected hamsters and humans (17). However, this observation has not been confirmed in 3 subsequent studies, which instead have suggested that the original immunoblot finding resulted from nonspecific cross-reaction either with contaminating bacterial proteins (18) or urinary IgG fragments (19,20). Recently, prion infectivity has been detected in urine from experimentally prion-infected animals, including hamsters (21,22), deer (13), and mice in association with lymphocytic nephritis (23). However, infectivity in urine from naturally prion-affected animals has never been reported.

It is difficult to extrapolate the animal data on urine infectivity to human urine, especially for sCJD, because this form of prion disease is believed to start spontaneously in the brain rather than being caused by exogenous infection. Nevertheless, the possibility that PrP^Sc is indeed present in urine of sCJD patients exists because small quantities of PrP^Sc have been identified in peripheral organs of sCJD patients (6,7,10).

Furthermore, we have recently shown that normal urine contains discrete amounts of a C-terminal fragment of PrP, matching the so-called C1 fragment but not full-length PrP (24). Although C1 may not be a good substrate for PrP^Sc replication, and its conversion to PrP^Sc has never been reported, the possibility of conversion may not be ruled out (25,26).

The presence of prions in urine of CJD patients would obviously pose serious risks relating to the medicinal use of urine-extracted proteins, hormones, and urokinase as well as collection and disposal of patient urine. Indeed, it has recently been reported that fragments of PrP, consistent with the urine PrP present in normal urine described above, co-purify with urine-derived gonadotropins (24,27). This finding has prompted the claim that PrP^Sc may also co-purify with urinary gonadotropins (27).

With the exception of early failed attempts to transmit prion disease to rodents and nonhuman primates with urine from CJD patients (28,29), no investigation on prion infectivity of human urine has been reported. We searched for prion infectivity in urine obtained from patients with sCJDMM1, the most common form of sCJD, by bioassay that used transgenic (Tg) mice expressing human PrP (30).